U.S. patent application number 13/051350 was filed with the patent office on 2012-03-22 for methods for identifying compounds of interest using encoded libraries.
This patent application is currently assigned to GlaxoSmithKline LLC. Invention is credited to Raksha A. Acharya, Christopher C. Arico-Muendel, Dennis Benjamin, Paolo A. Centrella, Matthew Clark, Steffen Phillip Creaser, George J. Franklin, Malcolm L. Gefter, Stephen Hale, Nils Jakob Vest Hansen, David I. Israel, Malcolm J. Kavarana, Barry Morgan, Richard Wagner.
Application Number | 20120071329 13/051350 |
Document ID | / |
Family ID | 38006378 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120071329 |
Kind Code |
A1 |
Morgan; Barry ; et
al. |
March 22, 2012 |
METHODS FOR IDENTIFYING COMPOUNDS OF INTEREST USING ENCODED
LIBRARIES
Abstract
The present invention provides a method for identifying a
compound of interest by screening libraries of molecules which
include an encoding oligonucleotide tag.
Inventors: |
Morgan; Barry; (Franklin,
MA) ; Hale; Stephen; (Belmont, MA) ;
Arico-Muendel; Christopher C.; (West Roxbury, MA) ;
Clark; Matthew; (Cambridge, MA) ; Wagner;
Richard; (Cambridge, MA) ; Israel; David I.;
(Concord, MA) ; Gefter; Malcolm L.; (Lincoln,
MA) ; Benjamin; Dennis; (Redmond, WA) ;
Hansen; Nils Jakob Vest; (Copenhagen V, DK) ;
Kavarana; Malcolm J.; (Burlington, MA) ; Creaser;
Steffen Phillip; (Cambridge, MA) ; Franklin; George
J.; (Auburn, MA) ; Centrella; Paolo A.;
(Acton, MA) ; Acharya; Raksha A.; (Bedford,
MA) |
Assignee: |
GlaxoSmithKline LLC
Philadelphia
PA
|
Family ID: |
38006378 |
Appl. No.: |
13/051350 |
Filed: |
March 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11584880 |
Oct 23, 2006 |
7989395 |
|
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13051350 |
|
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60731464 |
Oct 28, 2005 |
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Current U.S.
Class: |
506/4 |
Current CPC
Class: |
C07H 21/00 20130101;
C12N 15/1068 20130101 |
Class at
Publication: |
506/4 |
International
Class: |
C40B 20/04 20060101
C40B020/04 |
Claims
1. A method for identifying one or more compounds which bind to a
biological target, said method comprising: (A) synthesizing a
library of compounds, wherein the compounds comprise a functional
moiety comprising two or more building blocks which is operatively
linked to an initial oligonucleotide which identifies the structure
of the functional moiety by: (i) providing a solution comprising m
initiator compounds, wherein m is an integer of 1 or greater, where
the initiator compounds consist of a functional moiety comprising n
building blocks, where n is an integer of 1 or greater, which is
operatively linked to an initial oligonucleotide which identifies
the n building blocks; (ii) dividing the solution of step (i) into
r reaction vessels, wherein r is an integer of 2 or greater,
thereby producing r aliquots of the solution; (iii) reacting the
initiator compounds in each reaction vessel with one of r building
blocks, thereby producing r aliquots comprising compounds
consisting of a functional moiety comprising n+1 building blocks
operatively linked to the initial oligonucleotide; and (iv)
reacting the initial oligonucleotide in each aliquot with one of a
set of r distinct incoming oligonucleotides in the presence of an
enzyme which catalyzes the ligation of the incoming oligonucleotide
and the initial oligonucleotide, under conditions suitable for
enzymatic ligation of the incoming oligonucleotide and the initial
oligonucleotide; thereby producing r aliquots of molecules
consisting of a functional moiety comprising n+1 building blocks
operatively linked to an elongated oligonucleotide which encodes
the n+1 building blocks; (B) contacting the biological target with
the library of compounds, or a portion thereof, under conditions
suitable for at least one member of the library of compounds to
bind to the target; (C) removing library members that do not bind
to the target; (D) sequencing the encoding oligonucleotides of the
at least one member of the library of compounds which binds to the
target, and (E) using the sequences determined in step (D) to
determine the structure of the functional moieties of the members
of the library of compounds which bind to the biological target,
thereby identifying one or more compounds which bind to the
biological target.
2. The method of claim 1, further comprising amplifying the
encoding oligonucleotides of the at least one member of the library
of compounds which binds to the target.
3. The method of claim 2, wherein said amplifying step comprises:
(i) forming a water-in-oil emulsion to create a plurality of
aqueous microreactors, wherein at least one of the microreactors
comprises the at least one member of the library of compounds that
binds to the target, a single bead capable of binding to the
encoding oligonucleotide of the at least one member of the library
of compounds that binds to the target, and amplification reaction
solution containing reagents necessary to perform nucleic acid
amplification; (ii) amplifying the encoding oligonucleotide in the
microreactors to form amplified copies of said encoding
oligonucleotide; and (iii) binding the amplified copies of the
encoding oligonucleotide to the beads in the microreactors.
4. The method of claim 1, wherein said sequencing step (D)
comprises: (i) annealing an effective amount of a sequencing primer
to the amplified copies of the encoding oligonucleotide and
extending the sequencing primer with a polymerase and a
predetermined nucleotide triphosphate to yield a sequencing product
and, if the predetermined nucleotide triphosphate is incorporated
onto a 3' end of said sequencing primer, a sequencing reaction
byproduct; and (ii) identifying the sequencing reaction byproduct,
thereby determining the sequence of the encoding
oligonucleotide.
5. A method for identifying one or more compounds which bind to a
biological target, said method comprising: (A) synthesizing a
library of compounds, wherein the compounds comprise a functional
moiety comprising two or more building blocks which is operatively
linked to an initial oligonucleotide which identifies the structure
of the functional moiety by: (i) providing a solution comprising m
initiator compounds, wherein m is an integer of 1 or greater, where
the initiator compounds consist of a functional moiety comprising n
building blocks, where n is an integer of 1 or greater, which is
operatively linked to an initial oligonucleotide which identifies
the n building blocks; (ii) dividing the solution of step (i) into
r reaction vessels, wherein r is an integer of 2 or greater,
thereby producing r aliquots of the solution; (iii) reacting the
initiator compounds in each reaction vessel with one of r building
blocks, thereby producing r aliquots comprising compounds
consisting of a functional moiety comprising n+1 building blocks
operatively linked to the initial oligonucleotide; and (iv)
reacting the initial oligonucleotide in each aliquot with one of a
set of r distinct incoming oligonucleotides in the presence of an
enzyme which catalyzes the ligation of the incoming oligonucleotide
and the initial oligonucleotide, under conditions suitable for
enzymatic ligation of the incoming oligonucleotide and the initial
oligonucleotide; thereby producing r aliquots of molecules
consisting of a functional moiety comprising n+1 building blocks
operatively linked to an elongated oligonucleotide which encodes
the n+1 building blocks; (B) contacting the biological target with
the library of compounds, or a portion thereof, under conditions
suitable for at least one member of the library of compounds to
bind to the target; (C) removing library members that do not bind
to the target; (D) sequencing the encoding oligonucleotides of the
at least one member of the library of compounds which binds to the
target, wherein said sequencing comprises: (i) annealing an
effective amount of a sequencing primer to the amplified copies of
the encoding oligonucleotide and extending the sequencing primer
with a polymerase and a predetermined nucleotide triphosphate to
yield a sequencing product and, if the predetermined nucleotide
triphosphate is incorporated onto a 3' end of said sequencing
primer, a sequencing reaction byproduct; and (ii) identifying the
sequencing reaction byproduct, thereby determining the sequence of
the encoding oligonucleotide; and (E) using the sequence of the
encoding oligonucleotide determined in step (D) to determine the
structure of the functional moieties of the members of the library
of compounds which bind to the biological target, thereby
identifying one or more compounds which bind to the biological
target.
6. The method of claim 5, further comprising amplifying the
encoding oligonucleotides of the at least one member of the library
of compounds which binds to the target.
7. The method of claim 6, wherein said amplification of the
encoding oligonucleotides is carried out by a method selected from
the group consisting of: the polymerase chain reaction (PCR);
transcription-based amplification, rapid amplification of cDNA
ends, continuous flow amplification, and rolling circle
amplification.
8. The method of any one of claim 1, 4, or 5, wherein said
sequencing of the encoding oligonucleotides is carried out by a
pyrophosphate-based sequencing reaction or a single molecule
sequencing by synthesis method.
9. The method of claim 8, wherein the sequencing reaction byproduct
is PPi and a coupled sulfurylase/luciferase reaction is used to
generate light for detection.
10. The method of any one of claim 1 or 5, further comprising the
step of enriching for beads which bind amplified copies of the
encoding oligonucleotide away from beads to which no encoding
oligonucleotide is bound.
11. The method of claim 10, wherein the method for said enrichment
step is selected from the group consisting of affinity
purification, and electrophoresis.
12. The method of claim 3, further comprising breaking the emulsion
to retrieve one or more of the amplified copies of the encoding
oligonucleotide.
13. The method of claim 1 or 5, further comprising the step of
(A)(v) combining two or more of the r aliquots, thereby producing a
solution comprising molecules consisting of a functional moiety
comprising n+1 building blocks, which is operatively linked to an
elongated oligonucleotide which encodes the n+1 building
blocks.
14. The method of claim 13, wherein r aliquots are combined.
15. The method of claim 13, wherein the steps (A)(i) to (A)(v) are
conducted one or more times to yield cycles 1 to i, where i is an
integer of 2 or greater, wherein in cycle s+1, where s is an
integer of i-1 or less, the solution comprising m initiator
compounds of step (a) is the solution of step (e) of cycle s.
16. The method of claim 1 or 5, wherein at least one of building
blocks is an amino acid.
17. The method of claim 1 or 5, wherein the initial oligonucleotide
is a covalently coupled double-stranded oligonucleotide.
18. The method of claim 17, wherein the incoming oligonucleotide is
a double-stranded oligonucleotide.
19. The method of claim 1 or 5, wherein the initiator compounds
comprise a linker moiety comprising a first functional group
adapted to bond with a building block, a second functional group
adapted to bond to the 5' end of an oligonucleotide, and a third
functional group adapted to bond to the 3'-end of an
oligonucleotide.
20. The method of claim 19, wherein the linker moiety is of the
structure ##STR00115## wherein A is a functional group adapted to
bond to a building block; B is a functional group adapted to bond
to the 5'-end of an oligonucleotide; C is a functional group
adapted to bond to the 3'-end of an oligonucleotide; S is an atom
or a scaffold; D is a chemical structure that connects A to S; E is
a chemical structure that connects B to S; and F is a chemical
structure that connects C to S.
21. The method of claim 20, wherein: A is an amino group; B is a
phosphate group; and C is a phosphate group.
22. The method of claim 20, wherein D, E and F are each,
independently, an alkylene group or an oligo(ethylene glycol)
group.
23. The method of claim 20, wherein S is a carbon atom, a nitrogen
atom, a phosphorus atom, a boron atom, a phosphate group, a cyclic
group or a polycyclic group.
24. The method of claim 23, wherein the linker moiety is of the
structure ##STR00116## wherein each of n, m and p is,
independently, an integer from 1 to about 20.
25. The method of claim 24, wherein each of n, m and p is
independently an integer from 2 to eight.
26. The method of claim 25, wherein each of n, m and p is
independently an integer from 3 to 6.
27. The method of claim 24, wherein the linker moiety has the
structure ##STR00117##
28. The method of claim 1 or 5, wherein each of said initiator
compounds comprises a reactive group and wherein each of said r
building blocks comprises a complementary reactive group which is
complementary to said reactive group.
29. The method of claim 28, wherein the reactive group and the
complementary reactive group are selected from the group consisting
of an amino group; a carboxyl group; a sulfonyl group; a phosphonyl
group; an epoxide group; an aziridine group; and an isocyanate
group.
30. The method of claim 28, wherein reactive group and the
complementary reactive group are selected from the group consisting
of a hydroxyl group; a carboxyl group; a sulfonyl group; a
phosphonyl group; an epoxide group; an aziridine group; and an
isocyanate group.
31. The method of claim 28, wherein the reactive group and the
complementary reactive group are selected from the group consisting
of an amino group and an aldehyde or ketone group.
32. The method of claim 28, wherein the reaction between the
reactive group and the complementary reactive group is conducted
under reducing conditions.
33. The method of claim 28, wherein the reactive group and the
complementary reactive group are selected from the group consisting
of a phosphorous ylide group and an aldehyde or ketone group.
34. The method of claim 28, wherein the reactive group and the
complementary reactive group react via cycloaddition to form a
cyclic structure.
35. The method of claim 34, wherein the reactive group and the
complementary reactive group are selected from the group consisting
of an alkyne and an azide.
36. The method of claim 28, wherein the reactive group and the
complementary functional group are selected from the group
consisting of a halogenated heteroaromatic group and a
nucleophile.
37. The method of claim 36, wherein the halogenated heteroaromatic
group is selected from the group consisting of chlorinated
pyrimidines, chlorinated triazines and chlorinated purines.
38. The method of claim 36, wherein the nucleophile is an amino
group.
39. The method of claim 13, further comprising following cycle i,
the step of: (A)(vi) cyclizing one or more of the functional
moieties.
40. The method of claim 39, wherein a functional moiety of step
(A)(vi) comprises an azido group and an alkynyl group.
41. The method of claim 40, wherein the functional moiety is
maintained under conditions suitable for cycloaddition of the azido
group and the alkynyl group to form a triazole group, thereby
forming a cyclic functional moiety.
42. The method of claim 41, wherein the cycloaddition reaction is
conducted in the presence of a copper catalyst.
43. The method of claim 42, wherein at least one of the one or more
functional moieties of step (f) comprises at least two sulfhydryl
groups, and said functional moiety is maintained under conditions
suitable for reaction of the two sulfhydryl groups to form a
disulfide group, thereby cyclicizing the functional moiety.
44. The method of claim 1 or 5, wherein the initial oligonucleotide
comprises a PCR primer sequence.
45. The method of claim 13, wherein the incoming oligonucleotide of
cycle i comprises a PCR closing primer.
46. The method of claim 13, further comprising following cycle i,
the step of (d) ligating an oligonucleotide comprising a closing
PCR primer sequence to the encoding oligonucleotide.
47. The method of claim 46, wherein the oligonucleotide comprising
a closing PCR primer sequence is ligated to the encoding
oligonucleotide in the presence of an enzyme which catalyzes said
ligation.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/584,880, filed Oct. 23, 2006, which claims
priority to U.S. Provisional Application No. 60/731,464, filed Oct.
28, 2005. This application is related to U.S. Patent Application
No. 60/689,466, filed Jun. 9, 2005, pending, and U.S. patent
application Ser. No. 11/015,458 filed Dec. 17, 2004. This
application is also related to U.S. Provisional Patent Application
Ser. No. 60/530,854, filed on Dec. 17, 2003; U.S. Provisional
Patent Application Ser. No. 60/540,681, filed on Jan. 30, 2004;
U.S. Provisional Patent Application Ser. No. 60/553,715 filed Mar.
15, 2004; and U.S. Provisional Patent Application Ser. No.
60/588,672 filed Jul. 16, 2004. The entire contents of each of the
foregoing applications are incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. The ASCII copy of the Sequence Listing,
created on Mar. 15, 2011, is named SeqList.txt, and is 219,888
bytes in size.
BACKGROUND OF THE INVENTION
[0003] The search for more efficient methods of identifying
compounds having useful biological activities has led to the
development of methods for screening vast numbers of distinct
compounds, present in collections referred to as combinatorial
libraries. Such libraries can include 10.sup.5 or more distinct
compounds. A variety of methods exist for producing combinatorial
libraries, and combinatorial syntheses of peptides, peptidomimetics
and small organic molecules have been reported.
[0004] The two major challenges in the use of combinatorial
approaches in drug discovery are the synthesis of libraries of
sufficient complexity and the identification of molecules which are
active in the screens used. It is generally acknowledged that
greater the degree of complexity of a library, i.e., the number of
distinct structures present in the library, the greater the
probability that the library contains molecules with the activity
of interest. Therefore, the chemistry employed in library synthesis
must be capable of producing vast numbers of compounds within a
reasonable time frame. However, for a given formal or overall
concentration, increasing the number of distinct members within the
library lowers the concentration of any particular library member.
This complicates the identification of active molecules from high
complexity libraries.
[0005] One approach to overcoming these obstacles has been the
development of encoded libraries, and particularly libraries in
which each compound includes an amplifiable tag. Such libraries
include DNA-encoded libraries, in which a DNA tag identifying a
library member can be amplified using techniques of molecular
biology, such as the polymerase chain reaction. However, the use of
such methods for producing very large libraries is yet to be
demonstrated, and it is clear that improved methods for producing
such libraries are required for the realization of the potential of
this approach to drug discovery.
SUMMARY OF THE INVENTION
[0006] Traditional drug discovery methods have relied on multi-step
selection processes, often involving the amplification (e.g., PCR
amplification) of nucleic acid molecules, and the sequencing of up
to 1,000 or more of the top clones. This multi-step selection
process and the nucleic acid amplification often lead to the
introduction of many biases (as discussed in, for example, Holt, L.
J., et al. (2000) Nucleic Acids Res. 28(15):E72). The presence of
these biases typically leads to the selection of compounds that
lack the desired biological activity.
[0007] The present invention provides improved methods as compared
to the prior art methods in that it provides methods which
eliminate the foregoing biases. For example, the present invention
provides methods of identifying a compound of interest using a
massively parallel sequencing approach which leads to the accurate
identification of a compound with a desired biological activity
using fewer selection steps. Moreover, as described herein, a
unique tagging system has been developed that eliminates biases
introduced by nucleic acid amplification, e.g., PCR amplification.
In addition, the methods described herein allow for an expansive
and extensive analysis of the selected compounds having a desired
biological property, which, in turn, allows for related compounds
with familial structural relationships to be identified (structure
activity relationships). In summary, using the methods of the
invention, a single step selection/enrichment cycle can be
performed and then sequencing can be performed at the single
molecule level, preferably without the need for any nucleic acid
amplification.
[0008] Accordingly, in one aspect, the invention provides a method
for identifying one or more compounds which bind to a biological
target. The method comprises synthesizing a library of compounds,
wherein the compounds comprise a functional moiety comprising two
or more building blocks which is operatively linked to an initial
oligonucleotide which identifies the structure of the functional
moiety by providing a solution comprising m initiator compounds,
wherein m is an integer of 1 or greater, where the initiator
compounds consist of a functional moiety comprising n building
blocks, where n is an integer of 1 or greater, which is operatively
linked to an initial oligonucleotide which identifies the n
building blocks, dividing the solution described above into r
reaction vessels, wherein r is an integer of 2 or greater, thereby
producing r aliquots of the solution, reacting the initiator
compounds in each reaction vessel with one of r building blocks,
thereby producing r aliquots comprising compounds consisting of a
functional moiety comprising n+1 building blocks operatively linked
to the initial oligonucleotide, and reacting the initial
oligonucleotide in each aliquot with one of a set of r distinct
incoming oligonucleotides in the presence of an enzyme which
catalyzes the ligation of the incoming oligonucleotide and the
initial oligonucleotide, under conditions suitable for enzymatic
ligation of the incoming oligonucleotide and the initial
oligonucleotide; thereby producing r aliquots of molecules
consisting of a functional moiety comprising n+1 building blocks
operatively linked to an elongated oligonucleotide which encodes
the n+1 building blocks; contacting the biological target with the
library of compounds, or a portion thereof, under conditions
suitable for at least one member of the library of compounds to
bind to the target, removing library members that do not bind to
the target, sequencing the encoding oligonucleotides of the at
least one member of the library of compounds which binds to the
target, and using the foregoing sequences to determine the
structure of the functional moieties of the members of the library
of compounds which bind to the biological target, thereby
identifying one or more compounds which bind to the biological
target.
[0009] In one embodiment, the methods of the invention may further
comprise amplifying the encoding oligonucleotide of the at least
one member of the library of compounds which binds to the target
prior to sequencing.
[0010] In one embodiment, the method of amplifying comprises
forming a water-in-oil emulsion to create a plurality of aqueous
microreactors, wherein at least one of the microreactors comprises
the at least one member of the library of compounds that binds to
the target, a single bead capable of binding to the encoding
oligonucleotide of the at least one member of the library of
compounds that binds to the target, and amplification reaction
solution containing reagents necessary to perform nucleic acid
amplification, amplifying the encoding oligonucleotide in the
microreactors to form amplified copies of the encoding
oligonucleotide, and binding the amplified copies of the encoding
oligonucleotide to the beads in the microreactors.
[0011] In one embodiment, the method of sequencing comprises
annealing an effective amount of a sequencing primer to the
amplified copies of the encoding oligonucleotide and extending the
sequencing primer with a polymerase and a predetermined nucleotide
triphosphate to yield a sequencing product and, if the
predetermined nucleotide triphosphate is incorporated onto a 3' end
of the sequencing primer, a sequencing reaction byproduct, and
identifying the sequencing reaction byproduct, thereby determining
the sequence of the encoding oligonucleotide.
[0012] In one embodiment, sequencing is performed using the
polymerase chain reaction. In another embodiment, sequencing is
performed using a pyrophosphate sequencing method or using a single
molecule sequencing by synthesis method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic representation of ligation of double
stranded oligonucleotides (SEQ ID NOS 921-926, respectively, in
order of appearance), in which the initial oligonucleotide has an
overhang which is complementary to the overhang of the incoming
oligonucleotide. The initial strand is represented as either free,
conjugated to an aminohexyl linker or conjugated to a phenylalanine
residue via an aminohexyl linker.
[0014] FIG. 2 is a schematic representation of oligonucleotide
ligation using a splint strand. In this embodiment, the splint is a
12-mer oligonucleotide with sequences complementary to the
single-stranded initial oligonucleotide and the single-stranded
incoming oligonucleotide.
[0015] FIG. 3 is a schematic representation of ligation of an
initial oligonucleotide and an incoming oligonucleotide, when the
initial oligonucleotide is double-stranded with covalently linked
strands, and the incoming oligonucleotide is double-stranded.
[0016] FIG. 4 is a schematic representation of oligonucleotide
elongation using a polymerase (SEQ ID NOS 921 & 927-929,
respectively, in order of appearance). The initial strand is
represented as either free, conjugated to an aminohexyl linker or
conjugated to a phenylalanine residue via an aminohexyl linker.
[0017] FIG. 5 is a schematic representation of the synthesis cycle
of one embodiment of the invention.
[0018] FIG. 6 is a schematic representation of a multiple round
selection process using the libraries of the invention.
[0019] FIG. 7 is a gel resulting from electrophoresis of the
products of each of cycles 1 to 5 described in Example 1 and
following ligation of the closing primer. Molecular weight
standards are shown in lane 1, and the indicated quantities of a
hyperladder, for DNA quantitation, are shown in lanes 9 to 12.
[0020] FIG. 8 is a schematic depiction of the coupling of building
blocks using azide-alkyne cycloaddition.
[0021] FIGS. 9 and 10 illustrate the coupling of building blocks
via nucleophilic aromatic substitution on a chlorinated
triazine.
[0022] FIG. 11 shows representative chlorinated heteroaromatic
structures suitable for use in the synthesis of functional
moieties.
[0023] FIG. 12 illustrates the cyclization of a linear peptide
using the azide/alkyne cycloaddition reaction.
[0024] FIG. 13a is a chromatogram of the library produced as
described in Example 2 following Cycle 4.
[0025] FIG. 13b is a mass spectrum of the library produced as
described in Example 2 following Cycle 4.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to methods of producing
compounds and combinatorial compound libraries, the compounds and
libraries produced via the methods of the invention, and methods of
using the libraries to identify compounds having a desired
property, such as a desired biological activity. The invention
further relates to the compounds identified using these
methods.
[0027] A variety of approaches have been taken to produce and
screen combinatorial chemical libraries. Examples include methods
in which the individual members of the library are physically
separated from each other, such as when a single compound is
synthesized in each of a multitude of reaction vessels. However,
these libraries are typically screened one compound at a time, or
at most, several compounds at a time and do not, therefore, result
in the most efficient screening process. In other methods,
compounds are synthesized on solid supports. Such solid supports
include chips in which specific compounds occupy specific regions
of the chip or membrane ("position addressable"). In other methods,
compounds are synthesized on beads, with each bead containing a
different chemical structure.
[0028] Two difficulties that arise in screening large libraries are
(1) the number of distinct compounds that can be screened; and (2)
the identification of compounds which are active in the screen. In
one method, the compounds which are active in the screen are
identified by narrowing the original library into ever smaller
fractions and subfractions, in each case selecting the fraction or
subfraction which contains active compounds and further subdividing
until attaining an active subfraction which contains a set of
compounds which is sufficiently small that all members of the
subset can be individually synthesized and assessed for the desired
activity. This is a tedious and time consuming activity.
[0029] Another method of deconvoluting the results of a
combinatorial library screen is to utilize libraries in which the
library members are tagged with an identifying label, that is, each
label present in the library is associated with a discreet compound
structure present in the library, such that identification of the
label tells the structure of the tagged molecule. One approach to
tagged libraries utilizes oligonucleotide tags, as described, for
example, in U.S. Pat. Nos. 5,573,905; 5,708,153; 5,723,598,
6,060,596 published PCT applications WO 93/06121; WO 93/20242; WO
94/13623; WO 00/23458; WO 02/074929 and WO 02/103008, and by
Brenner and Lerner (Proc. Natl. Acad. Sci. USA 89, 5381-5383
(1992); Nielsen and Janda (Methods: A Companion to Methods in
Enzymology 6, 361-371 (1994); and Nielsen, Brenner and Janda (J.
Am. Chem. Soc. 115, 9812-9813 (1993)), each of which is
incorporated herein by reference in its entirety. Such tags can be
amplified, using for example, polymerase chain reaction, to produce
many copies of the tag and identify the tag by sequencing. The
sequence of the tag then identifies the structure of the binding
molecule, which can be synthesized in pure form and tested. To
date, there has been no report of the use of the methodology
disclosed by Lerner et al. to prepare large libraries. The present
invention provides an improvement in methods to produce DNA-encoded
libraries, as well as the first examples of large (10.sup.5 members
or greater) libraries of DNA-encoded molecules in which the
functional moiety is synthesized using solution phase synthetic
methods.
[0030] The present invention provides methods which enable facile
synthesis of oligonucleotide-encoded combinatorial libraries, and
permit an efficient, high-fidelity means of adding such an
oligonucleotide tag to each member of a vast collection of
molecules.
[0031] The methods of the invention include methods for
synthesizing bifunctional molecules which comprise a first moiety
("functional moiety") which is made up of building blocks, and a
second moiety operatively linked to the first moiety, comprising an
oligonucleotide tag which identifies the structure of the first
moiety, i.e., the oligonucleotide tag indicates which building
blocks were used in the construction of the first moiety, as well
as the order in which the building blocks were linked. Generally,
the information provided by the oligonucleotide tag is sufficient
to determine the building blocks used to construct the active
moiety. In certain embodiments, the sequence of the oligonucleotide
tag is sufficient to determine the arrangement of the building
blocks in the functional moiety, for example, for peptidic
moieties, the amino acid sequence.
[0032] The term "functional moiety" as used herein, refers to a
chemical moiety comprising one or more building blocks. Preferably,
the building blocks in the functional moiety are not nucleic acids.
The functional moiety can be a linear or branched or cyclic polymer
or oligomer or a small organic molecule.
[0033] The term "building block", as used herein, is a chemical
structural unit which is linked to other chemical structural units
or can be linked to other such units. When the functional moiety is
polymeric or oligomeric, the building blocks are the monomeric
units of the polymer or oligomer. Building blocks can also include
a scaffold structure ("scaffold building block") to which is, or
can be, attached one or more additional structures ("peripheral
building blocks").
[0034] It is to be understood that the term "building block" is
used herein to refer to a chemical structural unit as it exists in
a functional moiety and also in the reactive form used for the
synthesis of the functional moiety. Within the functional moiety, a
building block will exist without any portion of the building block
which is lost as a consequence of incorporating the building block
into the functional moiety. For example, in cases in which the
bond-forming reaction releases a small molecule (see below), the
building block as it exists in the functional moiety is a "building
block residue", that is, the remainder of the building block used
in the synthesis following loss of the atoms that it contributes to
the released molecule.
[0035] The building blocks can be any chemical compounds which are
complementary, that is the building blocks must be able to react
together to form a, structure comprising two or more building
blocks. Typically, all of the building blocks used will have at
least two reactive groups, although it is possible that some of the
building blocks (for example the last building block in an
oligomeric functional moiety) used will have only one reactive
group each. Reactive groups on two different building blocks should
be complementary, i.e., capable of reacting together to form a
covalent bond, optionally with the concomitant loss of a small
molecule, such as water, HCl, HF, and so forth.
[0036] For the present purposes, two reactive groups are
complementary if they are capable of reacting together to form a
covalent bond. In a preferred embodiment, the bond forming
reactions occur rapidly under ambient conditions without
substantial formation of side products. Preferably, a given
reactive group will react with a given complementary reactive group
exactly once. In one embodiment, complementary reactive groups of
two building blocks react, for example, via nucleophilic
substitution, to form a covalent bond. In one embodiment, one
member of a pair of complementary reactive groups is an
electrophilic group and the other member of the pair is a
nucleophilic group.
[0037] Complementary electrophilic and nucleophilic groups include
any two groups which react via nucleophilic substitution under
suitable conditions to form a covalent bond. A variety of suitable
bond-forming reactions are known in the art. See, for example,
March, Advanced Organic Chemistry, fourth edition, New York: John
Wiley and Sons (1992), Chapters 10 to 16; Carey and Sundberg,
Advanced Organic Chemistry, Part B, Plenum (1990), Chapters 1-11;
and Collman et al., Principles and Applications of Organotransition
Metal Chemistry, University Science Books, Mill Valley, Calif.
(1987), Chapters 13 to 20; each of which is incorporated herein by
reference in its entirety. Examples of suitable electrophilic
groups include reactive carbonyl groups, such as acyl chloride
groups, ester groups, including carbonyl pentafluorophenyl esters
and succinimide esters, ketone groups and aldehyde groups; reactive
sulfonyl groups, such as sulfonyl chloride groups, and reactive
phosphonyl groups. Other electrophilic groups include terminal
epoxide groups, isocyanate groups and alkyl halide groups. Suitable
nucleophilic groups include primary and secondary amino groups and
hydroxyl groups and carboxyl groups.
[0038] Suitable complementary reactive groups are set forth below.
One of skill in the art can readily determine other reactive group
pairs that can be used in the present method, and the examples
provided herein are not intended to be limiting.
[0039] In a first embodiment, the complementary reactive groups
include activated carboxyl groups, reactive sulfonyl groups or
reactive phosphonyl groups, or a combination thereof, and primary
or secondary amino groups. In this embodiment, the complementary
reactive groups react under suitable conditions to form an amide,
sulfonamide or phosphonamidate bond.
[0040] In a second embodiment, the complementary reactive groups
include epoxide groups and primary or secondary amino groups. An
epoxide-containing building block reacts with an amine-containing
building block under suitable conditions to form a carbon-nitrogen
bond, resulting in a .beta.-amino alcohol.
[0041] In another embodiment, the complementary reactive groups
include aziridine groups and primary or secondary amino groups.
Under suitable conditions, an aziridine-containing building block
reacts with an amine-containing building block to form a
carbon-nitrogen bond, resulting in a 1,2-diamine. In a third
embodiment, the complementary reactive groups include isocyanate
groups and primary or secondary amino groups. An
isocyanate-containing building block will react with an
amino-containing building block under suitable conditions to form a
carbon-nitrogen bond, resulting in a urea group.
[0042] In a fourth embodiment, the complementary reactive groups
include isocyanate groups and hydroxyl groups. An
isocyanate-containing building block will react with an
hydroxyl-containing building block under suitable conditions to
form a carbon-oxygen bond, resulting in a carbamate group.
[0043] In a fifth embodiment, the complementary reactive groups
include amino groups and carbonyl-containing groups, such as
aldehyde or ketone groups. Amines react with such groups via
reductive amination to form a new carbon-nitrogen bond.
[0044] In a sixth embodiment, the complementary reactive groups
include phosphorous ylide groups and aldehyde or ketone groups. A
phosphorus-ylide-containing building block will react with an
aldehyde or ketone-containing building block under suitable
conditions to form a carbon-carbon double bond, resulting in an
alkene.
[0045] In a seventh embodiment, the complementary reactive groups
react via cycloaddition to form a cyclic structure. One example of
such complementary reactive groups are alkynes and organic azides,
which react under suitable conditions to form a triazole ring
structure. An example of the use of this reaction to link two
building blocks is illustrated in FIG. 8. Suitable conditions for
such reactions are known in the art and include those disclosed in
WO 03/101972, the entire contents of which are incorporated by
reference herein.
[0046] In an eighth embodiment, the complementary reactive groups
are an alkyl halide and a nucleophile, such as an amino group, a
hydroxyl group or a carboxyl group. Such groups react under
suitable conditions to form a carbon-nitrogen (alkyl halide plus
amine) or carbon oxygen (alkyl halide plus hydroxyl or carboxyl
group).
[0047] In a ninth embodiment, the complementary functional groups
are a halogenated heteroaromatic group and a nucleophile, and the
building blocks are linked under suitable conditions via aromatic
nucleophilic substitution. Suitable halogenated heteroaromatic
groups include chlorinated pyrimidines, triazines and purines,
which react with nucleophiles, such as amines, under mild
conditions in aqueous solution. Representative examples of the
reaction of an oligonucleotide-tagged trichlorotriazine with amines
are shown in FIGS. 9 and 10. Examples of suitable chlorinated
heteroaromatic groups are shown in FIG. 11.
[0048] Additional bond-forming reactions that can be used to join
building blocks in the synthesis of the molecules and libraries of
the invention include those shown below. The reactions shown below
emphasize the reactive functional groups. Various substituents can
be present in the reactants, including those labeled R.sub.1,
R.sub.2, R.sub.3 and R.sub.4. The possible positions which can be
substituted include, but are not limited, to those indicated by
R.sub.1, R.sub.2, R.sub.3 and R.sub.4. These substituents can
include any suitable chemical moieties, but are preferably limited
to those which will not interfere with or significantly inhibit the
indicated reaction, and, unless otherwise specified, can include
hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,
heteroaryl, substituted heteroaryl, alkoxy, aryloxy, arylalkyl,
substituted arylalkyl, amino, substituted amino and others as are
known in the art. Suitable substituents on these groups include
alkyl, aryl, heteroaryl, cyano, halogen, hydroxyl, nitro, amino,
mercapto, carboxyl, and carboxamide. Where specified, suitable
electron-withdrawing groups include nitro, carboxyl, haloalkyl,
such as trifluoromethyl and others as are known in the art.
Examples of suitable electron-donating groups include alkyl,
alkoxy, hydroxyl, amino, halogen, acetamido and others as are known
in the art.
Addition of a Primary Amine to an Alkene:
##STR00001##
[0049] Nucleophilic Substitution:
##STR00002##
[0050] Reductive Alkylation of an Amine:
##STR00003##
[0051] Palladium Catalyzed Carbon-Carbon Bond Forming
Reactions:
##STR00004##
[0052] Ugi Condensation Reactions:
##STR00005##
[0053] Electrophilic Aromatic Substitution Reactions:
##STR00006##
[0054] X is an electron-donating group.
Imine/Iminium/Enamine Forming Reactions:
##STR00007##
[0055] Cycloaddition Reactions:
##STR00008##
[0057] Diels-Alder Cycloaddition
##STR00009##
[0058] 1,3-dipolar cycloaddition, X--Y--Z.dbd.C--N--O, C--N--S,
N.sub.3.
Nucleophilic Aromatic Substitution Reactions:
##STR00010##
[0059] W is an electron withdrawing group
##STR00011##
[0060] Examples of suitable substituents X and Y include
substituted or unsubstituted amino, substituted or unsubstituted
alkoxy, substituted or unsubstituted thioalkoxy, substituted or
unsubstituted aryloxy and substituted and unsubstituted
thioaryloxy.
##STR00012##
Heck Reaction:
##STR00013##
[0061] Acetal Formation:
##STR00014##
[0063] Examples of suitable substituents X and Y include
substituted and unsubstituted amino, hydroxyl and sulhydryl; Y is a
linker that connects X and Y and is suitable for forming the ring
structure found in the product of the reaction.
Aldol Reactions:
##STR00015##
[0064] Examples of suitable substituents X include O, S and
NR.sub.3.
[0065] Scaffold building blocks which can be used to form the
molecules and libraries of the invention include those which have
two or more functional groups which can participate in bond forming
reactions with peripheral building block precursors, for example,
using one or more of the bond forming reactions discussed above.
Scaffold moieties may also be synthesized during construction of
the libraries and molecules of the invention, for example, using
building block precursors which can react in specific ways to form
molecules comprising a central molecular moiety to which are
appended peripheral functional groups. In one embodiment, a library
of the invention comprises molecules comprising a constant scaffold
moiety, but different peripheral moieties or different arrangements
of peripheral moieties. In certain libraries, all library members
comprise a constant scaffold moiety; other libraries can comprise
molecules having two or more different scaffold moieties. Examples
of scaffold moiety-forming reactions that can be used in the
construction of the molecules and libraries of the invention are
set forth in the Table. The references cited in the table are
incorporated herein by reference in their entirety. The groups
R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are limited only in that they
should not interfere with, or significantly inhibit, the indicated
reaction, and can include hydrogen, alkyl, substituted alkyl,
heteroalkyl, substituted heteroalkyl, cycloalkyl, heterocycloalkyl,
substituted cycloalkyl, substituted heterocycloalkyl, aryl,
substituted aryl, arylalkyl, heteroarylalkyl, substituted
arylalkyl, substituted heteroarylalkyl, heteroaryl, substituted
heteroaryl, halogen, alkoxy, aryloxy, amino, substituted amino and
others as are known in the art. Suitable substituents include, but
are not limited to, alkyl, alkoxy, thioalkoxy, nitro, hydroxyl,
sulfhydryl, aryloxy, aryl-S--, halogen, carboxy, amino, alkylamino,
dialkylamino, arylamino, cyano, cyanate, nitrile, isocyanate,
thiocyanate, carbamyl, and substituted carbamyl.
[0066] It is to be understood that the synthesis of a functional
moiety can proceed via one particular type of coupling reaction,
such as, but not limited to, one of the reactions discussed above,
or via a combination of two or more coupling reactions, such as two
or more of the coupling reactions discussed above. For example, in
one embodiment, the building blocks are joined by a combination of
amide bond formation (amino and carboxylic acid complementary
groups) and reductive amination (amino and aldehyde or ketone
complementary groups). Any coupling chemistry can be used, provided
that it is compatible with the presence of an oligonucleotide.
Double stranded (duplex) oligonucleotide tags, as used in certain
embodiments of the present invention, are chemically more robust
than single stranded tags, and, therefore, tolerate a broader range
of reaction conditions and enable the use of bond-forming reactions
that would not be possible with single-stranded tags.
[0067] A building block can include one or more functional groups
in addition to the reactive m group or groups employed to form the
functional moiety. One or more of these additional functional
groups can be protected to prevent undesired reactions of these
functional groups. Suitable protecting groups are known in the art
for a variety of functional groups (Greene and Wuts, Protective
Groups in Organic Synthesis, second edition, New York: John Wiley
and Sons (1991), incorporated herein by reference). Particularly
useful protecting groups include t-butyl esters and ethers,
acetals, trityl ethers and amines, acetyl esters, trimethylsilyl
ethers, trichloroethyl ethers and esters and carbamates.
[0068] In one embodiment, each building block comprises two
reactive groups, which can be the same or different. For example,
each building block added in cycle s can comprise two reactive
groups which are the same, but which are both complementary to the
reactive groups of the building blocks added at steps s-1 and s+1.
In another embodiment, each building block comprises two reactive
groups which are themselves complementary. For example, a library
comprising polyamide molecules can be produced via reactions
between building blocks comprising two primary amino groups and
building blocks comprising two activated carboxyl groups. In the
resulting compounds there is no N- or C-terminus, as alternate
amide groups have opposite directionality. Alternatively, a
polyamide library can be produced using building blocks that each
comprise an amino group and an activated carboxyl group. In this
embodiment, the building blocks added in step n of the cycle will
have a free reactive group which is complementary to the available
reactive group on the n-1 building block, while, preferably, the
other reactive group on the nth building block is protected. For
example, if the members of the library are synthesized from the C
to N direction, the building blocks added will comprise an
activated carboxyl group and a protected amino group.
[0069] The functional moieties can be polymeric or oligomeric
moieties, such as peptides, peptidomimetics, peptide nucleic acids
or peptoids, or they can be small non-polymeric molecules, for
example, molecules having a structure comprising a central scaffold
and structures arranged about the periphery of the scaffold. Linear
polymeric or oligomeric libraries will result from the use of
building blocks having two reactive groups, while branched
polymeric or oligomeric libraries will result from the use of
building blocks having three or more reactive groups, optionally in
combination with building blocks having only two reactive groups.
Such molecules can be represented by the general formula
X.sub.1X.sub.2 . . . X.sub.n, where each X is a monomeric unit of a
polymer comprising n monomeric units, where n is an integer greater
than 1 In the case of oligomeric or polymeric compounds, the
terminal building blocks need not comprise two functional groups.
For example, in the case of a polyamide library, the C-terminal
building block can comprise an amino group, but the presence of a
carboxyl group is optional. Similarly, the building block at the
N-terminus can comprise a carboxyl group, but need not contain an
amino group.
[0070] Branched oligomeric or polymeric compounds can also be
synthesized provided that at least one building block comprises
three functional groups which are reactive with other building
blocks. A library of the invention can comprise linear molecules,
branched molecules or a combination thereof.
[0071] Libraries can also be constructed using, for example, a
scaffold building block having two or more reactive groups, in
combination with other building blocks having only one available
reactive group, for example, where any additional reactive groups
are either protected or not reactive with the other reactive groups
present in the scaffold building block. In one embodiment, for
example, the molecules synthesized can be represented by the
general formula X(Y).sub.n, where X is a scaffold building block;
each Y is a building block linked to X and n is an integer of at
least two, and preferably an integer from 2 to about 6. In one
preferred embodiment, the initial building block of cycle 1 is a
scaffold building block. In molecules of the formula X(Y).sub.n,
each Y can be the same or different, but in most members of a
typical library, each Y will be different.
[0072] In one embodiment, the libraries of the invention comprise
polyamide compounds. The polyamide compounds can be composed of
building blocks derived from any amino acids, including the twenty
naturally occurring .alpha.-amino acids, such as alanine (Ala; A),
glycine (Gly; G), asparagine (Asn; N), aspartic acid (Asp; D),
glutamic acid (Glu; E), histidine (His; H), leucine (Leu; L),
lysine (Lys; K), phenylalanine (Phe; F), tyrosine (Tyr; Y),
threonine (Thr; T), serine (Ser; S), arginine (Arg; R), valine
(Val; V), glutamine (Gln; Q), isoleucine (Ile; I), cysteine (Cys;
C), methionine (Met; M), proline (Pro; P) and tryptophan (Trp; W),
where the three-letter and one-letter codes for each amino acid are
given. In their naturally occurring form, each of the foregoing
amino acids exists in the L-configuration, which is to be assumed
herein unless otherwise noted. In the present method, however, the
D-configuration forms of these amino acids can also be used. These
D-amino acids are indicated herein by lower case three- or
one-letter code, i.e., ala (a), gly (g), leu (I), gln (q), thr (t),
ser (s), and so forth. The building blocks can also be derived from
other .alpha.-amino acids, including, but not limited to,
3-arylalanines, such as naphthylalanine, phenyl-substituted
phenylalanines, including 4-fluoro-, 4-chloro, 4-bromo and
4-methylphenylalanine; 3-heteroarylalanines, such as
3-pyridylalanine, 3-thienylalanine, 3-quinolylalanine, and
3-imidazolylalanine; ornithine; citrulline; homocitrulline;
sarcosine; homoproline; homocysteine; substituted proline, such as
hydroxyproline and fluoroproline; dehydroproline; norleucine;
O-methyltyrosine; O-methylserine; O-methylthreonine and
3-cyclohexylalanine. Each of the preceding amino acids can be
utilized in either the D- or L-configuration.
[0073] The building blocks can also be amino acids which are not
.alpha.-amino acids, such as .alpha.-azamino acids; .beta.,
.gamma., .delta., .epsilon.,-amino acids, and N-substituted amino
acids, such as N-substituted glycine, where the N-substituent can
be, for example, a substituted or unsubstituted alkyl, aryl,
heteroaryl, arylalkyl or heteroarylalkyl group. In one embodiment,
the N-substituent is a side chain from a naturally-occurring or
non-naturally occurring .alpha.-amino acid.
[0074] The building block can also be a peptidomimetic structure,
such as a dipeptide, tripeptide, tetrapeptide or pentapeptide
mimetic. Such peptidomimetic building blocks are preferably derived
from amino acyl compounds, such that the chemistry of addition of
these building blocks to the growing poly(aminoacyl) group is the
same as, or similar to, the chemistry used for the other building
blocks. The building blocks can also be molecules which are capable
of forming bonds which are isosteric with a peptide bond, to form
peptidomimetic functional moieties comprising a peptide backbone
modification, such as .psi.[CH.sub.2S], .psi.[CH.sub.2NH],
.psi.[CSNH.sub.2], .psi.[NHCO], .psi.[COCH.sub.2], and .psi.[(E) or
(Z) CH.dbd.CH]. In the nomenclature used above, .psi. indicates the
absence of an amide bond. The structure that replaces the amide
group is specified within the brackets.
[0075] In one embodiment, the invention provides a method of
synthesizing a compound comprising or consisting of a functional
moiety which is operatively linked to an encoding oligonucleotide.
The method includes the steps of: (1) providing an initiator
compound consisting of an initial functional moiety comprising n
building blocks, where n is an integer of 1 or greater, wherein the
initial functional moiety comprises at least one reactive group,
and wherein the initial functional moiety is operatively linked to
an initial oligonucleotide which encodes the n building blocks; (2)
reacting the initiator compound with a building block comprising at
least one complementary reactive group, wherein the at least one
complementary reactive group is complementary to the reactive group
of step (1), under suitable conditions for reaction of the reactive
group and the complementary reactive group to form a covalent bond;
(3) reacting the initial oligonucleotide with an incoming
oligonucleotide in the presence of an enzyme which catalyzes
ligation of the initial oligonucleotide and the incoming
oligonucleotide, under conditions suitable for ligation of the
incoming oligonucleotide and the initial oligonucleotide, thereby
producing a molecule which comprises or consists of a functional
moiety comprising n+1 building blocks which is operatively linked
to an encoding oligonucleotide. If the functional moiety of step
(3) comprises a reactive group, steps 1-3 can be repeated one or
more times, thereby forming cycles 1 to i, where i is an integer of
2 or greater, with the product of step (3) of a cycle s-1, where s
is an integer of i or less, becoming the initiator compound of step
(1) of cycle s. In each cycle, one building block is added to the
growing functional moiety and one oligonucleotide sequence, which
encodes the new building block, is added to the growing encoding
oligonucleotide.
[0076] In one embodiment, the initial initiator compound(s) is
generated by reacting a first building block with an
oligonucleotide (e.g., an oligonucleotide which includes PCR primer
sequences or an initial oligonucleotide) or with a linker to which
such an oligonucleotide is attached. In the embodiment set forth in
FIG. 5, the linker comprises a reactive group for attachment of a
first building block and is attached to an initial oligonucleotide.
In this embodiment, reaction of a building block, or in each of
multiple aliquots, one of a collection of building blocks, with the
reactive group of the linker and addition of an oligonucleotide
encoding the building block to the initial oligonucleotide produces
the one or more initial initiator compounds of the process set
forth above.
[0077] In a preferred embodiment, each individual building block is
associated with a distinct oligonucleotide, such that the sequence
of nucleotides in the oligonucleotide added in a given cycle
identifies the building block added in the same cycle.
[0078] The coupling of building blocks and ligation of
oligonucleotides will generally occur at similar concentrations of
starting materials and reagents. For example, concentrations of
reactants on the order of micromolar to millimolar, for example
from about 10 .mu.M to about 10 mM, are preferred in order to have
efficient coupling of building blocks.
[0079] In certain embodiments, the method further comprises,
following step (2), the step of scavenging any unreacted initial
functional moiety. Scavenging any unreacted initial functional
moiety in a particular cycle prevents the initial functional moiety
of the cycle from reacting with a building block added in a later
cycle. Such reactions could lead to the generation of functional
moieties missing one or more building blocks, potentially leading
to a range of functional moiety structures which correspond to a
particular oligonucleotide sequence. Such scavenging can be
accomplished by reacting any remaining initial functional moiety
with a compound which reacts with the reactive group of step (2).
Preferably, the scavenger compound reacts rapidly with the reactive
group of step (2) and includes no additional reactive groups that
can react with building blocks added in later cycles. For example,
in the synthesis of a compound where the reactive group of step (2)
is an amino group, a suitable scavenger compound is an
N-hydroxysuccinimide ester, such as acetic acid
N-hydroxysuccinimide ester.
[0080] In another embodiment, the invention provides a method of
producing a library of compounds, wherein each compound comprises a
functional moiety comprising two or more building block residues
which is operatively linked to an oligonucleotide. In a preferred
embodiment, the oligonucleotide present in each molecule provides
sufficient information to identify the building blocks within the
molecule and, optionally, the order of addition of the building
blocks. In this embodiment, the method of the invention comprises a
method of synthesizing a library of compounds, wherein the
compounds comprise a functional moiety comprising two or more
building blocks which is operatively linked to an oligonucleotide
which identifies the structure of the functional moiety. The method
comprises the steps of (1) providing a solution comprising m
initiator compounds, wherein m is an integer of 1 or greater, where
the initiator compounds consist of a functional moiety comprising n
building blocks, where n is an integer of 1 or greater, which is
operatively linked to an initial oligonucleotide which identifies
the n building blocks; (2) dividing the solution of step (1) into
at least r fractions, wherein r is an integer of 2 or greater; (3)
reacting each fraction with one of r building blocks, thereby
producing r fractions comprising compounds consisting of a
functional moiety comprising n+1 building blocks operatively linked
to the initial oligonucleotide; (4) reacting each of the r
fractions of step (3) with one of a set of r distinct incoming
oligonucleotides under conditions suitable for enzymatic ligation
of the incoming oligonucleotide to the initial oligonucleotide,
thereby producing r fractions comprising molecules consisting of a
functional moiety comprising n+1 building blocks operatively linked
to an elongated oligonucleotide which encodes the n+1 building
blocks. Optionally, the method can further include the step of (5)
recombining the r fractions, produced in step (4), thereby
producing a solution comprising molecules consisting of a
functional moiety comprising n+1 building blocks, which is
operatively linked to an elongated oligonucleotide which encodes
the n+1 building blocks. Steps (1) to (5) can be conducted one or
more times to yield cycles 1 to i, where i is an integer of 2 or
greater. In cycle s+1, where s is an integer of i-1 or less, the
solution comprising m initiator compounds of step (1) is the
solution of step (5) of cycle s. Likewise, the initiator compounds
of step (1) of cycle s+1 are the products of step (4) in cycle
s.
[0081] Preferably the solution of step (2) is divided into r
fractions in each cycle of the library synthesis. In this
embodiment, each fract is reated with a unique building block.
[0082] In the methods of the invention, the order of addition of
the building block and the incoming oligonucleotide is not
critical, and steps (2) and (3) of the synthesis of a molecule, and
steps (3) and (4) in the library synthesis can be reversed, i.e.,
the incoming oligonucleotide can be ligated to the initial
oligonucleotide before the new building block is added. In certain
embodiments, it may be possible to conduct these two steps
simultaneously.
[0083] In certain embodiments, the method further comprises,
following step (2), the step of scavenging any unreacted initial
functional moiety. Scavenging any unreacted initial functional
moiety in a particular cycle prevents the initial functional moiety
of a the cycle from reacting with a building block added in a later
cycle. Such reactions could lead to the generation of functional
moieties missing one or more building blocks, potentially leading
to a range of functional moiety structures which correspond to a
particular oligonucleotide sequence. Such scavenging can be
accomplished by reacting any remaining initial functional moiety
with a compound which reacts with the reactive group of step (2).
Preferably, the scavenger compound reacts rapidly with the reactive
group of step (2) and includes no additional reactive groups that
can react with building blocks added in later cycles. For example,
in the synthesis of a compound where the reactive group of step (2)
is an amino group, a suitable scavenger compound is an
N-hydroxysuccinimide ester, such as acetic acid
N-hydroxysuccinimide ester.
[0084] In one embodiment, the building blocks used in the library
synthesis are selected from a set of candidate building blocks by
evaluating the ability of the candidate building blocks to react
with appropriate complementary functional groups under the
conditions used for synthesis of the library. Building blocks which
are shown to be suitably reactive under such conditions can then be
selected for incorporation into the library. The products of a
given cycle can, optionally, be purified. When the cycle is an
intermediate cycle, i.e., any cycle prior to the final cycle, these
products are intermediates and can be purified prior to initiation
of the next cycle. If the cycle is the final cycle, the products of
the cycle are the final products, and can be purified prior to any
use of the compounds. This purification step can, for example,
remove unreacted or excess reactants and the enzyme employed for
oligonucleotide ligation. Any methods which are suitable for
separating the products from other species present in solution can
be used, including liquid chromatography, such as high performance
liquid chromatography (HPLC) and precipitation with a suitable
co-solvent, such as ethanol. Suitable methods for purification will
depend upon the nature of the products and the solvent system used
for synthesis.
[0085] The reactions are, preferably, conducted in aqueous
solution, such as a buffered aqueous solution, but can also be
conducted in mixed aqueous/organic media consistent with the
solubility properties of the building blocks, the oligonucleotides,
the intermediates and final products and the enzyme used to
catalyze the oligonucleotide ligation.
[0086] It is to be understood that the theoretical number of
compounds produced by a given cycle in the method described above
is the product of the number of different initiator compounds, m,
used in the cycle and the number of distinct building blocks added
in the cycle, r. The actual number of distinct compounds produced
in the cycle can be as high as the product of r and m (r.times.m),
but could be lower, given differences in reactivity of certain
building blocks with certain other building blocks. For example,
the kinetics of addition of a particular building block to a
particular initiator compound may be such that on the time scale of
the synthetic cycle, little to none of the product of that reaction
may be produced.
[0087] In certain embodiments, a common building block is added
prior to cycle 1, following the last cycle or in between any two
cycles. For example, when the functional moiety is a polyamide, a
common N-terminal capping building block can be added after the
final cycle. A common building block can also be introduced between
any two cycles, for example, to add a functional group, such as an
alkyne or azide group, which can be utilized to modify the
functional moieties, for example by cyclization, following library
synthesis.
[0088] The term "operatively linked", as used herein, means that
two chemical structures are linked together in such a way as to
remain linked through the various manipulations they are expected
to undergo. Typically the functional moiety and the encoding
oligonucleotide are linked covalently via an appropriate linking
group. The linking group is a bivalent moiety with a site of
attachment for the oligonucleotide and a site of attachment for the
functional moiety. For example, when the functional moiety is a
polyamide compound, the polyamide compound can be attached to the
linking group at its N-terminus, its C-terminus or via a functional
group on one of the side chains. The linking group is sufficient to
separate the polyamide compound and the oligonucleotide by at least
one atom, and preferably, by more than one atom, such as at least
two, at least three, at least four, at least five or at least six
atoms. Preferably, the linking group is sufficiently flexible to
allow the polyamide compound to bind target molecules in a manner
which is independent of the oligonucleotide.
[0089] In one embodiment, the linking group is attached to the
N-terminus of the polyamide compound and the 5'-phosphate group of
the oligonucleotide. For example, the linking group can be derived
from a linking group precursor comprising an activated carboxyl
group on one end and an activated ester on the other end. Reaction
of the linking group precursor with the N-terminal nitrogen atom
will form an amide bond connecting the linking group to the
polyamide compound or N-terminal building block, while reaction of
the linking group precursor with the 5'-hydroxy group of the
oligonucleotide will result in attachment of the oligonucleotide to
the linking group via an ester linkage. The linking group can
comprise, for example, a polymethylene chain, such as a
--(CH.sub.2).sub.n-- chain or a poly(ethylene glycol) chain, such
as a --(CH.sub.2CH.sub.2O).sub.n chain, where in both cases n is an
integer from 1 to about 20. Preferably, n is from 2 to about 12,
more preferably from about 4 to about 10. In one embodiment, the
linking group comprises a hexamethylene (--(CH.sub.2).sub.6--)
group.
[0090] When the building blocks are amino acid residues, the
resulting functional moiety is a polyamide. The amino acids can be
coupled using any suitable chemistry for the formation of amide
bonds. Preferably, the coupling of the amino acid building blocks
is conducted under conditions which are compatible with enzymatic
ligation of oligonucleotides, for example, at neutral or
near-neutral pH and in aqueous solution. In one embodiment, the
polyamide compound is synthesized from the C-terminal to N-terminal
direction. In this embodiment, the first, or C-terminal, building
block is coupled at its carboxyl group to an oligonucleotide via a
suitable linking group. The first building block is reacted with
the second building block, which preferably has an activated
carboxyl group and a protected amino group. Any
activating/protecting group strategy which is suitable for solution
phase amide bond formation can be used. For example, suitable
activated carboxyl species include acyl fluorides (U.S. Pat. No.
5,360,928, incorporated herein by reference in its entirety),
symmetrical anhydrides and N-hydroxysuccinimide esters. The acyl
groups can also be activated in situ, as is known in the art, by
reaction with a suitable activating compound. Suitable activating
compounds include dicyclohexylcarbodiimide (DCC),
diisopropylcarbodiimide (DIC),
1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),
n-propane-phosphonic anhydride (PPA),
N,N-bis(2-oxo-3-oxazolidinyl)imido-phosphoryl chloride (BOP-Cl),
bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrop),
diphenylphosphoryl azide (DPPA), Castro's reagent (BOP, PyBop),
O-benzotriazolyl-N,N,N',N'-tetramethyluronium salts (HBTU),
diethylphosphoryl cyanide (DEPCN),
2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxy-thiophene dioxide
(Steglich's reagent; HOTDO), 1,1'-carbonyl-diimidazole (CDI), and
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMT-MM). The coupling reagents can be employed alone or in
combination with additives such as N,N-dimethyl-4-aminopyridine
(DMAP), N-hydroxy-benzotriazole (HOBt), N-hydroxybenzotriazine
(HOOBt), N-hydroxysuccinimide (HOSu) N-hydroxyazabenzotriazole
(HOAt), azabenzotriazolyl-tetramethyluronium salts (HATU, HAPyU) or
2-hydroxypyridine. In certain embodiments, synthesis of a library
requires the use of two or more activation strategies, to enable
the use of a structurally diverse set of building blocks. For each
building block, one skilled in the art can determine the
appropriate activation strategy.
[0091] The N-terminal protecting group can be any protecting group
which is compatible with the conditions of the process, for
example, protecting groups which are suitable for solution phase
synthesis conditions. A preferred protecting group is the
fluorenylmethoxycarbonyl ("Fmoc") group. Any potentially reactive
functional groups on the side chain of the aminoacyl building block
may also need to be suitably protected. Preferably the side chain
protecting group is orthogonal to the N-terminal protecting group,
that is, the side chain protecting group is removed under
conditions which are different than those required for removal of
the N-terminal protecting group. Suitable side chain protecting
groups include the nitroveratryl group, which can be used to
protect both side chain carboxyl groups and side chain amino
groups. Another suitable side chain amine protecting group is the
N-pent-4-enoyl group.
[0092] The building blocks can be modified following incorporation
into the functional moiety, for example, by a suitable reaction
involving a functional group on one or more of the building blocks.
Building block modification can take place following addition of
the final building block or at any intermediate point in the
synthesis of the functional moiety, for example, after any cycle of
the synthetic process. When a library of bifunctional molecules of
the invention is synthesized, building block modification can be
carried out on the entire library or on a portion of the library,
thereby increasing the degree of complexity of the library.
Suitable building block modifying reactions include those reactions
that can be performed under conditions compatible with the
functional moiety and the encoding oligonucleotide. Examples of
such reactions include acylation and sulfonation of amino groups or
hydroxyl groups, alkylation of amino groups, esterification or
thioesterification of carboxyl groups, amidation of carboxyl
groups, epoxidation of alkenes, and other reactions as are known
the art. When the functional moiety includes a building block
having an alkyne or an azide functional group, the azide/alkyne
cycloaddition reaction can be used to derivatize the building
block. For example, a building block including an alkyne can be
reacted with an organic azide, or a building block including an
azide can be reacted with an alkyne, in either case forming a
triazole. Building block modification reactions can take place
after addition of the final building block or at an intermediate
point in the synthetic process, and can be used to append a variety
of chemical structures to the functional moiety, including
carbohydrates, metal binding moieties and structures for targeting
certain biomolecules or tissue types.
[0093] In another embodiment, the functional moiety comprises a
linear series of building blocks and this linear series is cyclized
using a suitable reaction. For example, if at least two building
blocks in the linear array include sulfhydryl groups, the
sulfhydryl groups can be oxidized to form a disulfide linkage,
thereby cyclizing the linear array. For example, the functional
moieties can be oligopeptides which include two or more L or
D-cysteine and/or L or D-homocysteine moieties. The building blocks
can also include other functional groups capable of reacting
together to cyclize the linear array, such as carboxyl groups and
amino or hydroxyl groups.
[0094] In a preferred embodiment, one of the building blocks in the
linear array comprises an alkyne group and another building block
in the linear array comprises an azide group. The azide and alkyne
groups can be induced to react via cycloaddition, resulting in the
formation of a macrocyclic structure. In the example illustrated in
FIG. 9, the functional moiety is a polypeptide comprising a
propargylglycine building block at its C-terminus and an
azidoacetyl group at its N-terminus. Reaction of the alkyne and the
azide group under suitable conditions results in formation of a
cyclic compound, which includes a triazole structure within the
macrocycle. In the case of a library, in one embodiment, each
member of the library comprises alkyne- and azide-containing
building blocks and can be cyclized in this way. In a second
embodiment, all members of the library comprises alkyne- and
azide-containing building blocks, but only a portion of the library
is cyclized. In a third embodiment, only certain functional
moieties include alkyne- and azide-containing building blocks, and
only these molecules are cyclized. In the forgoing second and third
embodiments, the library, following the cycloaddition reaction,
will include both cyclic and linear functional moieties.
[0095] In some embodiments of the invention in which the same
functional moiety, e.g., triazine, is added to each and all of the
fractions of the library during a particular synthesis step, it may
not be necessary to add an oligonucleotide tag encoding that
function moiety.
[0096] Oligonucleotides may be ligated by chemical or enzymatic
methods. In one embodiment, oligonucleotides are ligated by
chemical means. Chemical ligation of DNA and RNA may be performed
using reagents such as water soluble carbodiimide and cyanogen
bromide as taught by, for example, Shabarova, et al. (1991) Nucleic
Acids Research, 19, 4247-4251), Federova, et al. (1996) Nucleosides
and Nucleotides, 15, 1137-1147, and Carriero and Damha (2003)
Journal of Organic Chemistry, 68, 8328-8338. In one embodiment,
chemical ligation is performed using cyanogen bromide, 5 M in
acetonitrile, in a 1:10 v/v ratio with 5' phosphorylated
oligonucleotide in a pH 7.6 buffer (1 M MES+20 mM MgCl.sub.2) at 0
degrees for 1-5 minutes. The oligonucleotides may be double
stranded, preferably with an overhang of about 5 to about 14 bases.
The oligonucleotide may also be single stranded, in which case a
splint with an overlap of about 6 bases with each of the
oligonucleotides to be ligated is employed to position the reactive
5' and 3' moieties in proximity with each other.
[0097] In another embodiment, the oligonucleotides are ligated
using enzymatic methods. In one embodiment, the initial building
block is operatively linked to an initial oligonucleotide. Prior to
or following coupling of a second building block to the initial
building block, a second oligonucleotide sequence which identifies
the second building block is ligated to the initial
oligonucleotide. Methods for ligating the initial oligonucleotide
sequence and the incoming oligonucleotide sequence are set forth in
FIGS. 1 and 2. In FIG. 1, the initial oligonucleotide is
double-stranded, and one strand includes an overhang sequence which
is complementary to one end of the second oligonucleotide and
brings the second oligonucleotide into contact with the initial
oligonucleotide. Preferably the overhanging sequence of the initial
oligonucleotide and the complementary sequence of the second
oligonucleotide are both at least about 4 bases; more preferably
both sequences are both the same length. The initial
oligonucleotide and the second oligonucleotide can be ligated using
a suitable enzyme. If the initial oligonucleotide is linked to the
first building block at the 5' end of one of the strands (the "top
strand"), then the strand which is complementary to the top strand
(the "bottom strand") will include the overhang sequence at its 5'
end, and the second oligonucleotide will include a complementary
sequence at its 5' end. Following ligation of the second
oligonucleotide, a strand can be added which is complementary to
the sequence of the second oligonucleotide which is 3' to the
overhang complementary sequence, and which includes additional
overhang sequence.
[0098] In one embodiment, the oligonucleotide is elongated as set
forth in FIG. 2. The oligonucleotide bound to the growing
functional moiety and the incoming oligonucleotide are positioned
for ligation by the use of a "splint" sequence, which includes a
region which is complementary to the 3' end of the initial
oligonucleotide and a region which is complementary to the 5' end
of the incoming oligonucleotide. The splint brings the 5' end of
the oligonucleotide into proximity with the 3' end of the incoming
oligo and ligation is accomplished using enzymatic ligation. In the
example illustrated in FIG. 2, the initial oligonucleotide consists
of 16 nucleobases and the splint is complementary to the 6 bases at
the 3' end. The incoming oligonucleotide consists of 12
nucleobases, and the splint is complementary to the 6 bases at the
5' terminus. The length of the splint and the lengths of the
complementary regions are not critical. However, the complementary
regions should be sufficiently long to enable stable dimer
formation under the conditions of the ligation, but not so long as
to yield an excessively large encoding nucleotide in the final
molecules. It is preferred that the complementary regions are from
about 4 bases to about 12 bases, more preferably from about 5 bases
to about 10 bases, and most preferably from about 5 bases to about
8 bases in length.
[0099] The split-and-pool methods used for the methods for library
synthesis set forth herein assure that each unique functional
moiety is operatively linked to at least one unique oligonucleotide
sequence which identifies the functional moiety. If 2 or more
different oligonucleotide tags are used for at least one building
bock in at least one of the synthetic cycles, each distinct
functional moiety comprising that building block will be encoded by
multiple oligonucleotides. For example, if 2 oligonucleotide tags
are used for each building block during the synthesis of a 4 cycle
library, there will be 16 DNA sequences (2.sup.4) that encode each
unique functional moiety. There are several potential advantages
for encoding each unique functional moiety with multiple sequences.
First, selection of a different combination of tag sequences
encoding the same functional moiety assures that those molecules
were independently selected. Second, selection of a different
combination of tag sequences encoding the same functional moiety
eliminates the possibility that the selection was based on the
sequence of the oligonucleotide. Third, technical artifact can be
recognized if sequence analysis suggests that a particular
functional moiety is highly enriched, but only one sequence
combination out of many possibilities appears. Multiple tagging can
be accomplished by having independent split reactions with the same
building block but a different oligonucleotide tag. Alternatively,
multiple tagging can be accomplished by mixing an appropriate ratio
of each tag in a single tagging reaction with an individual
building block.
[0100] In one embodiment, the initial oligonucleotide is
double-stranded and the two strands are covalently joined. One
means of covalently joining the two strands is shown in FIG. 3, in
which a linking moiety is used to link the two strands and the
functional moiety. The linking moiety can be any chemical structure
which comprises a first functional group which is adapted to react
with a building block, a second functional group which is adapted
to react with the 3'-end of an oligonucleotide, and a third
functional group which is adapted to react with the 5'-end of an
oligonucleotide. Preferably, the second and third functional groups
are oriented so as to position the two oligonucleotide strands in a
relative orientation that permits hybridization of the two strands.
For example, the linking moiety can have the general structure
(I):
##STR00016##
where A, is a functional group that can form a covalent bond with a
building block, B is a functional group that can form a bond with
the 5'-end of an oligonucleotide, and C is a functional group that
can form a bond with the 3'-end of an oligonucleotide. D, F and E
are chemical groups that link functional groups A, C and B toS,
which is a core atom or scaffold. Preferably, D, E and F are each
independently a chain of atoms, such as an alkylene chain or an
oligo(ethylene glycol) chain, and D, E and F can be the same or
different, and are preferably effective to allow hybridization of
the two oligonucleotides and synthesis of the functional moiety. In
one embodiment, the trivalent linker has the structure
##STR00017##
In this embodiment, the NH group is available for attachment to a
building block, while the terminal phosphate groups are available
for attachment to an oligonucleotide.
[0101] In embodiments in which the initial oligonucleotide is
double-stranded, the incoming oligonucleotides are also
double-stranded. As shown in FIG. 3, the initial oligonucleotide
can have one strand which is longer than the other, providing an
overhang sequence. In this embodiment, the incoming oligonucleotide
includes an overhang sequence which is complementary to the
overhang sequence of the initial oligonucleotide. Hybridization of
the two complementary overhang sequences brings the incoming
oligonucleotide into position for ligation to the initial
oligonucleotide. This ligation can be performed enzymatically using
a DNA or RNA ligase. The overhang sequences of the incoming
oligonucleotide and the initial oligonucleotide are preferably the
same length and consist of two or more nucleotides, preferably from
2 to about 10 nucleotides, more preferably from 2 to about 6
nucleotides. In one preferred embodiment, the incoming
oligonucleotide is a double-stranded oligonucleotide having an
overhang sequence at each end. The overhang sequence at one end is
complementary to the overhang sequence of the initial
oligonucleotide, while, after ligation of the incoming
oligonucleotide and the initial oligonucleotide, the overhang
sequence at the other end becomes the overhang sequence of initial
oligonucleotide of the next cycle. In one embodiment, the three
overhang sequences are all 2 to 6 nucleotides in length, and the
encoding sequence of the incoming oligonucleotide is from 3 to 10
nucleotides in length, preferably 3 to 6 nucleotides in length. In
a particular embodiment, the overhang sequences are all 2
nucleotides in length and the encoding sequence is 5 nucleotides in
length.
[0102] In the embodiment illustrated in FIG. 4, the incoming strand
has a region at its 3' end which is complementary to the 3' end of
the initial oligonucleotide, leaving overhangs at the 5' ends of
both strands. The 5' ends can be filled in using, for example, a
DNA polymerase, such as vent polymerase, resulting in a
double-stranded elongated oligonucleotide. The bottom strand of
this oligonucleotide can be removed, and additional sequence added
to the 3' end of the top strand using the same method.
[0103] The encoding oligonucleotide tag is formed as the result of
the successive addition of oligonucleotides that identify each
successive building block. In one embodiment of the methods of the
invention, the successive oligonucleotide tags may be coupled by
enzymatic ligation to produce an encoding oligonucleotide.
[0104] Enzyme-catalyzed ligation of oligonucleotides can be
performed using any enzyme that has the ability to ligate nucleic
acid fragments. Exemplary enzymes include ligases, polymerases, and
topoisomerases. In specific embodiments of the invention, DNA
ligase (EC 6.5.1.1), DNA polymerase (EC 2.7.7.7), RNA polymerase
(EC 2.7.7.6) or topoisomerase (EC 5.99.1.2) are used to ligate the
oligonucleotides. Enzymes contained in each EC class can be found,
for example, as described in Bairoch (2000) Nucleic Acids Research
28:304-5.
[0105] In a preferred embodiment, the oligonucleotides used in the
methods of the invention are oligodeoxynucleotides and the enzyme
used to catalyze the oligonucleotide ligation is DNA ligase. In
order for ligation to occur in the presence of the ligase, i.e.,
for a phosphodiester bond to be formed between two
oligonucleotides, one oligonucleotide must have a free 5' phosphate
group and the other oligonucleotide must have a free 3' hydroxyl
group. Exemplary DNA ligases that may be used in the methods of the
invention include T4 DNA ligase, Taq DNA ligase, T.sub.4 RNA
ligase, DNA ligase (E. coli) (all available from, for example, New
England Biolabs, MA).
[0106] One of skill in the art will understand that each enzyme
used for ligation has optimal activity under specific conditions,
e.g., temperature, buffer concentration, pH and time. Each of these
conditions can be adjusted, for example, according to the
manufacturer's instructions, to obtain optimal ligation of the
oligonucleotide tags.
[0107] The incoming oligonucleotide can be of any desirable length,
but is preferably at least three nucleobases in length. More
preferably, the incoming oligonucleotide is 4 or more nucleobases
in length. In one embodiment, the incoming oligonucleotide is from
3 to about 12 nucleobases in length. It is preferred that the
oligonucleotides of the molecules in the libraries of the invention
have a common terminal sequence which can serve as a primer for
PCR, as is known in the art. Such a common terminal sequence can be
incorporated as the terminal end of the incoming oligonucleotide
added in the final cycle of the library synthesis, or it can be
added following library synthesis, for example, using the enzymatic
ligation methods disclosed herein.
[0108] A preferred embodiment of the method of the invention is set
forth in FIG. 5. The process begins with a synthesized DNA sequence
which is attached at its 5' end to a linker which terminates in an
amino group. In step 1, this starting DNA sequence is ligated to an
incoming DNA sequence in the presence of a splint DNA strand, DNA
ligase and dithiothreitol in Tris buffer. This yields a tagged DNA
sequence which can then be used directly in the next step or
purified, for example, using HPLC or ethanol precipitation, before
proceeding to the next step. In step 2 the tagged DNA is reacted
with a protected activated amino acid, in this example, an
Fmoc-protected amino acid fluoride, yielding a protected amino
acid-DNA conjugate. In step 3, the protected amino acid-DNA
conjugate is deprotected, for example, in the presence of
piperidine, and the resulting deprotected conjugate is, optionally,
purified, for example, by HPLC or ethanol precipitation. The
deprotected conjugate is the product of the first synthesis cycle,
and becomes the starting material for the second cycle, which adds
a second amino acid residue to the free amino group of the
deprotected conjugate.
[0109] In embodiments in which PCR is to be used to amplify and/or
sequence the encoding oligonucleotides of selected molecules, the
encoding oligonucleotides may include, for example, PCR primer
sequences and/or sequencing primers (e.g., primers such as, for
example, 3'-GACTACCGCGCTCCCTCCG-5' (SEQ ID NO: 891) and
3'-GACTCGCCCGACCGTTCCG-5' (SEQ ID NO: 892)). A PCR primer sequence
can be included, for example, in the initial oligonucleotide prior
to the first cycle of synthesis, and/or it can be included with the
first incoming oligonucleotide, and/or it can be ligated to the
encoding oligonucleotide following the final cycle of library
synthesis, and/or it can be included in the incoming
oligonucleotide of the final cycle. The PCR primer sequences added
following the final cycle of library synthesis and/or in the
incoming oligonucleotide of the final cycle are referred to herein
as "capping sequences".
[0110] In one embodiment, the PCR primer sequence is designed into
the encoding oligonucleotide tag. For example, a PCR primer
sequence may be incorporated into the initial oligonucleotide tag
and/or it may be incorporated into the final oligonucleotide tag.
In one embodiment the same PCR primer sequence is incorporated into
the initial and final oligonucleotide tag. In another embodiment, a
first PCR sequence is incorporated into the initial oligonucleotide
tag and a second PCR primer sequence is incorporated in the final
oligonucleotide tag. Alternatively, the second PCR primer sequence
may be incorporated into the capping sequence as described herein.
In preferred embodiments, the PCR primer sequence is at least about
5, 7, 10, 13, 15, 17, 20, 22, or 25 nucleotides in length.
[0111] PCR primer sequences suitable for use in the libraries of
the invention are known in the art; suitable primers and methods
are set forth, for example, in Innis, et al., eds., PCR Protocols:
A Guide to Methods and Applications, San Diego: Academic Press
(1990), the contents of which are incorporated herein by reference
in their entirety. Other suitable primers for use in the
construction of the libraries described herein are those primers
described in PCT Publications WO 2004/069849 and WO 2005/003375,
the contents of which are expressly incorporated herein by
reference.
[0112] The term "polynucleotide" as used herein in reference to
primers, probes and nucleic acid fragments or segments to be
synthesized by primer extension is defined as a molecule comprised
of two or more deoxyribonucleotides, preferably more than
three.
[0113] The term "primer" as used herein refers to a polynucleotide
whether purified from a nucleic acid restriction digest or produced
synthetically, which is capable of acting as a point of initiation
of nucleic acid synthesis when placed under conditions in which
synthesis of a primer extension product which is complementary to a
nucleic acid strand is induced, i.e., in the presence of
nucleotides and an agent for polymerization such as DNA polymerase,
reverse transcriptase and the like, and at a suitable temperature
and pH. The primer is preferably single stranded for maximum
efficiency, but may alternatively be in double stranded form. If
double stranded, the primer is first treated to separate it from
its complementary strand before being used to prepare extension
products. Preferably, the primer is a polydeoxyribonucleotide. The
primer must be sufficiently long to prime the synthesis of
extension products in the presence of the agents for
polymerization. The exact lengths of the primers will depend on
many factors, including temperature and the source of primer.
[0114] The primers used herein are selected to be "substantially"
complementary to the different strands of each specific sequence to
be amplified. This means that the primer must be sufficiently
complementary so as to non-randomly hybridize with its respective
template strand. Therefore, the primer sequence may or may not
reflect the exact sequence of the template.
[0115] The polynucleotide primers can be prepared using any
suitable method, such as, for example, the phosphotriester or
phosphodiester methods described in Narang et al., (1979) Meth.
Enzymol., 68:90; U.S. Pat. No. 4,356,270, U.S. Pat. No. 4,458,066,
U.S. Pat. No. 4,416,988, U.S. Pat. No. 4,293,652; and Brown et al.,
(1979) Meth. Enzymol., 68:109. The contents of all the foregoing
documents are incorporated herein by reference.
[0116] In cases in which the PCR primer sequences are included in
an incoming oligonucleotide, these incoming oligonucleotides will
preferably be significantly longer than the incoming
oligonucleotides added in the other cycles, because they will
include both an encoding sequence and a PCR primer sequence.
[0117] In one embodiment, the capping sequence is added after the
addition of the final building block and final incoming
oligonucleotide, and the synthesis of a library as set forth herein
includes the step of ligating the capping sequence to the encoding
oligonucleotide, such that the oligonucleotide portion of
substantially all of the library members terminates in a sequence
that includes a PCR primer sequence. Preferably, the capping
sequence is added by ligation to the pooled fractions which are
products of the final synthetic cycle. The capping sequence can be
added using the enzymatic process used in the construction of the
library.
[0118] In one embodiment, the same capping sequence is ligated to
every member of the library. In another embodiment, a plurality of
capping sequences are used. In this embodiment, oligonucleotide
capping sequences containing variable bases are, for example,
ligated onto library members following the final synthetic cycle.
In one embodiment, following the final synthetic cycle, the
fractions are pooled and then split into fractions again, with each
fraction having a different capping sequence added. Alternatively,
multiple capping sequences can be added to the pooled library
following the final synthesis cycle. In both embodiments, the final
library members will include molecules comprising specific
functional moieties linked to identifying oligonucleotides
including two or more different capping sequences.
[0119] In one embodiment, the capping primer comprises an
oligonucleotide sequence containing variable, i.e., degenerate,
nucleotides. Such degenerate bases within the capping primers
permit the identification of library molecules of interest by
determining whether a combination of building blocks is the
consequence of PCR duplication (identical sequence) or independent
occurrences of the molecule (different sequence). For example, such
degenerate bases may reduce the potential number of false positives
identified during the biological screening of the encoded
library.
[0120] In one embodiment, a degenerate capping primer comprises or
has the following sequence:
##STR00018##
where N can be any of the 4 bases, permitting 1024 different
sequences (4.sup.5). The primer has the following sequence after
its ligation onto the library and primer-extension:
TABLE-US-00001 (SEQ ID NO: 895) 5'-CAGCGTTCGA
N'N'N'N'N'CAGACAAGCTTCACCTGC-3' (SEQ ID NO: 896) 3'-AA GTCGCAAGCT N
N N N N GTCTGTTCGAAGTGGACG-5'
[0121] In another embodiment, the capping primer comprises or has
the following sequence:
##STR00019##
[0122] where B can be any of C, G or T, permitting 19,683 different
sequences (3.sup.9). The design of the degenerate region in this
primer improves DNA sequence analysis, as the A bases that flank
and punctuate the degenerate B bases prevent homopolymeric
stretches of greater than 3 bases, and facilitate sequence
alignment.
[0123] In one embodiment, the degenerate capping oligonucleotide is
ligated to the members of the library using a suitable enzyme and
the upper strand of the degenerate capping oligonucleotide is
subsequently polymerized using a suitable enzyme, such as a DNA
polymerase.
[0124] In another embodiment, the PCR priming sequence is a
"universal adaptor" or "universal primer". As used herein, a
"universal adaptor" or "universal primer" is an oligonucleotide
that contains a unique PCR priming region, that is, for example,
about 5, 7, 10, 13, 15, 17, 20, 22, or 25 nucleotides in length,
and is located adjacent to a unique sequencing priming region that
is, for example, about 5, 7, 10, 13, 15, 17, 20, 22, or 25
nucleotides in length, and is optionally followed by a unique
discriminating key sequence (or sample identifier sequence)
consisting of at least one of each of the four deoxyribonucleotides
(i.e., A, C, G, T).
[0125] As used herein, the term "discriminating key sequence" or
"sample identifier sequence" refers to a sequence that may be used
to uniquely tag a population of molecules from a sample. Multiple
samples, each containing a unique sample identifier sequence, can
be mixed, sequenced and re-sorted after DNA sequencing for analysis
of individual samples. The same discriminating sequence can be used
for an entire library or, alternatively, different discriminating
key sequences can be used to track different libraries. In one
embodiment, the discriminating key sequence is on either the 5' PCR
primer, the 3' PCR primer, or on both primers. If both PCR primers
contain a sample identifier sequence, the number of different
samples that can be pooled with unique sample identifier sequences
is the product of the number of sample identifier sequences on each
primer. Thus, 10 different 5' sample identifier sequence primers
can be combined with 10 different 3' sample identifier sequence
primers to yield 100 different sample identifier sequence
combinations.
[0126] Non-limiting examples of 5' and 3' unique PCR primers
containing discriminating key sequences include the following:
TABLE-US-00002 5' primers (variable positions bold and italicized):
(SEQ ID NO: 898) 5' A-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ
ID NO: 899) 5' C-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ ID
NO: 900) 5' G-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ ID NO:
901) 5' T-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ ID NO: 902)
5' AA-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ ID NO: 903) 5'
AC-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ ID NO: 904) 5'
AG-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; (SEQ ID NO: 905) 5'
AT-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG; and (SEQ ID NO: 906) 5'
CA-GCCTTGCCAGCCCGCTCAG TGACTCCCAAATCGATGTG. 3' SID primers
(variable positions bold and italicized): (SEQ ID NO: 907) 3'
A-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 908) 3'
C-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 909) 3'
G-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 910) 3'
T-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 911) 3'
AA-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 912) 3'
AC-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 913) 3'
AG-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; (SEQ ID NO: 914) 3'
AT-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG; and (SEQ ID NO: 915) 3'
CA-GCCTCCCTCGCGCCATCAG GCAGGTGAAGCTTGTCTG
[0127] In one embodiment, the discriminating key sequence is about
4, 5, 6, 7, 8, 9, or nucleotides in length. In another embodiment,
the discriminating key sequence is a combination of about 1-4
nucleotides. In yet another embodiment, each universal adaptor is
about forty-four nucleotides in length. In one embodiment the
universal adaptors are ligated, using T4 DNA ligase, onto the end
of the encoding oligonucleotide. Different universal adaptors may
be designed specifically for each library preparation and will,
therefore, provide a unique identifier for each library. The size
and sequence of the universal adaptors may be modified as deemed
necessary by one of skill in the art.
[0128] In one embodiment, the universal adaptor added as a capping
sequence is linked to a support binding moiety. For example, a
5'-biotin is added to the universal adaptor to allow, for example,
isolation of single-stranded DNA template as well as non-covalent
coupling of the universal adaptor to the surface of a solid support
that is saturated with a biotin-binding protein (i.e.,
streptavidin, neutravidin or avidin). Other linkages are well known
in the art and may be used in place of biotin-streptavidin (for
example antibody/antigen-epitope, receptor/ligand and
oligonucleotide pairing or complimentarity).
[0129] In another embodiment, the capping sequence contains anchor
primer sequences such that the members of the library may be
attached to a solid substrate. In one embodiment, the anchor primer
sequences are annealed to the capping sequences using recognized
techniques (see, e.g., Hatch, et al. (1999) Genet. Anal Biomol
Engineer 15: 35-40; U.S. Pat. No. 5,714,320, and U.S. Pat. No.
5,854,033). In general, any procedure for annealing the anchor
primers to the capping sequences is suitable as long as it results
in formation of specific, i.e., perfect or nearly perfect,
complementarity between the adapter region or regions in the anchor
primer sequence and a sequence present in the capping sequences.
The anchoring of the encoding oligonucleotide to the solid surface
may be reversible or irreversible, e.g., the anchor to the solid
surface may be cleavable or non-cleavable.
[0130] In one embodiment, the universal primer, is annealed to a
solid support that contains oligonucleotide capture primers that
are complementary to the PCR priming regions of the universal
adaptor ends.
[0131] In one embodiment, the solid support is a bead, for example,
a sepharose bead. The beads may be of any convenient size and
fabricated from any number of known materials. Example of such
materials include: inorganics, natural polymers, and synthetic
polymers. Specific examples of these materials include: cellulose,
cellulose derivatives, acrylic resins, glass; silica gels,
polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl
and acrylamide, polystyrene cross-linked with divinylbenzene or the
like (see, Merrifield (1964) Biochemistry 3:1385-1390),
polyacrylamides, latex gels, polystyrene, dextran, rubber, silicon,
plastics, nitrocellulose, celluloses, natural sponges, silica gels,
glass, metals plastic, cellulose, cross-linked dextrans (e.g.,
Sephadex.TM.) and agarose gel (Sepharose.TM.) and solid phase
supports known to those of skill in the art.
[0132] The encoding oligonucleotides may be attached to the solid
support capture bead ("DNA capture bead") in any manner known in
the art. Any suitable coupling agent known in the art can be used,
such as, for example, water-soluble carbodiimide, to link the
5'-phosphate on the DNA to amine-coated capture beads through a
phosphoamidate bond, coupling specific oligonucleotide linkers to
the bead using similar chemistry, and using DNA ligase to link the
DNA to the linker on the bead, joining the oligonucleotide to the
beads using N-hydroxysuccinamide (NHS) and its derivatives, such
that one end of the oligonucleotide may contain a reactive group
(such as an amide group) which forms a covalent bond with the solid
support, while the other end of the linker contains a second
reactive group that can bond with the oligonucleotide to be
immobilized.
[0133] In another embodiment, the oligonucleotide is bound to the
DNA capture bead by non-covalent linkage, such as chelation or
antigen-antibody complexes, may also be used to join the
oligonucleotide to the bead. Oligonucleotide linkers can be
employed which specifically hybridize to unique sequences at the
end of the DNA fragment, such as the overlapping end from a
restriction enzyme site or the "sticky ends" of bacteriophage
lambda based cloning vectors, but blunt-end ligations can also be
used beneficially. These methods are described in detail in U.S.
Pat. No. 5,674,743. It is preferred that any method used to
immobilize the beads will continue to bind the immobilized
oligonucleotide throughout the steps in the methods of the
invention.
[0134] In one embodiment, the oligonucleotide is attached to a
solid support manufactured from, for example, glass, plastic, a
nylon membrane, a gel matrix, ceramics, silica, silicon, or any
other non-reactive material as described in U.S. Pat. No.
6,787,308, the entire contents of which are incorporated by
reference. The supports generally comprise a flat, i.e., planar,
surface, or at least an array in which the molecules to be analysed
are in the same plane. The oligonucleotide may be attached by
specific covalent or non-covalent interactions. In one embodiment
of the invention, the surface of a solid support is coated with
streptavidin or avidin. In another embodiment of the invention, the
solid surface is coated with an epoxide and the molecules are
coupled via an amine linkage. In yet another embodiment, the
encoding oligonucleotide may be attached to a solid support via
hybridization to a complementary nucleic acid molecule previously
attached to the solid support.
[0135] In one embodiment, the solid support is pretreated to create
surface chemistry that facilitates oligonucleotide attachment and
subsequent sequence analysis. In one embodiment, the solid support
is coated with a polyelectrolyte multilayer (PEM). In another
embodiment, the encoding oligonucleotide is attached to the surface
of a microfabricated channel or to the surface of reaction chambers
that are disposed along a microfabricated flow channel, optionally
with streptavidin-biotin links. The methods of each of these
attachment methods are described in PCT Publication No. WO
2005/080605, the entire contents of which are incorporated by
reference.
[0136] In one embodiment, the encoding oligonucleotide is attached
to a solid surface at high density and at single molecule
resolution. In one embodiment, the encoding oligonucleotide is
attached to a solid surface at an individually-addressable location
(see, e.g., PCT Publication No. WO 2005/080605).
[0137] Attachment of the encoding oligonucleotide to any suitable
solid surface can occur prior to the hybridization of a primer for
amplification and/or sequencing or alternatively, the encoding
oligonucleotide can be attached to any suitable solid surface after
the hybridization of a primer for amplification and/or
sequencing.
[0138] In another embodiment, the oligonucleotide is attached to a
particle, such as a microsphere, which is itself attached to a
solid support. The microspheres may be of any suitable size,
typically in the range of from 10 nm to 100 nm in diameter.
[0139] In one embodiment, the universal adaptors are not
5'-phosphorylated. Accordingly, "gaps" or "nicks" can be filled in
by using a DNA polymerase enzyme that can bind to, strand displace
and extend the nicked DNA fragments according to techniques
recognized in the art. DNA polymerases that lack 3'->5'
exonuclease activity but exhibit 5'->3' exonuclease activity
have the ability to recognize nicks, displace the nicked strands,
and extend the strand in a manner that results in the repair of the
nicks and in the formation of non-nicked double-stranded DNA
(Hamilton, et al. (2001) BioTechniques 31:370).
[0140] Several modifying enzymes are utilized for the nick repair
step, including but not limited to polymerase, ligase and kinase.
DNA polymerases that can be used for this application include, for
example, E. coli DNA pol I, Thermoanaerobacter
thermohydrosulfuricus pol I, and bacteriophage phi 29. In one
embodiment, the strand displacing enzyme Bacillus
stearothermophilus pol I (Bst DNA polymerase I) is used to repair
the nicked dsDNA and results in non-nicked dsDNA. In another
embodiment, the ligase is T4 and the kinase is polynucleotide
kinase.
[0141] The invention further relates to the compounds which can be
produced using the methods of the invention, and collections of
such compounds, either as isolated species or pooled to form a
library of chemical structures. Compounds of the invention include
compounds of the formula
##STR00020##
where X is a functional moiety comprising one or more building
blocks, Z is an oligonucleotide attached at its 3' terminus to B
and Y is an oligonucleotide which is attached to C at its 5'
terminus. A is a functional group that forms a covalent bond with
X, B is a functional group that forms a bond with the 3'-end of Z
and C is a functional group that forms a bond with the 5'-end of Y.
D, F and E are chemical groups that link functional groups A, C and
B to S, which is a core atom or scaffold. Preferably, D, E and F
are each independently a chain of atoms, such as an alkylene chain
or an oligo(ethylene glycol) chain, and D, E and F can be the same
or different, and are preferably effective to allow hybridization
of the two oligonucleotides and synthesis of the functional
moiety.
[0142] Preferably, Y and Z are substantially complementary and are
oriented in the compound so as to enable Watson-Crick base pairing
and duplex formation under suitable conditions. Y and Z are the
same length or different lengths. Preferably, Y and Z are the same
length, or one of Y and Z is from 1 to 10 bases longer than the
other. In a preferred embodiment, Y and Z are each 10 or more bases
in length and have complementary regions of ten or more base pairs.
More preferably, Y and Z are substantially complementary throughout
their length, i.e., they have no more than one mismatch per every
ten base pairs. Most preferably, Y and Z are complementary
throughout their length, i.e., except for any overhang region on Y
or Z, the strands hybridize via Watson-Crick base pairing with no
mismatches throughout their entire length.
[0143] S can be a single atom or a molecular scaffold. For example,
S can be a carbon atom, a boron atom, a nitrogen atom or a
phosphorus atom, or a polyatomic scaffold, such as a phosphate
group or a cyclic group, such as a cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl group. In
one embodiment, the linker is a group of the structure
##STR00021##
where each of n, m and p is, independently, an integer from 1 to
about 20, preferably from 2 to eight, and more preferably from 3 to
6. In one particular embodiment, the linker has the structure shown
below.
##STR00022##
[0144] In one embodiment, the libraries of the invention include
molecules consisting of a functional moiety composed of building
blocks, where each functional moiety is operatively linked to an
encoding oligonucleotide. The nucleotide sequence of the encoding
oligonucleotide is indicative of the building blocks present in the
functional moiety, and in some embodiments, the connectivity or
arrangement of the building blocks. The invention provides the
advantage that the methodology used to construct the functional
moiety and that used to construct the oligonucleotide tag can be
performed in the same reaction medium, preferably an aqueous
medium, thus simplifying the method of preparing the library
compared to methods in the prior art. In certain embodiments in
which the oligonucleotide ligation steps and the building block
addition steps can both be conducted in aqueous media, each
reaction will have a different pH optimum. In these embodiments,
the building block addition reaction can be conducted at a suitable
pH and temperature in a suitable aqueous buffer. The buffer can
then be exchanged for an aqueous buffer which provides a suitable
pH for oligonucleotide ligation.
[0145] In another embodiment, the invention provides compounds, and
libraries comprising such compounds, of Formula II
Z-L-A.sub.t-X(Y).sub.n (II)
where X is a molecular scaffold, each Y is independently, a
peripheral moiety, and n is an integer from 1 to 6. Each A is
independently, a building block and n is an integer from 0 to about
5. L is a linking moiety and Z is a single-stranded or
double-stranded oligonucleotide which identifies the structure
-A.sub.t-X(Y).sub.n. The structure X(Y).sub.n can be, for example,
one of the scaffold structures set forth in Table 8 (see below). In
one embodiment, the invention provides compounds, and libraries
comprising such compounds, of Formula III:
##STR00023##
where t is an integer from 0 to about 5, preferably from 0 to 3,
and each A is, independently, a building block. L is a linking
moiety and Z is a single-stranded or double-stranded
oligonucleotide which identifies each A and R.sub.1, R.sub.2,
R.sub.3 and R.sub.4. R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each
independently a substituent selected from hydrogen, alkyl,
substituted alkyl, heteroalkyl, substituted heteroalkyl,
cycloalkyl, heterocycloalkyl, substituted cycloalkyl, substituted
heterocycloalkyl, aryl, substituted aryl, arylalkyl,
heteroarylalkyl, substituted arylalkyl, substituted
heteroarylalkyl, heteroaryl, substituted heteroaryl, alkoxy,
aryloxy, amino, and substituted amino. In one embodiment, each A is
an amino acid residue.
[0146] Libraries which include compounds of Formula II or Formula
III can comprise at least about 100; 1000; 10,000; 100,000;
1,000,000 or 10,000,000 compounds of Formula II or Formula III. In
one embodiment, the library is prepared via a method designed to
produce a library comprising at least about 100; 1000; 10,000;
100,000; 1,000,000 or 10,000,000 compounds of Formula II or Formula
III.
TABLE-US-00003 TABLE 8 Scaffolds Amine Aldehyde/Ketone Carboxylic
acid Other Reference ##STR00024## ##STR00025## ##STR00026##
##STR00027## Carranco, I., et al. (2005) J. Comb. Chem. 7: 33-41
##STR00028## amines benzaldehydes and furfural ##STR00029##
Rosamilia, A. E., et al. (2005) Organic Letters 7: 1525-1528
##STR00030## ##STR00031## ##STR00032## ##STR00033## ##STR00034##
Syeda Huma, H. Z., et al. (2002) Tet Lett 43: 6485- 6488
##STR00035## ##STR00036## R2--CHO ##STR00037## .ident.N--R3
Tempest, P., et al. (2001) Tet Lett 42: 4959-4962 ##STR00038##
##STR00039## ##STR00040## ##STR00041## ##STR00042## Paulvannan, K.
(1999) Tet Lett 40: 1851- 1854 ##STR00043## R1--CHO ##STR00044##
.ident.N--R4 Tempest, P., et al. (2001) Tet Lett 42: 4963-4968
##STR00045## R2--NH.sub.2 ##STR00046## ##STR00047## .ident.N--R3
Tempest, P., et al. (2003) Tet Lett 44: 1947-1950 ##STR00048##
R1--CHO R2--COOH ##STR00049## Nefzi, A., et al. (1999) Tet Lett 40:
4939- 4942 ##STR00050## ##STR00051## ##STR00052## ##STR00053##
##STR00054## Bose, A. K., et al. (2005) Tet Lett 46: 1901- 1903
##STR00055## ##STR00056## R1--CHO ##STR00057## Stadler, A. and
Kappe, C. O. (2001) J. Comb. Chem. 3: 624-630; Lengar, A. and
Kappe, C. O. (2004) Organic Letters 6: 771- 774 ##STR00058## wide
range of primary aliphatic amines ##STR00059## ##STR00060##
Ivachtchenko, A. V., et al. (2003) J. Comb. Chem. 5: 775-788
##STR00061## ##STR00062## ##STR00063## ##STR00064## Micheli, F., et
al. (2001) J. Comb. Chem. 3: 224- 228 ##STR00065## R1--HS
R1--NH.sub.2 ##STR00066## ##STR00067## ##STR00068## Sternson, S.
M., et al. (2001) Org. Lett. 3: 4239- 4242 ##STR00069##
##STR00070## ##STR00071## Cheng, W. -C., et al. (2002) J. Org.
Chem. 67: 5673- 5677; Park, K. -H., et al. (2001) J Comb Chem 3:
171-176 ##STR00072## ##STR00073## ##STR00074## Brown, B. J., et al.
(2000) Synlett 1: 131- 133 ##STR00075## R1--NH.sub.2 ##STR00076##
##STR00077## ##STR00078## Kilburn, J. P., et al. (2001) Tet Lett
42: 2583-2586 ##STR00079## amino acid amino acid ester del Fresno,
M., et al. (1998) Tet Lett 39: 2639- 2642 ##STR00080## amino acid
carboxylic acids Alvarez- Gutierrez, J. M., et al. (2000) Tet Lett
41: 609- 612 ##STR00081## R2--CHO ##STR00082## ##STR00083##
Rinnova, M., et al. (2002) J. Comb. Chem 4: 209-213 ##STR00084##
R1--NH.sub.2 ##STR00085## ##STR00086## Makara, G. M., et al. (2002)
Organic Lett 4: 1751-1754 ##STR00087## ##STR00088## Schell, P., et
al. (2005) J. Comb. Chem 7: 96-98 ##STR00089## amino acids Feliu,
L., et al. (2003) J. Comb. Chem. 5: 356-361 ##STR00090## Amines
Aldehydes ##STR00091## amino acids Hiroshige, M., et al. (1995) J.
Am. Chem. Soc. 117: 11590- 11591 ##STR00092## amino acids Bose, A.
K., et al. (2005) Tet Lett 46: 1901- 1903
[0147] One advantage of the methods of the invention is that they
can be used to prepare libraries comprising vast numbers of
compounds. The ability to amplify encoding oligonucleotide
sequences using known methods such as polymerase chain reaction
("PCR") means that selected molecules can be identified even if
relatively few copies are recovered. This allows the practical use
of very large libraries, which, as a consequence of their high
degree of complexity, either comprise relatively few copies of any
given library member, or require the use of very large volumes. For
example, a library consisting of 10.sup.8 unique structures in
which each structure has 1.times.10.sup.12 copies (about 1
picomole), requires about 100 L of solution at 1 .mu.M effective
concentration. For the same library, if each member is represented
by 1,000,000 copies, the volume required is 100 .mu.L at 1 .mu.M
effective concentration.
[0148] In a preferred embodiment, the library comprises from about
10.sup.3 to about 10.sup.15 copies of each library member. Given
differences in efficiency of synthesis among the library members,
it is possible that different library members will have different
numbers of copies in any given library. Therefore, although the
number of copies of each member theoretically present in the
library may be the same, the actual number of copies of any given
library member is independent of, the number of copies of any other
member. More preferably, the compound libraries of the invention
include at least about 10.sup.5, 10.sup.6 or 10.sup.7 copies of
each library member, or of substantially all library members. By
"substantially all" library members is meant at least about 85% of
the members of the library, preferably at least about 90%, and more
preferably at least about 95% of the members of the library.
[0149] Preferably, the library includes a sufficient number of
copies of each member that multiple rounds (i.e., two or more) of
selection against a biological target can be performed, with
sufficient quantities of binding molecules remaining following the
final round of selection to enable amplification of the
oligonucleotide tags of the remaining molecules and, therefore,
identification of the functional moieties of the binding molecules.
A schematic representation of such a selection process is
illustrated in FIG. 6, in which 1 and 2 represent library members,
B is a target molecule and X is a moiety operatively linked to B
that enables the removal of B from the selection medium. In this
example, compound 1 binds to B, while compound 2 does not bind to
B. The selection process, as depicted in Round 1, comprises (I)
contacting a library comprising compounds 1 and 2 with B--X under
conditions suitable for binding of compound 1 to B; (II) removing
unbound compound 2, (III) dissociating compound 1 from B and
removing BX from the reaction medium. The result of Round 1 is a
collection of molecules that is enriched in compound 1 relative to
compound 2. Subsequent rounds employing steps I-III result in
further enrichment of compound 1 relative to compound 2. Although
three rounds of selection are shown in FIG. 6, in practice any
number of rounds may be employed, for example from one round to ten
rounds, to achieve the desired enrichment of binding molecules
relative to non-binding molecules.
[0150] In the embodiment shown in FIG. 6, there is no amplification
(synthesis of more copies) of the compounds remaining after any of
the rounds of selection. Such amplification can lead to a mixture
of compounds which is not consistent with the relative amounts of
the compounds remaining after the selection. This inconsistency is
due to the fact that certain compounds may be more readily
synthesized that other compounds, and thus may be amplified in a
manner which is not proportional to their presence following
selection. For example, if compound 2 is more readily synthesized
than compound 1, the amplification of the molecules remaining after
Round 2 would result in a disproportionate amplification of
compound 2 relative to compound 1, and a resulting mixture of
compounds with a much lower (if any) enrichment of compound 1
relative to compound 2.
[0151] In one embodiment, the target is immobilized on a solid
support by any known immobilization technique. The solid support
can be, for example, a water-insoluble matrix contained within a
chromatography column or a membrane. The encoded library can be
applied to a water-insoluble matrix contained within a
chromatography column. The column is then washed to remove
non-specific binders. Target-bound compounds can then be
dissociated by changing the pH, salt concentration, organic solvent
concentration, or other methods, such as competition with a known
ligand to the target.
[0152] In another embodiment, the target is free in solution and is
incubated with the encoded library. Compounds which bind to the
target (also referred to herein as "ligands") are selectively
isolated by a size separation step such as gel filtration or
ultrafiltration. In one embodiment, the mixture of encoded
compounds and the target biomolecule are passed through a size
exclusion chromatography column (gel filtration), which separates
any ligand-target complexes from the unbound compounds. The
ligand-target complexes are transferred to a reverse-phase
chromatography column, which dissociates the ligands from the
target. The dissociated ligands are then analyzed by PCR
amplification and sequence analysis of the encoding
oligonucleotides. This approach is particularly advantageous in
situations where immobilization of the target may result in a loss
of activity.
[0153] Accordingly, in one aspect of the invention, methods are
provided for identifying one or more compounds in a library of
compounds, produced as described herein, that bind to a biological
target and subsequently determining the structure of the functional
moieties of the member(s) of the library of compounds that bind to
the biological target.
[0154] For example, in one embodiment, one or more compounds which
bind to a biological target can be identified by a method
comprising the steps of:
[0155] (A) synthesizing a library of compounds, wherein the
compounds comprise a functional moiety comprising two or more
building blocks which is operatively linked to an initial
oligonucleotide which identifies the structure of the functional
moiety by: [0156] (i) providing a solution comprising m initiator
compounds, wherein m is an integer of 1 or greater, where the
initiator compounds consist of a functional moiety comprising n
building blocks, where n is an integer of 1 or greater, which is
operatively linked to an initial oligonucleotide which identifies
the n building blocks; [0157] (ii) dividing the solution of step
(i) into r reaction vessels, wherein r is an integer of 2 or
greater, thereby producing r aliquots of the solution; [0158] (iii)
reacting the initiator compounds in each reaction vessel with one
of r building blocks, thereby producing r aliquots comprising
compounds consisting of a functional moiety comprising n+1 building
blocks operatively linked to the initial oligonucleotide; and
[0159] (iv) reacting the initial oligonucleotide in each aliquot
with one of a set of r distinct incoming oligonucleotides in the
presence of an enzyme which catalyzes the ligation of the incoming
oligonucleotide and the initial oligonucleotide, under conditions
suitable for enzymatic ligation of the incoming oligonucleotide and
the initial oligonucleotide; thereby producing r aliquots of
molecules consisting of a functional moiety comprising n+1 building
blocks operatively linked to an elongated oligonucleotide which
encodes the n+1 building blocks;
[0160] (B) contacting the biological target with the library of
compounds, or a portion thereof, under conditions suitable for at
least one member of the library of compounds to bind to the
target;
[0161] (C) removing library members that do not bind to the
target;
[0162] (D) sequencing the encoding oligonucleotides of the at least
one member of the library of compounds which binds to the target,
and
[0163] (E) using the sequences determined in step (D) to determine
the structure of the functional moieties of the members of the
library of compounds which bind to the biological target, thereby
identifying one or more compounds which bind to the biological
target.
[0164] In one embodiment, the method further comprises ligating a
degenerate capping oligonucleotide to the members of the library of
compounds in the presence of an enzyme which catalyzes the ligation
and polymerizing the degenerate capping oligonucleotide with an
enzyme that catalyzes the polymerization of DNA.
[0165] In one embodiment, the method may further comprise
amplifying the encoding oligonucleotide of the at least one member
of the library of compounds which binds to the target prior to
sequencing.
[0166] In one embodiment of the invention, the selection and
enrichment of the library is monitored using an oligonucleotide
array. For example, a library of compounds may be hybridized to a
solid surface, such as a chip comprising oligonucleotides, e.g., an
Affymetrix oligonucleotide chip, which is subsequently flouresced
to detect the oligonucleotide tags bound to the surface. This
hybridization can be repeated at each successive step of the
screening process for identifying a compound with a desired
biological activity.
[0167] In one embodiment, the library of compounds comprising
encoding oligonucleotides which are optionally attached to capture
beads as described above are emulsified as a heat stable
water-in-oil emulsion to form a microcapsule according to the
methods described in PCT Publications WO 2004/069849, WO
2005/003375, and WO 2005/073410. In one embodiment, the emulsion
can be generated by suspending the oligonucleotide tag, with or
without attached beads, in amplification solution, e.g., forming a
"microreactor." As used herein, the term "amplification solution"
means the sufficient mixture of reagents that is necessary to
perform amplification of template DNA. One example of an
amplification solution, is a PCR amplification solution, that one
of skill in the art can readily prepare.
[0168] In one embodiment of the invention, the library of compounds
comprising encoding oligonucleotides are amplified to increase the
copy number of encoding oligonucleotide molecules prior to
sequencing. Encoding oligonucleotides may be amplified by any
suitable method of DNA amplification including, for example,
temperature cycling-polymerase chain reaction (PCR) (see, e.g.,
Saiki, et al. (1995) Science 230:1350-1354; Gingeras, et al. WO
88/10315; Davey, et al. European Patent Application Publication No.
329,822; Miller, et al. WO 89/06700), ligase chain reaction (see,
e.g., Barany (1991) Proc. Natl Acad. Sci. USA 88:189-193;
Barringer, et al. (1990) Gene 89:117-122), transcription-based
amplification (see, e.g., Kwoh, et al. (1989) Proc. Natl. Acad.
Sci. USA 86:1173-1177) isothermal amplification
systems--self-sustaining, sequence replication (see, e.g.,
Guatelli, et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878);
the Qp replicase system (see, e.g., Lizardi, et al. (1988)
BioTechnology 6: 1197-1202); strand displacement amplification
(Walker, et al. (1992) Nucleic Acids Res 20(7):1691-6; the methods
described by Walker, et al. (Proc. Natl. Acad. Sci. USA (1992)
1:89(1):392-6; the methods described by Kievits, et al. (J Virol
Methods (1991) 35(3):273-86; "race" (Frohman, In: PCR Protocols: A
Guide to Methods and Applications, Academic Press, NY (1990));
"one-sided PCR" (Ohara, et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
86.5673-5677); "di-oligonucleotide" amplification, isothermal
amplification (Walker, et al. (1992) Proc. Natl. Acad. Sci. U.S.A.
89:392-396), and rolling circle amplification (reviewed in U.S.
Pat. No. 5,714,320).
[0169] In one embodiment, the library of compounds comprising
encoding oligonucleotides is amplified prior to sequence analysis
in order to minimize any potential skew in the population
distribution of DNA molecules present in the selected library mix.
For example, only a small amount of library is recovered after a
selection step and is typically amplified using PCR prior to
sequence analysis. PCR has the potential to produce a skew in the
population distribution of DNA molecules present in the selected
library mix. This is especially problematic when the number of
input molecules is small and the input molecules are poor PCR
templates. PCR products produced at early cycles are more efficient
templates than covalent duplex library, and therefore the frequency
of these molecules in the final amplified population may be much
higher than in original input template.
[0170] Accordingly, in order to minimize this potential PCR skew,
in one embodiment of the invention, a population of single-stranded
oligonucleotides corresponding to the individual library members is
produced by, for example, using one primer in a reaction, followed
by PCR amplification using two primers. By doing so, there is a
linear accumulation of single-stranded primer-extension product
prior to exponential amplification using PCR, and the diversity and
distribution of molecules in the accumulated primer-extension
product more accurately reflect the diversity and distribution of
molecules present in the original input template, since the
exponential phase of amplification occurs only after much of the
original molecular diversity present is represented in the
population of molecules produced during the primer-extension
reaction.
[0171] Preferably, DNA amplification is performed by PCR. PCR
amplification methods are described in detail in U.S. Pat. Nos.
4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in PCR
Technology: Principles and Applications for DNA Amplification, H.
Erlich, ed., Stockton Press, New York (1989); and PCR Protocols: A
Guide to Methods and Applications, Innis et al., eds., Academic
Press, San Diego, Calif. (1990). The contents of all the foregoing
documents are incorporated herein by reference. In one embodiment
of the invention, PCR amplification of the template is performed on
an oligonucleotide tag bound to a bead, and encapsulated with a PCR
solution comprising all the necessary reagents for a PCR reaction.
In another embodiment of the invention, PCR amplification of the
template is performed on a soluble oligonucleotide tag (i.e., not
bound to a bead) which is encapsulated with a PCR solution
comprising all the necessary reagents for a PCR reaction. PCR is
subsequently performed by exposing the emulsion to any suitable
thermocycling regimen known in the art. In one embodiment, between
30 and 50 cycles, preferably about 40 cycles, of amplification are
performed. It is desirable, bin not necessary, that following the
amplification procedure there be one or more hybridization and
extension cycles following the cycles of amplification. In a
another embodiment, between 10 and 30 cycles, or about 25 cycles,
of hybridization and extension are performed. In one embodiment,
the template DNA is amplified until about at least two million to
fifty million copies or about ten million to thirty million copies
of the template DNA are immobilized per bead.
[0172] Following amplification of the encoding oligonucleotide tag,
the emulsion is "broken" (also referred to as "demulsification" in
the art). There are many well known methods of breaking an emulsion
(see, e.g., U.S. Pat. No. 5,989,892 and references cited therein)
and one of skill in the art would be able to select the proper
method. For example, the emulsion may be broken by adding
additional oil to cause the emulsion to separate into two phases.
The oil phase is then removed, and a suitable organic solvent
(e.g., hexanes) is added. After mixing, the oil/organic solvent
phase is removed. This step may be repeated several times. Finally,
the aqueous layers is removed. If the encoding oligonucleotides are
attached to beads, the beads are then washed with an organic
solvent/annealing buffer mixture, and then washed again in
annealing buffer. Suitable organic solvents include alcohols such
as methanol, ethanol and the like.
[0173] The amplified encoding oligonucleotides may then be
resuspended in aqueous solution for use, for example, in a
sequencing reaction according to known technologies. (See, e.g.,
Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. U.S.A.
75:5463-5467; Maxam & Gilbert (1977) Proc Natl Acad Sci USA
74:560-564; Ronaghi, et al. (1998) Science 281:363, 365; Lysov, et
al. (1988) Dokl Akad Nauk SSSR 303:1508-1511; Bains & Smith
(1988) J TheorBiol 135:303-307; Drnanac, R. et al. (1989) Genomics
4:114-128; Khrapko, et al. (1989) FEBS Lett 256:118-122; Pevzner
(1989) J Biomol Struct Dyn 7:63-73; Southern, et al. (1992)
Genomics 13:1008-1017).
[0174] If the encoding oligonucleotide attached to a bead is to be
used in a pyrophosphate-based sequencing reaction (described, e.g.,
in U.S. Pat. Nos. 6,274,320, 6,258,568 and 6,210,891, and
incorporated herein by reference), then it is necessary to remove
the second strand of the PCR product and anneal a sequencing primer
to the single stranded template that is bound to the bead.
[0175] Briefly, the second strand is melted away using any number
of commonly known methods such as NaOH, low ionic (e.g., salt)
strength, or heat processing. Following this melting step, the
beads are pelleted and the supernatant is discarded. The beads are
resuspended in an annealing buffer, the sequencing primer added,
and annealed to the bead-attached single stranded template using a
standard annealing cycle.
[0176] The amplified encoding oligonucleotide, optionally on a
bead, may be sequenced either directly or in a different reaction
vessel. In one embodiment of the present invention, the encoding
oligonucleotide is sequenced directly on the bead by transferring
the bead to a reaction vessel and subjecting the DNA to a
sequencing reaction (e.g., pyrophosphate or Sanger sequencing).
Alternatively, the beads may be isolated and the encoding
oligonucleotide may be removed from each bead and sequenced.
Nonetheless, the sequencing steps may be performed on each
individual bead and/or the beads that contain no nucleic acid
template may be removed prior to distribution to a reaction vessel
by, for example, biotin-streptavidin magnetic beads. Other suitable
methods to separate beads are described in, for example, Bauer, J.
(1999) J. Chromatography B, 722:55-69 and in Brody et al. (1999)
Applied Physics Lett. 74:144-146.
[0177] Once the encoding oligonucleotide tag has been amplified,
the sequence of the tag, and ultimately the composition of the
selected molecule, can be determined using nucleic acid sequence
analysis, a well known procedure for determining the sequence of
nucleotide sequences. Nucleic acid sequence analysis is approached
by a combination of (a) physiochemical techniques, based on the
hybridization or denaturation of a probe strand plus its
complementary target, and (b) enzymatic reactions with
polymerases.
[0178] The nucleotide sequence of the oligonucleotide tag comprised
of polynucleotides that identify the building blocks that make up
the functional moiety as described herein, may be determined by the
use of any sequencing method known to one of skill in the art.
Suitable methods are described in, for example, Sanger, F. et al.
(1977) Proc. Natl. Acad. Sci. U.S.A. 75:5463-5467; Maxam &
Gilbert (1977) Proc Natl Acad Sci USA 74:560-564; Ronaghi, et al.
(1998) Science 281:363, 365; Lysov, et al. (1988) Dokl Akad Nauk
SSSR 303:1508-1511; Bains & Smith (1988) J TheorBiol
135:303-307; Drnanac, R. et al. (1989) Genomics 4:114-128; Khrapko,
et al. (1989) FEBS Lett 256:118-122; Pevzner (1989) J Biomol Struct
Dyn 7:63-73; Southern, et al. (1992) Genomics 13:1008-1017).
[0179] In a preferred embodiment, the oligonucleotide tags are
sequenced using the apparati and methods described in PCT
publications WO 2004/069849, WO 2005/003375, WO 2005/073410, and WO
2005/054431, the entire contents of each of which are incorporated
herein by this reference.
[0180] In one embodiment, a region of the sequence product is
determined by annealing a sequencing primer to a region of the
template nucleic acid, and then contacting the sequencing primer
with a DNA polymerase and a known nucleotide triphosphate, i.e.,
dATP, dCTP, dGTP, dTTP, or an analog of one of these nucleotides,
such as, for example, .alpha.-thio-dATP. The sequence can be
determined by detecting a sequence reaction byproduct, using
methods known in the art.
[0181] In some embodiments, the nucleotide is modified to contain a
disulfide-derivative of a hapten, such as biotin. The addition of
the modified nucleotide to the nascent primer annealed to an
anchored substrate is analyzed by a suitable post-polymerization
method. Such methods enable a nucleotide to be identified in a
given target position, and the DNA to be sequenced simply and
rapidly while avoiding the need for electrophoresis and the use of
potentially dangerous radiolabels.
[0182] Examples of suitable haptens include, for example, biotin,
digoxygenin, the fluorescent dye molecules cy3 and cy5, and
fluorescein. The attachment of the hapten can occur through
linkages via the sugar, the base, and/or via the phosphate moiety
on the nucleotide. Exemplary means for signal amplification
following polymerization and extension of the encoding
oligonucleotide include fluorescent, electrochemical and enzymatic
means. In one embodiment using enzymatic amplification, the enzyme
is one for which light-generating substrates are known, such as,
for example, alkaline phosphatase (AP), horse-radish peroxidase
(HRP), beta-galactosidase, or luciferase, and the means for the
detection of these light-generating (chemiluminescent) substrates
can include a CCD camera.
[0183] A sequencing primer can be of any length or base
composition, as long as it is capable of specifically annealing to
a region of the nucleic acid template (i.e., the oligonucleotide
tag). The oligonucleotide primers of the present invention may be
synthesized by conventional technology, e.g., with a commercial
oligonucleotide synthesizer and/or by ligating together
subfragments that have been so synthesized. No particular structure
for the sequencing primer is required so long as it is able to
specifically prime a region on the template nucleic acid. The
sequencing primer is extended with the DNA polymerase to form a
sequence product. The extension is performed in the presence of one
or more types of nucleotide triphosphates, and if desired,
auxiliary binding proteins. Incorporation of the dNTP is determined
by, for example, assaying for the presence of a sequencing
byproduct.
[0184] In one embodiment, the nucleic acid sequence of the
oligonucleotide tag is determined by the use of the polymerase
chain reaction (PCR). Briefly, the oligonucleotide tag (optionally
attached to a bead) is subjected to a PCR reaction as follows. The
appropriate sample is contacted with a PCR primer pair, each member
of the pair having a pre-selected nucleotide sequence. The PCR
primer pair is capable of initiating primer extension reactions by
hybridizing to a PCR primer binding site on the encoding
oligonucleotide tag.
[0185] The PCR reaction is performed by mixing the PCR primer pair,
preferably a predetermined amount thereof, with the nucleic acids
of the encoding oligonucleotide tag, preferably a predetermined
amount thereof, in a PCR buffer to form a PCR reaction admixture.
The admixture is thermocycled for a number of cycles, which is
typically predetermined, sufficient for the formation of a PCR
reaction product. A sufficient amount of product is one that can be
isolated in a sufficient amount to allow for DNA sequence
determination.
[0186] PCR is typically carried out by thermocycling i.e.,
repeatedly increasing and decreasing the temperature of a PCR
reaction admixture within a temperature range whose lower limit is
about 30.degree. C. to about 55.degree. C. and whose upper limit is
about 90.degree. C. to about 100.degree. C. The increasing and
decreasing can be continuous, but is preferably phasic with time
periods of relative temperature stability at each of temperatures
favoring polynucleotide synthesis, denaturation and
hybridization.
[0187] The PCR reaction is performed using any suitable method.
Generally it occurs in a buffered aqueous solution, i.e., a PCR
buffer, preferably at a pH of 7-9. Preferably, a molar excess of
the primer is present. A large molar excess is preferred to improve
the efficiency of the process.
[0188] The PCR buffer also contains the deoxyribonucleotide
triphosphates (polynucleotide synthesis substrates) dATP, dCTP,
dGTP, and dTTP and a polymerase, typically thermostable, all in
adequate amounts for primer extension (polynucleotide synthesis)
reaction. The resulting solution (PCR admixture) is heated to about
90.degree. C.-100.degree. C. for about 1 to 10 minutes, preferably
from 1 to 4 minutes. After this heating period the solution is
allowed to cool to 54.degree. C., which is preferable for primer
hybridization. The synthesis reaction may occur at a temperature
ranging from room temperature up to a temperature above which the
polymerase (inducing agent) no longer functions efficiently. Thus,
for example, if DNA polymerase is used, the temperature is
generally no greater than about 40.degree. C. The thermocycling is
repeated until the desired amount of PCR product is produced. An
exemplary PCR buffer comprises the following reagents: 50 mM KCl;
10 mM Tris-HCl at pH 8.3; 1.5 mM MgCl.sub.2; 0.001% (wt/vol)
gelatin, 200 .mu.M dATP; 200 .mu.M dTTP; 200 dCTP; 200 .mu.M dGTP;
and 2.5 units Thermus aquaticus (Taq) DNA polymerase I per 100
microliters of buffer.
[0189] Suitable enzymes for elongating the primer sequences
include, for example, E. coli DNA polymerase I, Taq DNA polymerase,
Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase,
other available DNA polymerases, reverse transcriptase, and other
enzymes, including heat-stable enzymes, which will facilitate
combination of the nucleotides in the proper manner to form the
primer extension products which are complementary to each nucleic
acid strand. Generally, the synthesis will be initiated at the 3'
end of each primer and proceed in the 5' direction along the
template strand, until synthesis terminates, producing molecules of
different lengths. The newly synthesized DNA strand and its
complementary strand form a double-stranded molecule which can be
used in the succeeding steps of the analysis process.
[0190] In one embodiment, the nucleotide sequence of the
oligonucleotide tag is determined by measuring inorganic
pyrophosphate (PPi) liberated from a nucleotide triphosphate (dNTP)
as the dNMP is incorporated into an extended sequence primer. This
method of sequencing, termed Pyrosequencing.TM. technology
(PyroSequencing AB, Stockholm, Sweden) can be performed in solution
(liquid phase) or as a solid phase technique. PPi-based sequencing
methods are described in, e.g., U.S. Pat. Nos. 6,274,320, 6,258,568
and 6,210,891, WO9813523A1, Ronaghi, et al. (1996) Anal Biochem.
242:84-89, Ronaghi, et al. (1998) Science 281:363-365, and USSN
2001/0024790. These disclosures of PPi sequencing are incorporated
herein in their entirety, by reference. See also, e.g., U.S. Pat.
Nos. 6,210,891 and 6,258,568, each of which are fully incorporated
herein by this reference.
[0191] Pyrophosphate can be detected by a number of different
methodologies, and various enzymatic methods have been previously
described (see e.g., Reeves, et al. (1969) Anal Biochem.
28:282-287; Guillory, et al. (1971) Anal Biochem. 39:170-180;
Johnson, et al. (1968) Anal Biochem. 15:273; Cook, et al. 1978.
Anal Biochem. 91:557-565; and Drake, et al. (1979) Anal Biochem.
94: 117-120).
[0192] In one embodiment, PPi is detected enzymatically (e.g., by
the generation of light). Such methods enable a nucleotide to be
identified in a given target position, and the DNA to be sequenced
simply and rapidly while avoiding the need for electrophoresis and
the use of potentially dangerous radiolabels.
[0193] In one embodiment, the PPi and a coupled
luciferase-luciferin reaction is used to generate light for
detection. In another embodiment, the PPi and a coupled
sulfurylase/luciferase reaction is used to generate light for
detection as described in U.S. Pat. No. 6,902,921, the contents of
which are hereby expressly incorporated herein by reference. In one
embodiment, the sulfurylase is thermostable. In some embodiments,
either or both the sulfurylase and luciferase are immobilized on
one or more mobile solid supports disposed at each reaction
site.
[0194] In another embodiment, the nucleotide sequence of the
oligonucleotide tag may be determined according to the methods
described in PCT Publication No. WO 01/23610, the contents of which
are incorporated herein by reference. Briefly, a target nucleotide
sequence can be determined by generating its complement using the
polymerase reaction to extend a suitable primer, and characterizing
the successive incorporation of bases that generate the complement
sequence. The target sequence is, typically, immobilized on a solid
support. Each of the different bases A, T, G, or C is then brought,
by sequential addition, into contact with the target, and any
incorporation events are detected via a suitable label attached to
the base.
[0195] A labeled base is incorporated into the complementary
sequence by the use of a polymerase, e.g., a polymerase with a 3'
to 5' exonuclease activity (e.g., DNA polymerase I, the Klenow
fragment, DNA polymerase III, T4 DNA polymerase, and T7 DNA
polymerase). Following detection of the incorporated labeled base,
the polymerase replaces the terminally labeled base with a
corresponding unlabelled base, thus permitting further sequencing
to occur.
[0196] In yet another embodiment, the nucleotide sequence of the
oligonucleotide tag is determined by the use of single molecule
sequencing by synthesis methods described in, for example, PCT
Publication No. WO 2005/080605, the entire contents of which are
expressly incorporated by reference. The benefit of using this
technology is that it eliminates the need for DNA amplification
prior to sequencing, thus, abolishing the introduction of
amplification errors and bias. Briefly, the encoding
oligonucleotide is hybridized to a universal primer immobilized on
a solid surface. The oligonucleotide:primer duplexes are visualized
by, e.g., illuminating the surface with a laser and imaging with a
digital TV camera connected to a microscope, and the positions of
all the duplexes on the surface are recorded. DNA polymerase and
one type of fluorescently labeled nucleotide, e.g., A, is added to
the surface and incorporated into the appropriate primer.
Subsequently, the polymerase and the unincorporated nucleotides are
washed from the surface and the incorporated nucleotide is
visualized by, e.g., illuminating the surface with a laser and
imaging with a camera as before to record the positions of the
incorporated nucleotides. The fluorescent label is removed from
each incorporated nucleotide and the process is repeated with the
next nucleotide, e.g., G, stepping through A, C, G, T, until the
desired read-length is achieved.
[0197] One group of fluorescent dyes suitable for this method of
sequencing is fluorescence resonance energy transfer (FRET) dyes,
including donor and acceptor energy fluorescent dyes and linkers
such as, for example, Cy3 and Cy5. FRET is a phenomenon described
in, for example, Selvin (1995) Methods in Enzym. 246:300. FRET can
detect the incorporation of multiple nucleotides into a single
oligonucleotide molecule and is, thus, useful for sequencing the
encoding oligonucleotides of the invention. Sequencing methods
using FRET are described in, for example, PCT Publication No. WO
2005/080605, the entire contents of which are expressly
incorporated by reference. Alternatively, quantum dots can be used
as a labeling moiety on the different types of nucleotides for use
in sequencing reactions.
[0198] Once single ligands are identified by the above-described
process, various levels of analysis can be applied to yield
structure-activity relationship information and to guide further
optimization of the affinity, specificity and bioactivity of the
ligand. For ligands derived from the same scaffold,
three-dimensional molecular modeling can be employed to identify
significant structural features common to the ligands, thereby
generating families of small-molecule ligands that presumably bind
at a common site on the target biomolecule.
[0199] A variety of screening approaches can be used to obtain
ligands that possess high affinity for one target but significantly
weaker affinity for another closely related target. One screening
strategy is to identify ligands for both biomolecules in parallel
experiments and to subsequently eliminate common ligands by a
cross-referencing comparison. In this method, ligands for each
biomolecule can be separately identified as disclosed above. This
method is compatible with both immobilized target biomolecules and
target biomolecules free in solution.
[0200] For immobilized target biomolecules, another strategy is to
add a preselection step that eliminates all ligands that bind to
the non-target biomolecule from the library. For example, a first
biomolecule can be contacted with an encoded library as described
above. Compounds which do not bind to the first biomolecule are
then separated from any first biomolecule-ligand complexes which
form. The second biomolecule is then contacted with the compounds
which did not bind to the first biomolecule. Compounds which bind
to the second biomolecule can be identified as described above and
have significantly greater affinity for the second biomolecule than
to the first biomolecule.
[0201] A ligand for a biomolecule of unknown function which is
identified by the method disclosed above can also be used to
determine the biological function of the biomolecule. This is
advantageous because although new gene sequences continue to be
identified, the functions of the proteins encoded by these
sequences and the validity of these proteins as targets for new
drug discovery and development are difficult to determine and
represent perhaps the most significant obstacle to applying genomic
information to the treatment of disease. Target-specific ligands
obtained through the process described in this invention can be
effectively employed in whole cell biological assays or in
appropriate animal models to understand both the function of the
target protein and the validity of the target protein for
therapeutic intervention. This approach can also confirm that the
target is specifically amenable to small molecule drug
discovery.
[0202] In one embodiment, one or more compounds within a library of
the invention are identified as ligands for a particular
biomolecule. These compounds can then be assessed in an in vitro
assay for the ability to bind to the biomolecule. Preferably, the
functional moieties of the binding compounds are synthesized
without the oligonucleotide tag or linker moiety, and these
functional moieties are assessed for the ability to bind to the
biomolecule.
[0203] The effect of the binding of the functional moieties to the
biomolecule on the function of the biomolecule can also be assessed
using in vitro cell-free or cell-based assays. For a biomolecule
having a known function, the assay can include a comparison of the
activity of the biomolecule in the presence and absence of the
ligand, for example, by direct measurement of the activity, such as
enzymatic activity, or by an indirect measure, such as a cellular
function that is influenced by the biomolecule. If the biomolecule
is of unknown function, a cell which expresses the biomolecule can
be contacted with the ligand and the effect of the ligand on the
viability, function, phenotype, and/or gene expression of the cell
is assessed. The in vitro assay can be, for example, a cell death
assay, a cell proliferation assay or a viral replication assay. For
example, if the biomolecule is a protein expressed by a virus, a
cell infected with the virus can be contacted with a ligand for the
protein. The affect of the binding of the ligand to the protein on
viral viability can then be assessed.
[0204] A ligand identified by the method of the invention can also
be assessed in an in vivo model or in a human. For example, the
ligand can be evaluated in an animal or organism which produces the
biomolecule. Any resulting change in the health status (e.g.,
disease progression) of the animal or organism can be
determined.
[0205] For a biomolecule, such as a protein or a nucleic acid
molecule, of unknown function, the effect of a ligand which binds
to the biomolecule on a cell or organism which produces the
biomolecule can provide information regarding the biological
function of the biomolecule. For example, the observation that a
particular cellular process is inhibited in the presence of the
ligand indicates that the process depends, at least in part, on the
function of the biomolecule.
[0206] Ligands identified using the methods of the invention can
also be used as affinity reagents for the biomolecule to which they
bind. In one embodiment, such ligands are used to effect affinity
purification of the biomolecule, for example, via chromatography of
a solution comprising the biomolecule using a solid phase to which
one or more such ligands are attached.
[0207] In addition to the screening of encoded libraries as
described herein, other traditional drug discovery methods, such as
phage display, differential display (mRNA display), and
aptamer/SELEX, could benefit from the methods of the invention
which eliminate the introduction of amplification errors and
biases. For example, multiple rounds of selection using phage
display (described in, for example, PCT Publication Nos.
WO91/18980, WO91/19818, and WO92/18619, and U.S. Pat. No.
5,223,409, the entire contents of each of which are incorporated
herein by reference) can cause host toxicity and, consequently,
loss or under-representation of desired library members (see, e.g.,
Daugherty, P. S., et al. (1999) Protein Engineering 12(7):613-621
and Holt, L. J., et al. (2000) Nucleic Acids Res. 28(15):E72).
Moreover, methods such as Systematic Evolution of Ligands by
EXponential enrichment (also known as SELEX which is described in,
for example, U.S. Pat. Nos. 5,654,151, 5,503,978, 5,567,588 and
5270163, as well as PCT Publication Nos. WO 96/38579 and
WO9927133A1, the entire contents of each of which are incorporated
herein by reference) introduce biases due to the need for multiple
rounds of selection, i.e., partitioning unbound nucleic acids from
those nucleic acids which have bound specifically to a target
molecule, and multiple rounds of amplification of the nucleic acids
that have bound to the target by reverse transcription and PCR.
Similarly, methods of selection like differential display
(described in, for example, U.S. Pat. Nos. 5,580,726 and 5,700,644,
the entire contents of each of which are incorporated herein by
reference) rely on multiple rounds of PCR amplification which also
leads to unequal representation of the clones in the library. Thus,
the foregoing multi-step selection processes may benefit from the
methods described herein which employ massively parallel sequencing
approaches (such as, for example, a pyrophosphate-based sequencing
method or a single molecule sequencing by synthesis method) which
leads to the accurate identification of a compound with a desired
biological activity without the need for any nucleic acid
amplification.
[0208] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Figures and the
Sequence Listing, are hereby incorporated in reference.
EXAMPLES
Example 1
Synthesis and Characterization of a Library on the Order of
10.sup.5 Members
[0209] The synthesis of a library comprising on the order of
10.sup.5 distinct members was accomplished using the following
reagents:
Compound 1 (SEQ ID NOS 916-917, Respectively, in Order of
Appearance):
##STR00093##
[0210] Single Letter Codes for Deoxyribonucleotides:
[0211] A=adenosine C=cytidine G=guanosine T=thymidine
Building Block Precursors:
##STR00094## ##STR00095##
[0212] Oligonucleotide Tags:
TABLE-US-00004 [0213] Tag Sequence number 5'-PO.sub.4-GCAACGAAG
(SEQ ID NO: 1) 1.1 ACCGTTGCT-PO.sub.3-5' (SEQ ID NO: 2)
5'-PO.sub.3-GCGTACAAG (SEQ ID NO: 3) 1.2 ACCGCATGT-PO.sub.3-5' (SEQ
ID NO: 4) 5'-PO.sub.3-GCTCTGTAG (SEQ ID NO: 5) 1.3
ACCGAGACA-PO.sub.3-5' (SEQ ID NO: 6) 5'-PO.sub.3-GTGCCATAG (SEQ ID
NO: 7) 1.4 ACCACGGTA-PO.sub.3-5' (SEQ ID NO: 8)
5'-PO.sub.3-GTTGACCAG (SEQ ID NO: 9) 1.5 ACCAACTGG-PO.sub.3-5' (SEQ
ID NO: 10) 5'-PO.sub.3-CGACTTGAC (SEQ ID NO: 11) 1.6
CAAGTCGCA-PO.sub.3-5' (SEQ ID NO: 12) 5'-PO.sub.3-CGTAGTCAG (SEQ ID
NO: 13) 1.7 ACGCATCAG-PO.sub.3-5' (SEQ ID NO: 14)
5'-PO.sub.3-CCAGCATAG (SEQ ID NO: 15) 1.8 ACGGTCGTA-PO.sub.3-5'
(SEQ ID NO: 16) 5'-PO.sub.3-CCTACAGAG (SEQ ID NO: 17) 1.9
ACGGATGTC-PO.sub.3-5' (SEQ ID NO: 18) 5'-PO.sub.3-CTGAACGAG (SEQ ID
NO: 19) 1.10 CGTTCAGCA-PO.sub.3-5' (SEQ ID NO: 20)
5'-PO.sub.3-CTCCAGTAG (SEQ ID NO: 21) 1.11 ACGAGGTCA-PO.sub.3-5'
(SEQ ID NO: 22) 5'-PO.sub.3-TAGGTCCAG (SEQ ID NO: 23) 1.12
ACATCCAGG-PO.sub.3-5' (SEQ ID NO: 24) 5'-PO.sub.3-GCGTGTTGT (SEQ ID
NO: 25) 2.1 TCCGCACAA-PO.sub.3-5' (SEQ ID NO: 26)
5'-PO.sub.3-GCTTGGAGT (SEQ ID NO: 27) 2.2 TCCGAACCT-PO.sub.3-5'
(SEQ ID NO: 28) 5'-PO.sub.3-GTCAAGCGT (SEQ ID NO: 29) 2.3
TCCAGTTCG-PO.sub.3-5' (SEQ ID NO: 30) 5'-PO.sub.3-CAAGAGCGT (SEQ ID
NO: 31) 2.4 TCGTTCTCG-PO.sub.3-5' (SEQ ID NO: 32)
5'-PO.sub.3-CAGTTCGGT (SEQ ID NO: 33) 2.5 TCGTCAAGC-PO.sub.3-5'
(SEQ ID NO: 34) 5'-PO.sub.3-CGAAGGAGT (SEQ ID NO: 35) 2.6
TCGCTTCCT-PO.sub.3-5' (SEQ ID NO: 36) 5'-PO.sub.3-CGGTGTTGT (SEQ ID
NO: 37) 2.7 TCGCCACAA-PO.sub.3-5' (SEQ ID NO: 38)
5'-PO.sub.3-CGTTGCTGT (SEQ ID NO: 39) 2.8 TCGCAACGA-PO.sub.3-5'
(SEQ ID NO: 40) 5'-PO.sub.3-CCGATCTGT (SEQ ID NO: 41) 2.9
TCGGCTAGA-PO.sub.3-5' (SEQ ID NO: 42) 5'-PO.sub.3-CCTTCTCGT (SEQ ID
NO: 43) 2.10 TCGGAAGAG-PO.sub.3-5' (SEQ ID NO: 44)
5'-PO.sub.3-TGAGTCCGT (SEQ ID NO: 45) 2.11 TCACTCAGG-PO.sub.3-5'
(SEQ ID NO: 46) 5'-PO.sub.3-TGCTACGGT (SEQ ID NO: 47) 2.12
TCAGATTGC-PO.sub.3-5' (SEQ ID NO: 48) 5'-PO.sub.3-GTGCGTTGA (SEQ ID
NO: 49) 3.1 CACACGCAA-PO.sub.3-5' (SEQ ID NO: 50)
5'-PO.sub.3-GTTGGCAGA (SEQ ID NO: 51) 3.2 CACAACCGT-PO.sub.3-5'
(SEQ ID NO: 52) 5'-PO.sub.3-CCTGTAGGA (SEQ ID NO: 53) 3.3
CAGGACATC-PO.sub.3-5' (SEQ ID NO: 54) 5'-PO.sub.3-CTGCGTAGA (SEQ ID
NO: 55) 3.4 CAGACGCAT-PO.sub.3-5' (SEQ ID NO: 56)
5'-PO.sub.3-CTTACGCGA (SEQ ID NO: 57) 3.5 CAGAATGCG-PO.sub.3-5'
(SEQ ID NO: 58) 5'-PO.sub.3-TGGTCACGA (SEQ ID NO: 59) 3.6
CAACCAGTG-PO.sub.3-5' (SEQ ID NO: 60) 5'-PO.sub.3-TCAGAGCGA (SEQ ID
NO: 61) 3.7 CAAGTCTCG-PO.sub.3-5' (SEQ ID NO: 62)
5'-PO.sub.3-TTGCTCGGA (SEQ ID NO: 63) 3.8 CAAACGAGC-PO.sub.3-5'
(SEQ ID NO: 64) 5'-PO.sub.3-GCAGTTGGA (SEQ ID NO: 65) 3.9
CACGTCAAC-PO.sub.3-5' (SEQ ID NO: 66) 5'-PO.sub.3-GCCTGAAGA (SEQ ID
NO: 67) 3.10 CACGGACTT-PO.sub.3-5' (SEQ ID NO: 68)
5'-PO.sub.3-GTAGCCAGA (SEQ ID NO: 69) 3.11 CACATCGGT-PO.sub.3-5'
(SEQ ID NO: 70) 5'-PO.sub.3-GTCGCTTGA (SEQ ID NO: 71) 3.12
CACAGCGAA-PO.sub.3-5' (SEQ ID NO: 72) 5'-PO.sub.3-GCCTAAGTT (SEQ ID
NO: 73) 4.1 CTCGGATTC-PO.sub.3-5' (SEQ ID NO: 74)
5'-PO.sub.3-GTAGTGCTT (SEQ ID NO: 75) 4.2 CTCATCACG-PO.sub.3-5'
(SEQ ID NO: 76) 5'-PO.sub.3-GTCGAAGTT (SEQ ID NO: 77) 4.3
CTCAGCTTC-PO.sub.3-5' (SEQ ID NO: 78) 5'-PO.sub.3-GTTTCGGTT (SEQ ID
NO: 79) 4.4 CTCAAAGCC-PO.sub.3-5' (SEQ ID NO: 80)
5'-PO.sub.3-CAGCGTTTT (SEQ ID NO: 81) 4.5 CTGTCGCAA-PO.sub.3-5'
(SEQ ID NO: 82) 5'-PO.sub.3-CATACGCTT (SEQ ID NO: 83) 4.6
CTGTATGCG-PO.sub.3-5' (SEQ ID NO: 84) 5'-PO.sub.3-CGATCTGTT (SEQ ID
NO: 85) 4.7 CTGCTAGAC-PO.sub.3-5' (SEQ ID NO: 86)
5'-PO.sub.3-CGCTTTGTT (SEQ ID NO: 87) 4.8 CTGCGAAAC-PO.sub.3-5'
(SEQ ID NO: 88) 5'-PO.sub.3-CCACAGTTT (SEQ ID NO: 89) 4.9
CTGGTGTCA-PO.sub.3-5' (SEQ ID NO: 90) 5'-PO.sub.3-CCTGAAGTT (SEQ ID
NO: 91) 4.10 CTGGACTTC-PO.sub.3-5' (SEQ ID NO: 92)
5'-PO.sub.3-CTGACGATT (SEQ ID NO: 93) 4.11 CTGACTGCT-PO.sub.3-5'
(SEQ ID NO: 94) 5'-PO.sub.3-CTCCACTTT (SEQ ID NO: 95) 4.12
CTGAGGTGA-PO.sub.3-5' (SEQ ID NO: 96) 5'-PO.sub.3-ACCAGAGCC (SEQ ID
NO: 97) 5.1 AATGGTCTC-PO.sub.3-5' (SEQ ID NO: 98)
5'-PO.sub.3-ATCCGCACC (SEQ ID NO: 99) 5.2 AATAGGCGT-PO.sub.3-5'
(SEQ ID NO: 100) 5'-PO.sub.3-GACGACACC (SEQ ID NO: 101) 5.3
AACTGCTGT-PO.sub.3-5' (SEQ ID NO: 102) 5'-PO.sub.3-GGATGGACC (SEQ
ID NO: 103) 5.4 AACCTACCT-PO.sub.3-5' (SEQ ID NO: 104)
5'-PO.sub.3-GCAGAAGCC (SEQ ID NO: 105) 5.5 AACGTCTTC-PO.sub.3-5'
(SEQ ID NO: 106) 5'-PO.sub.3-GCCATGTCC (SEQ ID NO: 107) 5.6
AACGGTACA-PO.sub.3-5' (SEQ ID NO: 108) 5'-PO.sub.3-GTCTGCTCC (SEQ
ID NO: 109) 5.7 AACAGACGA-PO.sub.3-5' (SEQ ID NO: 110)
5'-PO.sub.3-CGACAGACC (SEQ ID NO: 111) 5.8 AAGCTGTCT-PO.sub.3-5'
(SEQ ID NO: 112) 5'-PO.sub.3-CGCTACTCC (SEQ ID NO: 113) 5.9
AAGCGATGA-PO.sub.3-5' (SEQ ID NO: 114) 5'-PO.sub.3-CCACAGACC (SEQ
ID NO: 115) 5.10 AAGGTGTCT-PO.sub.3-5' (SEQ ID NO: 116)
5'-PO.sub.3-CCTCTCTCC (SEQ ID NO: 117) 5.11 AAGGAGAGA-PO.sub.3-5'
(SEQ ID NO: 118) 5'-PO.sub.3-CTCGTAGCC (SEQ ID NO: 119) 5.12
AAGAGCATC-PO.sub.3-5' (SEQ ID NO: 120)
1.times. ligase buffer: 50 mM Tris, pH 7.5; 10 mM dithiothreitol;
10 mM MgCl.sub.2; 2.5 mM ATP; 50 mM NaCl. 10.times. ligase buffer:
500 mM Tris, pH 7.5; 100 mM dithiothreitol; 100 mM MgCl.sub.2; 25
mM ATP; 500 mM NaCl
Cycle 1
[0214] To each of twelve PCR tubes was added 50 .mu.L of a 1 mM
solution of Compound 1 in water; 75 .mu.L of a 0.80 mM solution of
one of Tags 1.1-1.12; 15 .mu.L 10.times. ligase buffer and 10 .mu.L
deionized water. The tubes were heated to 95.degree. C. for 1
minute and then cooled to 16.degree. C. over 10 minutes. To each
tube was added 5,000 units T4 DNA ligase (2.5 .mu.L of a 2,000,000
unit/mL solution (New England Biolabs, Cat. No. M0202)) in 50 .mu.l
1.times. ligase buffer and the resulting solutions were incubated
at 16.degree. C. for 16 hours.
[0215] Following ligation, samples were transferred to 1.5 ml
Eppendorf tubes and treated with 20 .mu.L 5 M aqueous NaCl and 500
.mu.L cold (-20.degree. C.) ethanol, and held at -20.degree. C. for
1 hour. Following centrifugation, the supernatant was removed and
the pellet was washed with 70% aqueous ethanol at -20.degree. C.
Each of the pellets was then dissolved in 150 .mu.L of 150 mM
sodium borate buffer, pH 9.4.
[0216] Stock solutions comprising one each of building block
precursors BB1 to BB12, N,N-diisopropylethanolamine and
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate, each at a concentration of 0.25 M, were
prepared in DMF and stirred at room temperature for 20 minutes. The
building block precursor solutions were added to each of the pellet
solutions described above to provide a 10-fold excess of building
block precursor relative to linker. The resulting solutions were
stirred. An additional 10 equivalents of building block precursor
was added to the reaction mixture after 20 minute, and another 10
equivalents after 40 minutes. The final concentration of DMF in the
reaction mixture was 22%. The reaction solutions were then stirred
overnight at 4.degree. C. The reaction progress was monitored by
RP-HPLC using 50 mM aqueous tetraethylammonium acetate (pH=7.5) and
acetonitrile, and a gradient of 2-46% acetonitrile over 14 min.
Reaction was stopped when .about.95% of starting material (linker)
is acylated. Following acylation the reaction mixtures were pooled
and lyophilized to dryness. The lyophilized material was then
purified by HPLC, and the fractions corresponding to the library
(acylated product) were pooled and lyophilized.
[0217] The library was dissolved in 2.5 ml of 0.01M sodium
phosphate buffer (pH=8.2) and 0.1 ml of piperidine (4% v/v) was
added to it. The addition of piperidine results in turbidity which
does not dissolve on mixing. The reaction mixtures were stirred at
room temperature for 50 minutes, and then the turbid solution was
centrifuged (14,000 rpm), the supernatant was removed using a 200
.mu.l pipette, and the pellet was resuspended in 0.1 ml of water.
The aqueous wash was combined with the supernatant and the pellet
was discarded. The deprotected library was precipitated from
solution by addition of excess ice-cold ethanol so as to bring the
final concentration of ethanol in the reaction to 70% v/v.
Centrifugation of the aqueous ethanol mixture gave a white pellet
comprising the library. The pellet was washed once with cold 70%
aq. ethanol. After removal of solvent the pellet was dried in air
(.about.5 min.) to remove traces of ethanol and then used in cycle
2. The tags and corresponding building block precursors used in
Round 1 are set forth in Table 1, below.
TABLE-US-00005 TABLE 1 Building Block Precursor Tag BB1 1.11 BB2
1.6 BB3 1.2 BB4 1.8 BB5 1.1 BB6 1.10 BB7 1.12 BB8 1.5 BB9 1.4 BB10
1.3 BB11 1.7 BB12 1.9
Cycles 2-5
[0218] For each of these cycles, the combined solution resulting
from the previous cycle was divided into 12 equal aliquots of 50 ul
each and placed in PCR tubes. To each tube was added a solution
comprising a different tag, and ligation, purification and
acylation were performed as described for Cycle 1, except that for
Cycles 3-5, the HPLC purification step described for Cycle 1 was
omitted. The correspondence between tags and building block
precursors for Cycles 2-5 is presented in Table 2.
[0219] The products of Cycle 5 were ligated with the closing primer
shown below, using the method described above for ligation of
tags.
TABLE-US-00006 5'-PO.sub.3-GGCACATTGATTTGGGAGTCA (SEQ ID NO: 918)
GTGTAACTAAACCCTCAGT-PO.sub.3-5' (SEQ ID NO: 919)
TABLE-US-00007 TABLE 2 Building Block Cycle 2 Cycle 3 Cycle 4 Cycle
5 Precursor Tag Tag Tag Tag BB1 2.7 3.7 4.7 5.7 BB2 2.8 3.8 4.8 5.8
BB3 2.2 3.2 4.2 5.2 BB4 2.10 3.10 4.10 5.10 BB5 2.1 3.1 4.1 5.1 BB6
2.12 3.12 4.12 5.12 BB7 2.5 3.5 4.5 5.5 BB8 2.6 3.6 4.6 5.6 BB9 2.4
3.4 4.4 5.4 BB10 2.3 3.3 4.3 5.3 BB11 2.9 3.9 4.9 5.9 BB12 2.11
3.11 4.11 5.11
Results:
[0220] The synthetic procedure described above has the capability
of producing a library comprising 12.sup.5 (about 249,000)
different structures. The synthesis of the library was monitored
via gel electrophoresis of the product of each cycle. The results
of each of the five cycles and the final library following ligation
of the closing primer are illustrated in FIG. 7. The compound
labeled "head piece" is Compound 1. The figure shows that each
cycle results in the expected molecular weight increase and that
the products of each cycle are substantially homogeneous with
regard to molecular weight.
Example 2
Synthesis and Characterization of a Library on the Order of
10.sup.8 Members
[0221] The synthesis of a library comprising on the order of
10.sup.8 distinct members was accomplished using the following
reagents:
Compound 2: (Nucleotides 1-8 of SEQ ID NO:916 and Nucleotides 12-17
of SEQ ID NO:917, Respectively, in Order of Appearance):
##STR00096##
[0222] Single Letter Codes for Deoxyribonucleotides:
[0223] A=adenosine C=cytidine G=guanosine T=thymidine
Building Block Precursors:
##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101##
##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106##
##STR00107## ##STR00108## ##STR00109##
TABLE-US-00008 [0224] TABLE 3 Oligonucleotide tags used in cycle 1:
Tag Number Top Strand Sequence Bottom Strand Sequence 1.1 5'-PO3-
5'-PO3- AAATCGATGTGGTCACTCAG GAGTGACCACATCGATTTGG (SEQ ID NO: 121)
(SEQ ID NO: 122) 1.2 5'-PO3- 5'-PO3- AAATCGATGTGGACTAGGAG
CCTAGTCCACATCGATTTGG (SEQ ID NO: 123) (SEQ ID NO: 124) 1.3 5'-PO3-
5'-PO3- AAATCGATGTGCCGTATGAG CATACGGCACATCGATTTGG (SEQ ID NO: 125)
(SEQ ID NO: 126) 1.4 5'-PO3- 5'-PO3- AAATCGATGTGCTGAAGGAG
CCTTCAGCACATCGATTTGG (SEQ ID NO: 127) (SEQ ID NO: 128) 1.5 5'-PO3-
5'-PO3- AAATCGATGTGGACTAGCAG GCTAGTCCACATCGATTTGG (SEQ ID NO: 129)
(SEQ ID NO: 130) 1.6 5'-PO3- 5'-PO3- AAATCGATGTGCGCTAAGAG
CTTAGCGCACATCGATTTGG (SEQ ID NO: 131) (SEQ ID NO: 132) 1.7 5'-PO3-
5'-PO3- AAATCGATGTGAGCCGAGAG CTCGGCTCACATCGATTTGG (SEQ ID NO: 133)
(SEQ ID NO: 134) 1.8 5'-PO3- 5'-PO3- AAATCGATGTGCCGTATCAG
GATACGGCACATCGATTTGG (SEQ ID NO: 135) (SEQ ID NO: 136) 1.9 5'-PO3-
5'-PO3- AAATCGATGTGCTGAAGCAG GCTTCAGCACATCGATTTGG (SEQ ID NO: 137)
(SEQ ID NO: 138) 1.10 5'-PO3- 5'-PO3- AAATCGATGTGTGCGAGTAG
ACTCGCACACATCGATTTGG (SEQ ID NO: 139) (SEQ ID NO: 140) 1.11 5'-PO3-
5'-PO3- AAATCGATGTGTTTGGCGAG CGCCAAACACATCGATTTGG (SEQ ID NO: 141)
(SEQ ID NO: 142) 1.12 5'-PO3- 5'-PO3- AAATCGATGTGCGCTAACAG
GTTAGCGCACATCGATTTGG (SEQ ID NO: 143) (SEQ ID NO: 144) 1.13 5'-PO3-
5'-PO3- AAATCGATGTGAGCCGACAG GTCGGCTCACATCGATTTGG (SEQ ID NO: 145)
(SEQ ID NO: 146) 1.14 5'-PO3- 5'-PO3- AAATCGATGTGAGCCGAAAG
TTCGGCTCACATCGATTTGG (SEQ ID NO: 147) (SEQ ID NO: 148) 1.15 5'-PO3-
5'-PO3- AAATCGATGTGTCGGTAGAG CTACCGACACATCGATTTGG (SEQ ID NO: 149)
(SEQ ID NO: 150) 1.16 5'-PO3- 5'-PO3- AAATCGATGTGGTTGCCGAG
CGGCAACCACATCGATTTGG (SEQ ID NO: 151) (SEQ ID NO: 152) 1.17 5'-PO3-
5'-PO3- AAATCGATGTGAGTGCGTAG ACGCACTCACATCGATTTGG (SEQ ID NO: 153)
(SEQ ID NO: 154) 1.18 5'-PO3- 5'-PO3- AAATCGATGTGGTTGCCAAG
TGGCAACCACATCGATTTGG (SEQ ID NO: 155) (SEQ ID NO: 156) 1.19 5'-PO3-
5'-PO3- AAATCGATGTGTGCGAGGAG CCTCGCACACATCGATTTGG (SEQ ID NO: 157)
(SEQ ID NO: 158) 1.20 5'-PO3- 5'-PO3- AAATCGATGTGGAACACGAG
CGTGTTCCACATCGATTTGG (SEQ ID NO: 159) (SEQ ID NO: 160) 1.21 5'-PO3-
5'-PO3- AAATCGATGTGCTTGTCGAG CGACAAGCACATCGATTTGG (SEQ ID NO: 161)
(SEQ ID NO: 162) 1.22 5'-PO3- 5'-PO3- AAATCGATGTGTTCCGGTAG
A0CCGGAACACATCGATTTGG (SEQ ID NO: 163) (SEQ ID NO: 164) 1.23
5'-PO3- 5'-PO3- AAATCGATGTGTGCGAGCAG GCTCGCACACATCGATTTGG (SEQ ID
NO: 165) (SEQ ID NO: 166) 1.24 5'-PO3- 5'-PO3- AAATCGATGTGGTCAGGTAG
ACCTGACCACATCGATTTGG (SEQ ID NO: 167) (SEQ ID NO: 168) 1.25 5'-PO3-
5'-PO3- AAATCGATGTGGCCTGTTAG AACAGGCCACATCGATTTGG (SEQ ID NO: 169)
(SEQ ID NO: 170) 1.26 5'-PO3- 5'-PO3- AAATCGATGTGGAACACCAG
GGTGTTCCACATCGATTTGG (SEQ ID NO: 171) (SEQ ID NO: 172) 1.27
5'-PO3-AAATCGATGTGCTTGTCCAG 5'-PO3- (SEQ ID NO: 173)
GGACAAGCACATCGATTTGG (SEQ ID NO: 174) 1.28 5'-PO3- 5'-PO3-
AAATCGATGTGTGCGAGAAG TCTCGCACACATCGATTTGG (SEQ ID NO: 175) (SEQ ID
NO: 176) 1.29 5'-PO3- 5'-PO3- AAATCGATGTGAGTGCGGAG
CCGCACTCACATCGATTTGG (SEQ ID NO: 177) (SEQ ID NO: 178) 1.30 5'-PO3-
5'-PO3- AAATCGATGTGTTGTCCGAG CGGACAACACATCGATTTGG (SEQ ID NO: 179)
(SEQ ID NO: 180) 1.31 5'-PO3- 5'-PO3- AAATCGATGTGTGGAACGAG
CGTTCCACACATCGATTTGG (SEQ ID NO: 181) (SEQ ID NO: 182) 1.32 5'-PO3-
5'-PO3- AAATCGATGTGAGTGCGAAG TCGCACTCACATCGATTTGG (SEQ ID NO: 183)
(SEQ ID NO: 184) 1.33 5'-PO3- 5'-PO3- AAATCGATGTGTGGAACCAG
GGTTCCACACATCGATTTGG (SEQ ID NO: 185) (SEQ ID NO: 186) 1.34 5'-PO3-
5'-PO3- AAATCGATGTGTTAGGCGAG CGCCTAACACATCGATTTGG (SEQ ID NO: 187)
(SEQ ID NO: 188) 1.35 5'-PO3- 5'-PO3- AAATCGATGTGGCCTGTGAG
CACAGGCCACATCGATTTGG (SEQ ID NO: 189) (SEQ ID NO: 190) 1.36
5'-PO3-AAATCGATGTGCTCCTGTAG 5'-PO3- (SEQ ID NO: 191)
ACAGGAGCACATCGATTTGG (SEQ ID NO: 192) 1.37 5'-PO3- 5'-PO3-
AAATCGATGTGGTCAGGCAG GCCTGACCACATCGATTTGG (SEQ ID NO: 193) (SEQ ID
NO: 194) 1.38 5'-PO3- 5'-PO3- AAATCGATGTGGTCAGGAAG
TCCTGACCACATCGATTTGG (SEQ ID NO: 195) (SEQ ID NO: 196) 1.39 5'-PO3-
5'-PO3- AAATCGATGTGGTAGCCGAG CGGCTACCACATCGATTTGG (SEQ ID NO: 197)
(SEQ ID NO: 198) 1.40 5'-PO3- 5'-PO3- AAATCGATGTGGCCTGTAAG
TACAGGCCACATCGATTTGG (SEQ ID NO: 199) (SEQ ID NO: 200) 1.41 5'-PO3-
5'-PO3- AAATCGATGTGCTTTCGGAG CCGAAAGCACATCGATTTGG (SEQ ID NO: 201)
(SEQ ID NO: 202) 1.42 5'-PO3- 5'-PO3- AAATCGATGTGCGTAAGGAG
CCTTACGCACATCGATTTGG (SEQ ID NO: 203) (SEQ ID NO: 204) 1.43 5'-PO3-
5'-PO3- AAATCGATGTGAGAGCGTAG ACGCTCTCACATCGATTTGG (SEQ ID NO: 205)
(SEQ ID NO: 206) 1.44 5'-PO3- 5'-PO3- AAATCGATGTGGACGGCAAG
TGCCGTCCACATCGATTTGG (SEQ ID NO: 207) (SEQ ID NO: 208) 1.45
5'-PO3-AAATCGATGTGCTTTCGCAG 5'-PO3- (SEQ ID NO: 209)
GCGAAAGCACATCGATTTGG (SEQ ID NO: 210) 1.46 5'-PO3- 5'-PO3-
AAATCGATGTGCGTAAGCAG GCTTACGCACATCGATTTGG (SEQ ID NO: 211) (SEQ ID
NO: 212) 1.47 5'-PO3- 5'-PO3- AAATCGATGTGGCTATGGAG
CCATAGCCACATCGATTTGG (SEQ ID NO: 213) (SEQ ID NO: 214) 1.48 5'-PO3-
5'-PO3- AAATCGATGTGACTCTGGAG CCAGAGTCACATCGATTTGG (SEQ ID NO: 215)
(SEQ ID NO: 216) 1.49 5'-PO3-AAATCGATGTGCTGGAAAG 5'-PO3- (SEQ ID
NO: 217) TTCCAGCACATCGATTTGG (SEQ ID NO: 218) 1.50 5'-PO3- 5'-PO3-
AAATCGATGTGCCGAAGTAG ACTTCGGCACATCGATTTGG (SEQ ID NO: 219) (SEQ ID
NO: 220) 1.51 5'-PO3- 5'-PO3- AAATCGATGTGCTCCTGAAG
TCAGGAGCACATCGATTTGG (SEQ ID NO: 221) (SEQ ID NO: 222) 1.52 5'-PO3-
5'-PO3- AAATCGATGTGTCCAGTCAG GACTGGACACATCGATTTGG (SEQ ID NO: 223)
(SEQ ID NO: 224) 1.53 5'-PO3- 5'-PO3- AAATCGATGTGAGAGCGGAG
CCGCTCTCACATCGATTTGG (SEQ ID NO: 225) (SEQ ID NO: 226) 1.54 5'-PO3-
5'-PO3- AAATCGATGTGAGAGCGAAG TCGCTCTCACATCGATTTGG (SEQ ID NO: 227)
(SEQ ID NO: 228) 1.55 5'-PO3- 5'-PO3- AAATCGATGTGCCGAAGGAG
CCTTCGGCACATCGATTTGG (SEQ ID NO: 229) (SEQ ID NO: 230) 1.56 5'-PO3-
5'-PO3- AAATCGATGTGCCGAAGCAG GCTTCGGCACATCGATTTGG (SEQ ID NO: 231)
(SEQ ID NO: 232) 1.57 5'-PO3- 5'-PO3- AAATCGATGTGTGTTCCGAG
CGGAACACACATCGATTTGG (SEQ ID NO: 233) (SEQ ID NO: 234) 1.58 5'-PO3-
5'-PO3- AAATCGATGTGTCTGGCGAG CGCCAGACACATCGATTTGG (SEQ ID NO: 235)
(SEQ ID NO: 236) 1.59 5'-PO3- 5'-PO3- AAATCGATGTGCTATCGGAG
CCGATAGCACATCGATTTGG (SEQ ID NO: 237) (SEQ ID NO: 238) 1.60 5'-PO3-
5'-PO3- AAATCGATGTGCGAAAGGAG CCTTTCGCACATCGATTTGG (SEQ ID NO: 239)
(SEQ ID NO: 240) 1.61 5'-PO3- 5'-PO3- AAATCGATGTGCCGAAGAAG
TCTTCGGCACATCGATTTGG (SEQ ID NO: 241) (SEQ ID NO: 242)
1.62 5'-PO3- 5'-PO3- AAATCGATGTGGTTGCAGAG CTGCAACCACATCGATTTGG (SEQ
ID NO: 243) (SEQ ID NO: 244) 1.63 5'-PO3- 5'-PO3-
AAATCGATGTGGATGGTGAG- CACCATCCACATCGATTTGG (SEQ ID NO: 245) (SEQ ID
NO: 246) 1.64 5'-PO3- 5'-PO3- AAATCGATGTGCTATCGCAG
GCGATAGCACATCGATTTGG (SEQ ID NO: 247) (SEQ ID NO: 248) 1.65 5'-PO3-
5'-PO3- AAATCGATGTGCGAAAGCAG GCTTTCGCACATCGATTTGG (SEQ ID NO: 249)
(SEQ ID NO: 250) 1.66 5'-PO3- 5'-PO3- AAATCGATGTGACACTGGAG
CCAGTGTCACATCGATTTGG (SEQ ID NO: 251) (SEQ ID NO: 252) 1.67 5'-PO3-
5'-PO3- AAATCGATGTGTCTGGCAAG TGCCAGACACATCGATTTGG (SEQ ID NO: 253)
(SEQ ID NO: 254) 1.68 5'-PO3- 5'-PO3- AAATCGATGTGGATGGTCAG
GACCATCCACATCGATTTGG (SEQ ID NO: 255) (SEQ ID NO: 256) 1.69 5'-PO3-
5'-PO3- AAATCGATGTGGTTGCACAG GTGCAACCACATCGATTTGG (SEQ ID NO: 257)
(SEQ ID NO: 258) 1.70 5'-PO3- 5'-PO3-CGATGCCCCATCCGA
AAATCGATGTGGGCATCGAG TTT GG (SEQ ID NO: 259) (SEQ ID NO: 260) 1.71
5'-PO3- 5'-PO3- AAATCGATGTGTGCCTCCAG GGAGGCACACATCGATTTGG (SEQ ID
NO: 261) (SEQ ID NO: 262) 1.72 5'-PO3- 5'-PO3- AAATCGATGTGTGCCTCAAG
TGAGGCACACATCGATTTGG (SEQ ID NO: 263) (SEQ ID NO: 264) 1.73 5'-PO3-
5'-PO3- AAATCGATGTGGGCATCCAG GGATGCCCACATCGATTTGG (SEQ ID NO: 265)
(SEQ ID NO: 266) 1.74 5'-PO3- 5'-PO3-TGATGCCCA CAT CGA
AAATCGATGTGGGCATCAAG TTT GG (SEQ ID NO: 267) (SEQ ID NO: 268) 1.75
5'-PO3- 5'-PO3-CGA CAG GCA CAT AAATCGATGTGCCTGTCGAG CGA TTT GG (SEQ
ID NO: 269) (SEQ ID NO: 270) 1.76 5'-PO3- 5'-PO3-ATC CGT CCA CAT
AAATCGATGTGGACGGATAG CGA TTT GG (SEQ ID NO: 271) (SEQ ID NO: 272)
1.77 5'-PO3- 5'-PO3-GGA CAG GCA CAT AAATCGATGTGCCTGTCCAG CGA TTT GG
(SEQ ID NO: 273) (SEQ ID NO: 274) 1.78 5'-PO3- 5'-PO3-CGT GCT TCA
CAT AAATCGATGTGAAGCACGAG CGA TTT GG (SEQ ID NO: 275) (SEQ ID NO:
276) 1.79 5'-PO3- 5'-PO3-TGA CAG GCA CAT AAATCGATGTGCCTGTCAAG CGA
TTT GG (SEQ ID NO: 277) (SEQ ID NO: 278) 1.80 5'-PO3- 5'-PO3-GGT
GCT TCA CAT AAATCGATGTGAAGCACCAG CGA TTT GG (SEQ ID NO: 279) (SEQ
ID NO: 280) 1.81 5'-PO3-AAATCGATGTGCCTTCGTAG 5'-PO3-ACG AAG GCA CAT
(SEQ ID NO: 281) CGA TTT GG (SEQ ID NO: 282) 1.82 5'-PO3-
5'-PO3-CGG ACG ACA CAT AAATCGATGTGTCGTCCGAG CGA TTT GG (SEQ ID NO:
283) (SEQ ID NO: 284) 1.83 5'-PO3- 5'-PO3-CAG ACT CCA CAT
AAATCGATGTGGAGTCTGAG CGA TTT GG (SEQ ID NO: 285) (SEQ ID NO: 286)
1.84 5'-PO3- 5'-PO3-CGG ATC ACA CAT AAATCGATGTGTGATCCGAG CGA TTT GG
(SEQ ID NO: 287) (SEQ ID NO: 288) 1.85 5'-PO3- 5'-PO3-CGC CTG ACA
CAT AAATCGATGTGTCAGGCGAG CGA TTT GG (SEQ ID NO: 289) (SEQ ID NO:
290) 1.86 5'-PO3- 5'-PO3-TGG ACG ACA CAT AAATCGATGTGTCGTCCAAG CGA
TTT GG (SEQ ID NO: 291) (SEQ ID NO: 292) 1.87 5'-PO3- 5'-PO3-CTC
CGT CCA CAT AAATCGATGTGGACGGAGAG CGA TTT GG (SEQ ID NO: 293) (SEQ
ID NO: 294) 1.88 5'-PO3- 5'-PO3-CTG CTA CCA CAT
AAATCGATGTGGTAGCAGAG CGA TTT GG (SEQ ID NO: 295) (SEQ ID NO: 296)
1.89 5'-PO3- 5'-PO3- AAATCGATGTGGCTGTGTAG ACACAGCCACATCGATTTGG (SEQ
ID NO: 297) (SEQ ID NO: 298) 1.90 5'-PO3- 5'-PO3-GTC CGT CCA CAT
AAATCGATGTGGACGGACAG CGA TTT GG (SEQ ID NO: 299) (SEQ ID NO: 300)
1.91 5'-PO3- 5'-PO3-TGC CTG ACA CAT AAATCGATGTGTCAGGCAAG CGA TTT GG
(SEQ ID NO: 301) (SEQ ID NO: 302) 1.92 5'-PO3- 5'-PO3-
AAATCGATGTGGCTCGAAAG TTCGAGCCACATCGATTTGG (SEQ ID NO: 303) (SEQ ID
NO: 304) 1.93 5'-PO3- 5'-PO3-CCG AAG GCA CAT AAATCGATGTGCCTTCGGAG
CGA TTT GG (SEQ ID NO: 305) (SEQ ID NO: 306) 1.94 5'-PO3-
5'-PO3-GTG CTA CCA CAT AAATCGATGTGGTAGCACAG CGA TTT GG (SEQ ID NO:
307) (SEQ ID NO: 308) 1.95 5'-PO3- 5'-PO3-GAC CTT CCA CAT
AAATCGATGTGGAAGGTCAG CGA TTT GG (SEQ ID NO: 309) (SEQ ID NO: 310)
1.96 5'-PO3- 5'-PO3-ACA GCA CCA CAT AAATCGATGTGGTGCTGTAG CGA TTT GG
(SEQ ID NO: 311) (SEQ ID NO: 312)
TABLE-US-00009 TABLE 4 Oligonucleotide tags used in cycle 2: Tag
Number Top strand sequence Bottom strand sequence 2.1 5'-PO3-GTT
GCC TGT 5'-PO3-AGG CAA CCT (SEQ ID NO: 313) (SEQ ID NO: 314) 2.2
5'-PO3-CAG GAC GGT 5'-PO3-CGT CCT GCT (SEQ ID NO: 315) (SEQ ID NO:
316) 2.3 5'-PO3-AGA CGT GGT 5'-PO3-CAC GTC TCT (SEQ ID NO: 317)
(SEQ ID NO: 318) 2.4 5'-PO3-CAG GAC CGT 5'-PO3-GGT CCT GCT (SEQ ID
NO: 319) (SEQ ID NO: 320) 2.5 5'-PO3-CAG GAC AGT 5'-PO3-TGT CCT GCT
(SEQ ID NO: 321) (SEQ ID NO: 322) 2.6 5'-PO3-CAC TCT GGT 5'-PO3-CAG
AGT GCT (SEQ ID NO: 323) (SEQ ID NO: 324) 2.7 5'-PO3-GAC GGC TGT
5'-PO3-AGC CGT CCT (SEQ ID NO: 325) (SEQ ID NO: 326) 2.8 5'-PO3-CAC
TCT CGT 5'-PO3-GAG AGT GCT (SEQ ID NO: 327) (SEQ ID NO: 328) 2.9
5'-PO3-GTA GCC TGT 5'-PO3-AGG CTA CCT (SEQ ID NO: 329) (SEQ ID NO:
330) 2.10 5'-PO3-GCC ACT TGT 5'-PO3-AAG TGG CCT (SEQ ID NO: 331)
(SEQ ID NO: 332) 2.11 5'-PO3-CAT CGC TGT 5'-PO3-AGC GAT GCT (SEQ ID
NO: 333) (SEQ ID NO: 334) 2.12 5'-PO3-CAC TGG TGT 5'-PO3-ACC AGT
GCT (SEQ ID NO: 335) (SEQ ID NO: 336) 2.13 5'-PO3-GCC ACT GGT
5'-PO3-CAG TGG CCT (SEQ ID NO: 337) (SEQ ID NO: 338) 2.14
5'-PO3-TCT GGC TGT 5'-PO3-AGC CAG ACT (SEQ ID NO: 339) (SEQ ID NO:
340) 2.15 5'-PO3-GCC ACT CGT 5'-PO3-GAG TGG CCT (SEQ ID NO: 341)
(SEQ ID NO: 342) 2.16 5'-PO3-TGC CTC TGT 5'-PO3-AGA GGC ACT (SEQ ID
NO: 343) (SEQ ID NO: 344) 2.17 5'-PO3-CAT CGC AGT 5'-PO3-TGC GAT
GCT (SEQ ID NO: 345) (SEQ ID NO: 346) 2.18 5'-PO3-CAG GAA GGT
5'-PO3-CTT CCT GCT (SEQ ID NO: 347) (SEQ ID NO: 348) 2.19
5'-PO3-GGC ATC TGT 5'-PO3-AGA TGC CCT (SEQ ID NO: 349) (SEQ ID NO:
350) 2.20 5'-PO3-CGG TGC TGT 5'-PO3-AGC ACC GCT (SEQ ID NO: 351)
(SEQ ID NO: 352) 2.21 5'-PO3-CAC TGG CGT 5'-PO3-GCC AGT GCT (SEQ ID
NO: 353) (SEQ ID NO: 354) 2.22 5'-PO3-TCTCCTCGT 5'-PO3-GAGGAGACT
(SEQ ID NO: 355) (SEQ ID NO: 356) 2.23 5'-PO3-CCT GTC TGT
5'-PO3-AGA CAG GCT (SEQ ID NO: 357) (SEQ ID NO: 358) 2.24
5'-PO3-CAA CGC TGT 5'-PO3-AGC GTT GCT (SEQ ID NO: 359) (SEQ ID NO:
360) 2.25 5'-PO3-TGC CTC GGT 5'-PO3-CGA GGC ACT (SEQ ID NO: 361)
(SEQ ID NO: 362) 2.26 5'-PO3-ACA CTG CGT 5'-PO3-GCA GTG TCT (SEQ ID
NO: 363) (SEQ ID NO: 364) 2.27 5'-PO3-TCG TCC TGT 5'-PO3-AGG ACG
ACT (SEQ ID NO: 365) (SEQ ID NO: 366) 2.28 5'-PO3-GCT GCC AGT
5'-PO3-TGG CAG CCT (SEQ ID NO: 367) (SEQ ID NO: 368) 2.29
5'-PO3-TCA GGC TGT 5'-PO3-AGC CTG ACT (SEQ ID NO: 369) (SEQ ID NO:
370) 2.30 5'-PO3-GCC AGG TGT 5'-PO3-ACC TGG CCT (SEQ ID NO: 371)
(SEQ ID NO: 372) 2.31 5'-PO3-CGG ACC TGT 5'-PO3-AGG TCC GCT (SEQ ID
NO: 373) (SEQ ID NO: 374) 2.32 5'-PO3-CAA CGC AGT 5'-PO3-TGC GTT
GCT (SEQ ID NO: 375) (SEQ ID NO: 376) 2.33 5'-PO3-CAC ACG AGT
5'-PO3-TCG TGT GCT (SEQ ID NO: 377) (SEQ ID NO: 378) 2.34
5'-PO3-ATG GCC TGT 5'-PO3-AGG CCA TCT (SEQ ID NO: 379) (SEQ ID NO:
380) 2.35 5'-PO3-CCA GTC TGT 5'-PO3-AGA CTG GCT (SEQ ID NO: 381)
(SEQ ID NO: 382) 2.36 5'-PO3-GCC AGG AGT 5'-PO3-TCC TGG CCT (SEQ ID
NO: 383) (SEQ ID NO: 384) 2.37 5'-PO3-CGG ACC AGT 5'-PO3-TGG TCC
GCT (SEQ ID NO: 385) (SEQ ID NO: 386) 2.38 5'-PO3-CCT TCG CGT
5'-PO3-GCG AAG GCT (SEQ ID NO: 387) (SEQ ID NO: 388) 2.39
5'-PO3-GCA GCC AGT 5'-PO3-TGG CTG CCT (SEQ ID NO: 389) (SEQ ID NO:
390) 2.40 5'-PO3-CCA GTC GGT 5'-PO3-CGA CTG GCT (SEQ ID NO: 391)
(SEQ ID NO: 392) 2.41 5'-PO3-ACT GAG CGT 5'-PO3-GCT CAG TCT (SEQ ID
NO: 393) (SEQ ID NO: 394) 2.42 5'-PO3-CCA GTC CGT 5'-PO3-GGA CTG
GCT (SEQ ID NO: 395) (SEQ ID NO: 396) 2.43 5'-PO3-CCA GTC AGT
5'-PO3-TGA CTG GCT (SEQ ID NO: 397) (SEQ ID NO: 398) 2.44
5'-PO3-CAT CGA GGT 5'-PO3-CTC GAT GCT (SEQ ID NO: 399) (SEQ ID NO:
400) 2.45 5'-PO3-CCA TCG TGT 5'-PO3-ACG ATG GCT (SEQ ID NO: 401)
(SEQ ID NO: 402) 2.46 5'-PO3-GTG CTG CGT 5'-PO3-GCA GCA CCT (SEQ ID
NO: 403) (SEQ ID NO: 404) 2.47 5'-PO3-GAC TAC GGT 5'-PO3-CGT AGT
CCT (SEQ ID NO: 405) (SEQ ID NO: 406) 2.48 5'-PO3-GTG CTG AGT
5'-PO3-TCA GCA CCT (SEQ ID NO: 407) (SEQ ID NO: 408) 2.49
5'-PO3-GCTGCATGT 5'-PO3-ATGCAGCCT (SEQ ID NO: 409) (SEQ ID NO: 410)
2.50 5'-PO3-GAGTGGTGT 5'-PO3-ACCACTCCT (SEQ ID NO: 411) (SEQ ID NO:
412) 2.51 5'-PO3-GACTACCGT 5'-PO3-GGTAGTCCT (SEQ ID NO: 413) (SEQ
ID NO: 414) 2.52 5'-PO3-CGGTGATGT 5'-PO3-ATCACCGCT (SEQ ID NO: 415)
(SEQ ID NO: 416) 2.53 5'-PO3-TGCGACTGT 5'-PO3-AGTCGCACT (SEQ ID NO:
417) (SEQ ID NO: 418) 2.54 5'-PO3-TCTGGAGGT 5'-PO3-CTCCAGACT (SEQ
ID NO: 419) (SEQ ID NO: 420) 2.55 5'-PO3-AGCACTGGT 5'-PO3-CAGTGCTCT
(SEQ ID NO: 421) (SEQ ID NO: 422) 2.56 5'-PO3-TCGCTTGGT
5'-PO3-CAAGCGACT (SEQ ID NO: 423) (SEQ ID NO: 424) 2.57
5'-PO3-AGCACTCGT 5'-PO3-GAGTGCTCT (SEQ ID NO: 425) (SEQ ID NO: 426)
2.58 5'-PO3-GCGATTGGT 5'-PO3-CAATCGCCT (SEQ ID NO: 427) (SEQ ID NO:
428) 2.59 5'-PO3-CCATCGCGT 5'-PO3-GCGATGGCT (SEQ ID NO: 429) (SEQ
ID NO: 430) 2.60 5'-PO3-TCGCTTCGT 5'-PO3-GAAGCGACT (SEQ ID NO: 431)
(SEQ ID NO: 432) 2.61 5'-PO3-AGTGCCTGT 5'-PO3-AGGCACTCT (SEQ ID NO:
433) (SEQ ID NO: 434) 2.62 5'-PO3-GGCATAGGT 5'-PO3-CTATGCCCT (SEQ
ID NO: 435) (SEQ ID NO: 436) 2.63 5'-PO3-GCGATTCGT 5'-PO3-GAATCGCCT
(SEQ ID NO: 437) (SEQ ID NO: 438) 2.64 5'-PO3-TGCGACGGT
5'-PO3-CGTCGCACT (SEQ ID NO: 439) (SEQ ID NO: 440) 2.65
5'-PO3-GAGTGGCGT 5'-PO3-GCCACTCCT (SEQ ID NO: 441) (SEQ ID NO: 442)
2.66 5'-PO3-CGGTGAGGT 5'-PO3-CTCACCGCT (SEQ ID NO: 443) (SEQ ID NO:
444) 2.67 5'-PO3-GCTGCAAGT 5'-PO3-TTGCAGCCT (SEQ ID NO: 445) (SEQ
ID NO: 446) 2.68 5'-PO3-TTCCGCTGT 5'-PO3-AGCGGAACT (SEQ ID NO: 447)
(SEQ ID NO: 448) 2.69 5'-PO3-GAGTGGAGT 5'-PO3-TCCACTCCT (SEQ ID NO:
449) (SEQ ID NO: 450) 2.70 5'-PO3-ACAGAGCGT 5'-PO3-GCTCTGTCT (SEQ
ID NO: 451) (SEQ ID NO: 452) 2.71 5'-PO3-TGCGACCGT 5'-PO3-GGTCGCACT
(SEQ ID NO: 453) (SEQ ID NO: 454) 2.72 5'-PO3-CCTGTAGGT
5'-PO3-CTACAGGCT (SEQ ID NO: 455) (SEQ ID NO: 456) 2.73
5'-PO3-TAGCCGTGT 5'-PO3-ACGGCTACT (SEQ ID NO: 457) (SEQ ID NO: 458)
2.74 5'-PO3-TGCGACAGT 5'-PO3-TGTCGCACT (SEQ ID NO: 459) (SEQ ID NO:
460) 2.75 5'-PO3-GGTCTGTGT 5'-PO3-ACAGACCCT (SEQ ID NO: 461) (SEQ
ID NO: 462) 2.76 5'-PO3-CGGTGAAGT 5'-PO3-TTCACCGCT (SEQ ID NO: 463)
(SEQ ID NO: 464) 2.77 5'-PO3-CAACGAGGT 5'-PO3-CTCGTTGCT (SEQ ID NO:
465) (SEQ ID NO: 466) 2.78 5'-PO3-GCAGCATGT 5'-PO3-ATGCTGCCT (SEQ
ID NO: 467) (SEQ ID NO: 468) 2.79 5'-PO3-TCGTCAGGT 5'-PO3-CTGACGACT
(SEQ ID NO: 469) (SEQ ID NO: 470) 2.80 5'-PO3-AGTGCCAGT
5'-PO3-TGGCACTCT (SEQ ID NO: 471) (SEQ ID NO: 472) 2.81
5'-PO3-TAGAGGCGT 5'-PO3-GCCTCTACT (SEQ ID NO: 473) (SEQ ID NO: 474)
2.82 5'-PO3-GTCAGCGGT 5'-PO3-CGCTGACCT
(SEQ ID NO: 475) (SEQ ID NO: 476) 2.83 5'-PO3-TCAGGAGGT
5'-PO3-CTCCTGACT (SEQ ID NO: 477) (SEQ ID NO: 478) 2.84
5'-PO3-AGCAGGTGT 5'-PO3-ACCTGCTCT (SEQ ID NO: 479 (SEQ ID NO: 480)
2.85 5'-PO3-TTCCGCAGT 5'-PO3-TGCGGAACT (SEQ ID NO: 481) (SEQ ID NO:
482) 2.86 5'-PO3-GTCAGCCGT 5'-PO3-GGCTGACCT (SEQ ID NO: 483) (SEQ
ID NO: 484) 2.87 5'-PO3-GGTCTGCGT 5'-PO3-GCAGACCCT (SEQ ID NO: 485)
(SEQ ID NO: 486) 2.88 5'-PO3-TAGCCGAGT 5'-PO3-TCGGCTACT (SEQ ID NO:
487) (SEQ ID NO: 488) 2.89 5'-PO3-GTCAGCAGT 5'-PO3-TGCTGACCT (SEQ
ID NO: 489) (SEQ ID NO: 490) 2.90 5'-PO3-GGTCTGAGT 5'-PO3-TCAGACCCT
(SEQ ID NO: 491) (SEQ ID NO: 492) 2.91 5'-PO3-CGGACAGGT
5'-PO3-CTGTCCGCT (SEQ ID NO: 493) (SEQ ID NO: 494) 2.92
5'-PO3-TTAGCCGGT5'- 5'-PO3-CGGCTAACT5'-PO3- PO3-3' 3' (SEQ ID NO:
495) (SEQ ID NO: 496) 2.93 5'-PO3-GAGACGAGT 5'-PO3-TCGTCTCCT (SEQ
ID NO: 497) (SEQ ID NO: 498) 2.94 5'-PO3-CGTAACCGT 5'-PO3-GGTTACGCT
(SEQ ID NO: 499) (SEQ ID NO: 500) 2.95 5'-PO3-TTGGCGTGT5'-
5'-PO3-ACGCCAACT5'-PO3- PO3-3' 3' (SEQ ID NO: 501) (SEQ ID NO: 502)
2.96 5'-PO3-ATGGCAGGT 5'-PO3-CTGCCATCT (SEQ ID NO: 503) (SEQ ID NO:
504)
TABLE-US-00010 TABLE 5 Oligonucleotide tags used in cycle 3 Tag
Bottom strand number Top strand sequence sequence 3.1 5'-PO3-CAG
CTA CGA 5'-PO3-GTA GCT GAC (SEQ ID NO: 505) (SEQ ID NO: 506) 3.2
5'-PO3-CTC CTG CGA 5'-PO3-GCA GGA GAC (SEQ ID NO: 507) (SEQ ID NO:
508) 3.3 5'-PO3-GCT GCC TGA 5'-PO3-AGG CAG CAC (SEQ ID NO: 509)
(SEQ ID NO: 510) 3.4 5'-PO3-CAG GAA CGA 5'-PO3-GTT CCT GAC (SEQ ID
NO: 511) (SEQ ID NO: 512) 3.5 5'-PO3-CAC ACG CGA 5'-PO3-GCG TGT GAC
(SEQ ID NO: 513) (SEQ ID NO: 514) 3.6 5'-PO3-GCA GCC TGA 5'-PO3-AGG
CTG CAC (SEQ ID NO: 515) (SEQ ID NO: 516) 3.7 5'-PO3-CTG AAC GGA
5'-PO3-CGT TCA GAC (SEQ ID NO: 517) (SEQ ID NO: 518) 3.8 5'-PO3-CTG
AAC CGA 5'-PO3-GGT TCA GAC (SEQ ID NO: 519) (SEQ ID NO: 520) 3.9
5'-PO3-TCT GGA CGA 5'-PO3-GTC CAG AAC (SEQ ID NO: 521) (SEQ ID NO:
522) 3.10 5'-PO3-TGC CTA CGA 5'-PO3-GTA GGC AAC (SEQ ID NO: 523)
(SEQ ID NO: 524) 3.11 5'-PO3-GGC ATA CGA 5'-PO3-GTA TGC CAC (SEQ ID
NO: 525) (SEQ ID NO: 526) 3.12 5'-PO3-CGG TGA CGA 5'-PO3-GTC ACC
GAC (SEQ ID NO: 527) (SEQ ID NO: 528) 3.13 5'-PO3-CAA CGA CGA
5'-PO3-GTC GTT GAC (SEQ ID NO: 529) (SEQ ID NO: 530) 3.14
5'-PO3-CTC CTC TGA 5'-PO3-AGA GGA GAC (SEQ ID NO: 531) (SEQ ID NO:
532) 3.15 5'-PO3-TCA GGA CGA 5'-PO3-GTC CTG AAC (SEQ ID NO: 533)
(SEQ ID NO: 534) 3.16 5'-PO3-AAA GGC GGA 5'-PO3-CGC CTT TAC (SEQ ID
NO: 535) (SEQ ID NO: 536) 3.17 5'-PO3-CTC CTC GGA 5'-PO3-CGA GGA
GAC (SEQ ID NO: 537) (SEQ ID NO: 538) 3.18 5'-PO3-CAG ATG CGA
5'-PO3-GCA TCT GAC (SEQ ID NO: 539) (SEQ ID NO: 540) 3.19
5'-PO3-GCA GCA AGA 5'-PO3-TTG CTG CAC (SEQ ID NO: 541) (SEQ ID NO:
542) 3.20 5'-PO3-GTG GAG TGA 5'-PO3-ACT CCA CAC (SEQ ID NO: 543)
(SEQ ID NO: 544) 3.21 5'-PO3-CCA GTA GGA 5'-PO3-CTA CTG GAC (SEQ ID
NO: 545) (SEQ ID NO: 546) 3.22 5'-PO3-ATG GCA CGA 5'-PO3-GTG CCA
TAC (SEQ ID NO: 547) (SEQ ID NO: 548) 3.23 5'-PO3-GGA CTG TGA
5'-PO3-ACA GTC CAC (SEQ ID NO: 549) (SEQ ID NO: 550) 3.24
5'-PO3-CCG AAC TGA 5'-PO3-AGT TCG GAC (SEQ ID NO: 551) (SEQ ID NO:
552) 3.25 5'-PO3-CTC CTC AGA 5'-PO3-TGA GGA GAC (SEQ ID NO: 553)
(SEQ ID NO: 554) 3.26 5'-PO3-CAC TGC TGA 5'-PO3-AGC AGT GAC (SEQ ID
NO: 555) (SEQ ID NO: 556) 3.27 5'-PO3-AGC AGG CGA 5'-PO3-GCC TGC
TAC (SEQ ID NO: 557) (SEQ ID NO: 558) 3.28 5'-PO3-AGC AGG AGA
5'-PO3-TCC TGC TAC (SEQ ID NO: 559) (SEQ ID NO: 560) 3.29
5'-PO3-AGA GCC AGA 5'-PO3-TGG CTC TAC (SEQ ID NO: 561) (SEQ ID NO:
562) 3.30 5'-PO3-GTC GTT GGA 5'-PO3-CAA CGA CAC (SEQ ID NO: 563)
(SEQ ID NO: 564) 3.31 5'-PO3-CCG AAC GGA 5'-PO3-CGT TCG GAC (SEQ ID
NO: 565) (SEQ ID NO: 566) 3.32 5'-PO3-CAC TGC GGA 5'-PO3-CGC AGT
GAC (SEQ ID NO: 567) (SEQ ID NO: 568) 3.33 5'-PO3-GTG GAG CGA
5'-PO3-GCT CCA CAC (SEQ ID NO: 569) (SEQ ID NO: 570) 3.34
5'-PO3-GTG GAG AGA 5'-PO3-TCT CCA CAC (SEQ ID NO: 571) (SEQ ID NO:
572) 3.35 5'-PO3-GGA CTG CGA 5'-PO3-GCA GTC CAC (SEQ ID NO: 573)
(SEQ ID NO: 574) 3.36 5'-PO3-CCG AAC CGA 5'-PO3-GGT TCG GAC (SEQ ID
NO: 575) (SEQ ID NO: 576) 3.37 5'-PO3-CAC TGC CGA 5'-PO3-GGC AGT
GAC (SEQ ID NO: 577) (SEQ ID NO: 578) 3.38 5'-PO3-CGA AAC GGA
5'-PO3-CGT TTC GAC (SEQ ID NO: 579) (SEQ ID NO: 580) 3.39
5'-PO3-GGA CTG AGA 5'-PO3-TCA GTC CAC (SEQ ID NO: 581) (SEQ ID NO:
582) 3.40 5'-PO3-CCG AAC AGA 5'-PO3-TGT TCG GAC (SEQ ID NO: 583)
(SEQ ID NO: 584) 3.41 5'-PO3-CGA AAC CGA 5'-PO3-GGT TTC GAC (SEQ ID
NO: 585) (SEQ ID NO: 586) 3.42 5'-PO3-CTG GCT TGA 5'-PO3-AAG CCA
GAC (SEQ ID NO: 587) (SEQ ID NO: 588) 3.43 5'-PO3-CAC ACC TGA
5'-PO3-AGG TGT GAC (SEQ ID NO: 589) (SEQ ID NO: 590) 3.44
5'-PO3-AAC GAC CGA 5'-PO3-GGT CGT TAC (SEQ ID NO: 591) (SEQ ID NO:
592) 3.45 5'-PO3-ATC CAG CGA 5'-PO3-GCT GGA TAC (SEQ ID NO: 593)
(SEQ ID NO: 594) 3.46 5'-PO3-TGC GAA GGA 5'-PO3-CTT CGC AAC (SEQ ID
NO: 595) (SEQ ID NO: 596) 3.47 5'-PO3-TGC GAA CGA 5'-PO3-GTT CGC
AAC (SEQ ID NO: 597) (SEQ ID NO: 598) 3.48 5'-PO3-CTG GCT GGA
5'-PO3-CAG CCA GAC (SEQ ID NO: 599) (SEQ ID NO: 600) 3.49
5'-PO3-CAC ACC GGA 5'-PO3-CGG TGT GAC (SEQ ID NO: 601) (SEQ ID NO:
602) 3.50 5'-PO3-AGT GCA GGA 5'-PO3-CTG CAC TAC (SEQ ID NO: 603)
(SEQ ID NO: 604) 3.51 5'-PO3-GAC CGT TGA 5'-PO3-AAC GGT CAC (SEQ ID
NO: 605) (SEQ ID NO: 606) 3.52 5'-PO3-GGT GAG TGA 5'-PO3-ACT CAC
CAC (SEQ ID NO: 607) (SEQ ID NO: 608) 3.53 5'-PO3-CCT TCC TGA
5'-PO3-AGG AAG GAC (SEQ ID NO: 609) (SEQ ID NO: 610) 3.54
5'-PO3-CTG GCT AGA 5'-PO3-TAG CCA GAC (SEQ ID NO: 611) (SEQ ID NO:
612) 3.55 5'-PO3-CAC ACC AGA 5'-PO3-TGG TGT GAC (SEQ ID NO: 613)
(SEQ ID NO: 614) 3.56 5'-PO3-AGC GGT AGA 5'-PO3-TAC CGC TAC (SEQ ID
NO: 615) (SEQ ID NO: 616) 3.57 5'-PO3-GTC AGA GGA 5'-PO3-CTC TGA
CAC (SEQ ID NO: 617) (SEQ ID NO: 618) 3.58 5'-PO3-TTC CGA CGA
5'-PO3-GTC GGA AAC (SEQ ID NO: 619) (SEQ ID NO: 620) 3.59
5'-PO3-AGG CGT AGA 5'-PO3-TAC GCC TAC (SEQ ID NO: 621) (SEQ ID NO:
622) 3.60 5'-PO3-CTC GAC TGA 5'-PO3-AGT CGA GAC (SEQ ID NO: 623)
(SEQ ID NO: 624) 3.61 5'-PO3-TAC GCT GGA 5'-PO3-CAG CGT AAC (SEQ ID
NO: 625) (SEQ ID NO: 626) 3.62 5'-PO3-GTT CGG TGA 5'-PO3-ACC GAA
CAC (SEQ ID NO: 627) (SEQ ID NO: 628) 3.63 5'-PO3-GCC AGC AGA
5'-PO3-TGC TGG CAC (SEQ ID NO: 629) (SEQ ID NO: 630) 3.64
5'-PO3-GAC CGT AGA 5'-PO3-TAC GGT CAC (SEQ ID NO: 631) (SEQ ID NO:
632) 3.65 5'-PO3-GTG CTC TGA 5'-PO3-AGA GCA CAC (SEQ ID NO: 633)
(SEQ ID NO: 634) 3.66 5'-PO3-GGT GAG CGA 5'-PO3-GCT CAC CAC (SEQ ID
NO: 635) (SEQ ID NO: 636) 3.67 5'-PO3-GGT GAG AGA 5'-PO3-TCT CAC
CAC (SEQ ID NO: 637) (SEQ ID NO: 638) 3.68 5'-PO3-CCT TCC AGA
5'-PO3-TGG AAG GAC (SEQ ID NO: 639) (SEQ ID NO: 640) 3.69
5'-PO3-CTC CTA CGA 5'-PO3-GTA GGA GAC (SEQ ID NO: 641) (SEQ ID NO:
642) 3.70 5'-PO3-CTC GAC GGA 5'-PO3-CGT CGA GAC (SEQ ID NO: 643)
(SEQ ID NO: 644) 3.71 5'-PO3-GCC GTT TGA 5'-PO3-AAA CGG CAC (SEQ ID
NO: 645) (SEQ ID NO: 646) 3.72 5'-PO3-GCG GAG TGA 5'-PO3-ACT CCG
CAC (SEQ ID NO: 647) (SEQ ID NO: 648) 3.73 5'-PO3-CGT GCT TGA
5'-PO3-AAG CAC GAC (SEQ ID NO: 649) (SEQ ID NO: 650) 3.74
5'-PO3-CTC GAC CGA 5'-PO3-GGT CGA GAC (SEQ ID NO: 651) (SEQ ID NO:
652) 3.75 5'-PO3-AGA GCA GGA 5'-PO3-CTG CTC TAC (SEQ ID NO: 653)
(SEQ ID NO: 654) 3.76 5'-PO3-GTG CTC GGA 5'-PO3-CGA GCA CAC (SEQ ID
NO: 655) (SEQ ID NO: 656) 3.77 5'-PO3-CTC GAC AGA 5'-PO3-TGT CGA
GAC (SEQ ID NO: 657) (SEQ ID NO: 658) 3.78 5'-PO3-GGA GAG TGA
5'-PO3-ACT CTC CAC (SEQ ID NO: 659) (SEQ ID NO: 660) 3.79
5'-PO3-AGG CTG TGA 5'-PO3-ACA GCC TAC (SEQ ID NO: 661) (SEQ ID NO:
662) 3.80 5'-PO3-AGA GCA CGA 5'-PO3-GTG CTC TAC (SEQ ID NO: 663)
(SEQ ID NO: 664) 3.81 5'-PO3-CCA TCC TGA 5'-PO3-AGG ATG GAC (SEQ ID
NO: 665) (SEQ ID NO: 666) 3.82 5'-PO3-GTT CGG AGA 5'-PO3-TCC GAA
CAC
(SEQ ID NO: 667) (SEQ ID NO: 668) 3.83 5'-PO3-TGG TAG CGA
5'-PO3-GCT ACC AAC (SEQ ID NO: 669) (SEQ ID NO: 670) 3.84
5'-PO3-GTG CTC CGA 5'-PO3-GGA GCA CAC (SEQ ID NO: 671) (SEQ ID NO:
672) 3.85 5'-PO3-GTG CTC AGA 5'-PO3-TGA GCA CAC (SEQ ID NO: 673)
(SEQ ID NO: 674) 3.86 5'-PO3-GCC GTT GGA 5'-PO3-CAA CGG CAC (SEQ ID
NO: 675) (SEQ ID NO: 676) 3.87 5'-PO3-GAG TGC TGA 5'-PO3-AGC ACT
CAC (SEQ ID NO: 677) (SEQ ID NO: 678) 3.88 5'-PO3-GCT CCT TGA
5'-PO3-AAG GAG CAC (SEQ ID NO: 679) (SEQ ID NO: 680) 3.89
5'-PO3-CCG AAA GGA 5'-PO3-CTT TCG GAC (SEQ ID NO: 681) (SEQ ID NO:
682) 3.90 5'-PO3-CAC TGA GGA 5'-PO3-CTC AGT GAC (SEQ ID NO: 683)
(SEQ ID NO: 684) 3.91 5'-PO3-CGT GCT GGA 5'-PO3-CAG CAC GAC (SEQ ID
NO: 685) (SEQ ID NO: 686) 3.92 5'-PO3-CCG AAA CGA 5'-PO3-GTT TCG
GAC (SEQ ID NO: 687) (SEQ ID NO: 688) 3.93 5'-PO3-GCG GAG AGA
5'-PO3-TCT CCG CAC (SEQ ID NO: 689) (SEQ ID NO: 690) 3.94
5'-PO3-GCC GTT AGA 5'-PO3-TAA CGG CAC (SEQ ID NO: 691) (SEQ ID NO:
692) 3.95 5'-PO3-TCT CGT GGA 5'-PO3-CAC GAG AAC (SEQ ID NO: 693)
(SEQ ID NO: 694) 3.96 5'-PO3-CGT GCT AGA 5'-PO3-TAG CAC GAC (SEQ ID
NO: 695) (SEQ ID NO: 696)
TABLE-US-00011 TABLE 6 Oligonucleotide tags used in cycle 4 Tag
Bottom strand number Top strand sequence sequence 4.1
5'-PO3-GCCTGTCTT 5'-PO3-GAC AGG CTC (SEQ ID NO: 697) (SEQ ID NO:
698) 4.2 5'-PO3-CTCCTGGTT 5'-PO3-CCA GGA GTC (SEQ ID NO: 699) (SEQ
ID NO: 700) 4.3 5'-PO3-ACTCTGCTT 5'-PO3-GCA GAG TTC (SEQ ID NO:
701) (SEQ ID NO: 702) 4.4 5'-PO3-CATCGCCTT 5'-PO3-GGC GAT GTC (SEQ
ID NO: 703) (SEQ ID NO: 704) 4.5 5'-PO3-GCCACTATT 5'-PO3-TAG TGG
CTC (SEQ ID NO: 705) (SEQ ID NO: 706) 4.6 5'-PO3-CACACGGTT
5'-PO3-CCG TGT GTC (SEQ ID NO: 707) (SEQ ID NO: 708) 4.7
5'-PO3-CAACGCCTT 5'-PO3-GGC GTT GTC (SEQ ID NO: 709) (SEQ ID NO:
710) 4.8 5'-PO3-ACTGAGGTT 5'-PO3-CCT CAG TTC (SEQ ID NO: 711) (SEQ
ID NO: 712) 4.9 5'-PO3-GTGCTGGTT 5'-PO3-CCA GCA CTC (SEQ ID NO:
713) (SEQ ID NO: 714) 4.10 5'-PO3-CATCGACTT 5'-PO3-GTC GAT GTC (SEQ
ID NO: 715) (SEQ ID NO: 716) 4.11 5'-PO3-CCATCGGTT 5'-PO3-CCG ATG
GTC (SEQ ID NO: 717) (SEQ ID NO: 718) 4.12 5'-PO3-GCTGCACTT
5'-PO3-GTG CAG CTC (SEQ ID NO: 719) (SEQ ID NO: 720) 4.13
5'-PO3-ACAGAGGTT 5'-PO3-CCT CTG TTC (SEQ ID NO: 721) (SEQ ID NO:
722) 4.14 5'-PO3-AGTGCCGTT 5'-PO3-CGG CAC TTC (SEQ ID NO: 723) (SEQ
ID NO: 724) 4.15 5'-PO3-CGGACATTT 5'-PO3-ATG TCC GTC (SEQ ID NO:
725) (SEQ ID NO: 726) 4.16 5'-PO3-GGTCTGGTT 5'-PO3-CCA GAC CTC (SEQ
ID NO: 727) (SEQ ID NO: 728) 4.17 5'-PO3-GAGACGGTT 5'-PO3-CCG TCT
CTC (SEQ ID NO: 729) (SEQ ID NO: 730) 4.18 5'-PO3-CTTTCCGTT
5'-PO3-CGG AAA GTC (SEQ ID NO: 731) (SEQ ID NO: 732) 4.19
5'-PO3-CAGATGGTT 5'-PO3-CCA TCT GTC (SEQ ID NO: 733) (SEQ ID NO:
734) 4.20 5'-PO3-CGGACACTT 5'-PO3-GTG TCC GTC (SEQ ID NO: 735) (SEQ
ID NO: 736) 4.21 5'-PO3-ACTCTCGTT 5'-PO3-CGA GAG TTC (SEQ ID NO:
737) (SEQ ID NO: 738) 4.22 5'-PO3-GCAGCACTT 5'-PO3-GTG CTG CTC (SEQ
ID NO: 739) (SEQ ID NO: 740) 4.23 5'-PO3-ACTCTCCTT 5'-PO3-GGA GAG
TTC (SEQ ID NO: 741) (SEQ ID NO: 742) 4.24 5'-PO3-ACCTTGGTT
5'-PO3-CCA AGG TTC (SEQ ID NO: 743) (SEQ ID NO: 744) 4.25
5'-PO3-AGAGCCGTT 5'-PO3-CGG CTC TTC (SEQ ID NO: 745) (SEQ ID NO:
746) 4.26 5'-PO3-ACCTTGCTT 5'-PO3-GCA AGG TTC (SEQ ID NO: 747) (SEQ
ID NO: 748) 4.27 5'-PO3-AAGTCCGTT 5'-PO3-CGG ACT TTC (SEQ ID NO:
749) (SEQ ID NO: 750) 4.28 5'-PO3-GGA CTG GTT 5'-PO3-CCA GTC CTC
(SEQ ID NO: 751) (SEQ ID NO: 752) 4.29 5'-PO3-GTCGTTCTT 5'-PO3-GAA
CGA CTC (SEQ ID NO: 753) (SEQ ID NO: 754) 4.30 5'-PO3-CAGCATCTT
5'-PO3-GAT GCT GTC (SEQ ID NO: 755) (SEQ ID NO: 756) 4.31
5'-PO3-CTATCCGTT 5'-PO3-CGG ATA GTC (SEQ ID NO: 757) (SEQ ID NO:
758) 4.32 5'-PO3-ACACTCGTT 5'-PO3-CGA GTG TTC (SEQ ID NO: 759) (SEQ
ID NO: 760) 4.33 5'-PO3-ATCCAGGTT 5'-PO3-CCT GGA TTC (SEQ ID NO:
761) (SEQ ID NO: 762) 4.34 5'-PO3-GTTCCTGTT 5'-PO3-CAG GAA CTC (SEQ
ID NO: 763) (SEQ ID NO: 764) 4.35 5'-PO3-ACACTCCTT 5'-PO3-GGA GTG
TTC (SEQ ID NO: 765) (SEQ ID NO: 766) 4.36 5'-PO3-GTTCCTCTT
5'-PO3-GAG GAA CTC (SEQ ID NO: 767) (SEQ ID NO: 768) 4.37
5'-PO3-CTGGCTCTT 5'-PO3-GAG CCA GTC (SEQ ID NO: 769) (SEQ ID NO:
770) 4.38 5'-PO3-ACGGCATTT 5'-PO3-ATG CCG TTC (SEQ ID NO: 771) (SEQ
ID NO: 772) 4.39 5'-PO3-GGTGAGGTT 5'-PO3-CCT CAC CTC (SEQ ID NO:
773) (SEQ ID NO: 774) 4.40 5'-PO3-CCTTCCGTT 5'-PO3-CGG AAG GTC (SEQ
ID NO: 775) (SEQ ID NO: 776) 4.41 5'-PO3 -TACGCTCTT 5'-PO3 -GAG CGT
ATC (SEQ ID NO: 777) (SEQ ID NO: 778) 4.42 5'-PO3-ACGGCAGTT
5'-PO3-CTG CCG TTC (SEQ ID NO: 779) (SEQ ID NO: 780 4.43
5'-PO3-ACTGACGTT 5'-PO3-CGT CAG TTC (SEQ ID NO: 781) (SEQ ID NO:
782) 4.44 5'-PO3-ACGGCACTT 5'-PO3-GTG CCG TTC (SEQ ID NO: 783) (SEQ
ID NO: 784) 4.45 5'-PO3-ACTGACCTT 5'-PO3-GGT CAG TTC (SEQ ID NO:
785) (SEQ ID NO: 786) 4.46 5'-PO3-TTTGCGGTT 5'-PO3-CCG CAA ATC (SEQ
ID NO: 787) (SEQ ID NO: 788) 4.47 5'-PO3-TGGTAGGTT 5'-PO3-CCT ACC
ATC (SEQ ID NO: 789) (SEQ ID NO: 790) 4.48 5'-PO3-GTTCGGCTT
5'-PO3-GCC GAA CTC (SEQ ID NO: 791) (SEQ ID NO: 792) 4.49
5'-PO3-GCC GTT CTT 5'-PO3-GAA CGG CTC (SEQ ID NO: 793) (SEQ ID NO:
794) 4.50 5'-PO3-GGAGAGGTT 5'-PO3-CCT CTC CTC (SEQ ID NO: 795) (SEQ
ID NO: 796) 4.51 5'-PO3-CACTGACTT 5'-PO3-GTC AGT GTC (SEQ ID NO:
797) (SEQ ID NO: 798) 4.52 5'-PO3-CGTGCTCTT 5'-PO3-GAG CAC GTC (SEQ
ID NO: 799) (SEQ ID NO: 800) 4.53 5'-PO3-AATCCGCTT 5'-PO3
-GCGGATTTC (SEQ ID NO: 801) (SEQ ID NO: 802) 4.54 5'-PO3-AGGCTGGTT
5'-PO3-CCA GCC TTC (SEQ ID NO: 803) (SEQ ID NO: 804) 4.55
5'-PO3-GCTAGTGTT 5'-PO3-CAC TAG CTC (SEQ ID NO: 805) (SEQ ID NO:
806) 4.56 5'-PO3-GGAGAGCTT 5'-PO3-GCT CTC CTC (SEQ ID NO: 807) (SEQ
ID NO: 808) 4.57 5'-PO3-GGAGAGATT 5'-PO3-TCT CTC CTC (SEQ ID NO:
809) (SEQ ID NO: 810) 4.58 5'-PO3-AGGCTGCTT 5'-PO3-GCA GCC TTC (SEQ
ID NO: 811) (SEQ ID NO: 812) 4.59 5'-PO3-GAGTGCGTT 5'-PO3-CGC ACT
CTC (SEQ ID NO: 813) (SEQ ID NO: 814) 4.60 5'-PO3-CCATCCATT
5'-PO3-TGG ATG GTC (SEQ ID NO: 815) (SEQ ID NO: 816) 4.61
5'-PO3-GCTAGTCTT 5'-PO3-GAC TAG CTC (SEQ ID NO: 817) (SEQ ID NO:
818) 4.62 5'-PO3-AGGCTGATT 5'-PO3-TCA GCC TTC (SEQ ID NO: 819) (SEQ
ID NO: 820) 4.63 5'-PO3-ACAGACGTT 5'-PO3-CGT CTG TTC (SEQ ID NO:
821) (SEQ ID NO: 822) 4.64 5'-PO3-GAGTGCCTT 5'-PO3-GGC ACT CTC (SEQ
ID NO: 823) (SEQ ID NO: 824) 4.65 5'-PO3-ACAGACCTT 5'-PO3-GGT CTG
TTC (SEQ ID NO: 825) (SEQ ID NO: 826) 4.66 5'-PO3-CGAGCTTTT
5'-PO3-AAG CTC GTC (SEQ ID NO: 827) (SEQ ID NO: 828) 4.67
5'-PO3-TTAGCGGTT 5'-PO3-CCG CTA ATC (SEQ ID NO: 829) (SEQ ID NO:
830) 4.68 5'-PO3-CCTCTTGTT 5'-PO3-CAA GAG GTC (SEQ ID NO: 831) (SEQ
ID NO: 832) 4.69 5'-PO3-GGTCTCTTT 5'-PO3-AGA GAC CTC (SEQ ID NO:
833) (SEQ ID NO: 834) 4.70 5'-PO3-GCCAGATTT 5'-PO3-ATC TGG CTC (SEQ
ID NO: 835) (SEQ ID NO: 836) 4.71 5'-PO3-GAGACCTTT 5'-PO3-AGG TCT
CTC (SEQ ID NO: 837) (SEQ ID NO: 838) 4.72 5'-PO3-CACACAGTT
5'-PO3-CTG TGT GTC (SEQ ID NO: 839) (SEQ ID NO: 840) 4.73
5'-PO3-CCTCTTCTT 5'-PO3-GAA GAG GTC (SEQ ID NO: 841) (SEQ ID NO:
842) 4.74 5'-PO3-TAGAGCGTT 5'-PO3-CGC TCT ATC (SEQ ID NO: 843) (SEQ
ID NO: 844) 4.75 5'-PO3-GCACCTTTT 5'-PO3-AAG GTG CTC (SEQ ID NO:
845) (SEQ ID NO: 846) 4.76 5'-PO3-GGCTTGTTT 5'-PO3-ACA AGC CTC (SEQ
ID NO: 847) (SEQ ID NO: 848) 4.77 5'-PO3-GACGCGATT 5'-PO3-TCG CGT
CTC (SEQ ID NO: 849) (SEQ ID NO: 850) 4.78 5'-PO3-CGAGCTGTT
5'-PO3-CAG CTC GTC (SEQ ID NO: 851) (SEQ ID NO: 852) 4.79
5'-PO3-TAGAGCCTT 5'-PO3-GGC TCT ATC (SEQ ID NO: 853) (SEQ ID NO:
854) 4.80 5'-PO3-CATCCGTTT 5'-PO3-ACG GAT GTC (SEQ ID NO: 855) (SEQ
ID NO: 856) 4.81 5'-PO3-GGTCTCGTT 5'-PO3-CGA GAC CTC (SEQ ID NO:
857) (SEQ ID NO: 858) 4.82 5'-PO3-GCCAGAGTT 5'-PO3-CTC TGG CTC
(SEQ ID NO: 859) (SEQ ID NO: 860) 4.83 5'-PO3-GAGACCGTT 5'-PO3-CGG
TCT CTC (SEQ ID NO: 861) (SEQ ID NO: 862) 4.84 5'-PO3-CGAGCTATT
5'-PO3-TAG CTC GTC (SEQ ID NO: 863) (SEQ ID NO: 864) 4.85
5'-PO3-GCAAGTGTT 5'-PO3-CAC TTG CTC (SEQ ID NO: 865) (SEQ ID NO:
866) 4.86 5'-PO3-GGTCTCCTT 5'-PO3-GGA GAC CTC (SEQ ID NO: 867) (SEQ
ID NO: 868) 4.87 5'-PO3-GCCAGACTT 5'-PO3-GTC TGG CTC (SEQ ID NO:
869) (SEQ ID NO: 870) 4.88 5'-PO3-GGTCTCATT 5'-PO3-TGA GAC CTC (SEQ
ID NO: 871) (SEQ ID NO: 872) 4.89 5'-PO3-GAGACCATT 5'-PO3-TGG TCT
CTC (SEQ ID NO: 873) (SEQ ID NO: 874) 4.90 5'-PO3-CCTTCAGTT
5'-PO3-CTG AAG GTC (SEQ ID NO: 875) (SEQ ID NO: 876) 4.91
5'-PO3-GCACCTGTT 5'-PO3-CAG GTG CTC (SEQ ID NO: 877) (SEQ ID NO:
878) 4.92 5'-PO3-AAAGGCGTT 5'-PO3-CGC CTT TTC (SEQ ID NO: 879) (SEQ
ID NO: 880) 4.93 5'-PO3-CAGATCGTT 5'-PO3-CGA TCT GTC (SEQ ID NO:
881) (SEQ ID NO: 882) 4.94 5'-PO3-CATAGGCTT 5'-PO3-GCC TAT GTC (SEQ
ID NO: 883) (SEQ ID NO: 884) 4.95 5'-PO3-CCTTCACTT 5'-PO3-GTG AAG
GTC (SEQ ID NO: 885) (SEQ ID NO: 886) 4.96 5'-PO3-GCACCTCTT
5'-PO3-GAG GTG CTC (SEQ ID NO: 887) (SEQ ID NO: 888)
TABLE-US-00012 TABLE 7 Correspondence between building blocks and
oligonucleotide tags for Cycles 1-4. Building block Cycle 1 Cycle 2
Cycle 3 Cycle 4 BB1 1.1 2.1 3.1 4.1 BB2 1.2 2.2 3.2 4.2 BB3 1.3 2.3
3.3 4.3 BB4 1.4 2.4 3.4 4.4 BB5 1.5 2.5 3.5 4.5 BB6 1.6 2.6 3.6 4.6
BB7 1.7 2.7 3.7 4.7 BB8 1.8 2.8 3.8 4.8 BB9 1.9 2.9 3.9 4.9 BB10
1.10 2.10 3.10 4.10 BB11 1.11 2.11 3.11 4.11 BB12 1.12 2.12 3.12
4.12 BB13 1.13 2.13 3.13 4.13 BB14 1.14 2.14 3.14 4.14 BB15 1.15
2.15 3.15 4.15 BB16 1.16 2.16 3.16 4.16 BB17 1.17 2.17 3.17 4.17
BB18 1.18 2.18 3.18 4.18 BB19 1.19 2.19 3.19 4.19 BB20 1.20 2.20
3.20 4.20 BB21 1.21 2.21 3.21 4.21 BB22 1.22 2.22 3.22 4.22 BB23
1.23 2.23 3.23 4.23 BB24 1.24 2.24 3.24 4.24 BB25 1.25 2.25 3.25
4.25 BB26 1.26 2.26 3.26 4.26 BB27 1.27 2.27 3.27 4.27 BB28 1.28
2.28 3.28 4.28 BB29 1.29 2.29 3.29 4.29 BB30 1.30 2.30 3.30 4.30
BB31 1.31 2.31 3.31 4.31 BB32 1.32 2.32 3.32 4.32 BB33 1.33 2.33
3.33 4.33 BB34 1.34 2.34 3.34 4.34 BB35 1.35 2.35 3.35 4.35 BB36
1.36 2.36 3.36 4.36 BB37 1.37 2.37 3.37 4.37 BB38 1.38 2.38 3.38
4.38 BB39 1.39 2.39 3.39 4.39 BB40 1.44 2.44 3.44 4.44 BB41 1.41
2.41 3.41 4.41 BB42 1.42 2.42 3.42 4.42 BB43 1.43 2.43 3.43 4.43
BB44 1.40 2.40 3.40 4.40 BB45 1.45 2.45 3.45 4.45 BB46 1.46 2.46
3.46 4.46 BB47 1.47 2.47 3.47 4.47 BB48 1.48 2.48 3.48 4.48 BB49
1.49 2.49 3.49 4.49 BB50 1.50 2.50 3.50 4.50 BB51 1.51 2.51 3.51
4.51 BB52 1.52 2.52 3.52 4.52 BB53 1.53 2.53 3.53 4.53 BB54 1.54
2.54 3.54 4.54 BB55 1.55 2.55 3.55 4.55 BB56 1.56 2.56 3.56 4.56
BB57 1.57 2.57 3.57 4.57 BB58 1.58 2.58 3.58 4.58 BB59 1.59 2.59
3.59 4.59 BB60 1.60 2.60 3.60 4.60 BB61 1.61 2.61 3.61 4.61 BB62
1.62 2.62 3.62 4.62 BB63 1.63 2.63 3.63 4.63 BB64 1.64 2.64 3.64
4.64 BB65 1.65 2.65 3.65 4.65 BB66 1.66 2.66 3.66 4.66 BB67 1.67
2.67 3.67 4.67 BB68 1.68 2.68 3.68 4.68 BB69 1.69 2.69 3.69 4.69
BB70 1.70 2.70 3.70 4.70 BB71 1.71 2.71 3.71 4.71 BB72 1.72 2.72
3.72 4.72 BB73 1.73 2.73 3.73 4.73 BB74 1.74 2.74 3.74 4.74 BB75
1.75 2.75 3.75 4.75 BB76 1.76 2.76 3.76 4.76 BB77 1.77 2.77 3.77
4.77 BB78 1.78 2.78 3.78 4.78 BB79 1.79 2.79 3.79 4.79 BB80 1.80
2.80 3.80 4.80 BB81 1.81 2.81 3.81 4.81 BB82 1.82 2.82 3.82 4.82
BB83 1.96 2.96 3.96 4.96 BB84 1.83 2.83 3.83 4.83 BB85 1.84 2.84
3.84 4.84 BB86 1.85 2.85 3.85 4.85 BB87 1.86 2.86 3.86 4.86 BB88
1.87 2.87 3.87 4.87 BB89 1.88 2.88 3.88 4.88 BB90 1.89 2.89 3.89
4.89 BB91 1.90 2.90 3.90 4.90 BB92 1.91 2.91 3.91 4.91 BB93 1.92
2.92 3.92 4.92 BB94 1.93 2.93 3.93 4.93 BB95 1.94 2.94 3.94 4.94
BB96 1.95 2.95 3.95 4.95
1.times. ligase buffer: 50 mM Tris, pH 7.5; 10 mM dithiothreitol;
10 mM MgCl.sub.2; 2 mM ATP; 50 mM NaCl. 10.times. ligase buffer:
500 mM Tris, pH 7.5; 100 mM dithiothreitol; 100 mM MgCl.sub.2; 20
mM ATP; 500 mM NaCl
Attachment of Water Soluble Spacer to Compound 2
[0225] To a solution of Compound 2 (60 mL, 1 mM) in sodium borate
buffer (150 mM, pH 9.4) that was chilled to 4.degree. C. was added
40 equivalents of N-Fmoc-15-amino-4,7,10,13-tetraoxaoctadecanoic
acid (S-Ado) in N,N-dimethylformamide (DMF) (16 mL, 0.15 M)
followed by 40 equivalents of
4-(4,6-dimethoxy[1.3.5]triazin-2-yl)-4-methylmorpholinium chloride
hydrate (DMTMM) in water (9.6 mL, 0.25 M). The mixture was gently
shaken for 2 hours at 4.degree. C. before an additional 40
equivalents of S-Ado and DMTMM were added and shaken for a further
16 hours at 4.degree. C.
[0226] Following acylation, a 0.1.times. volume of 5 M aqueous NaCl
and a 2.5.times. volume of cold (-20.degree. C.) ethanol was added
and the mixture was allowed to stand at -20.degree. C. for at least
one hour. The mixture was then centrifuged for 15 minutes at 14,000
rpm in a 4.degree. C. centrifuge to give a white pellet which was
washed with cold EtOH and then dried in a lyophilizer at room
temperature for 30 minutes. The solid was dissolved in 40 mL of
water and purified by Reverse Phase HPLC with a Waters Xterra
RP.sub.18 column. A binary mobile phase gradient profile was used
to elute the product using a 50 mM aqueous triethylammonium acetate
buffer at pH 7.5 and 99% acetonitrile/1% water solution. The
purified material was concentrated by lyophilization and the
resulting residue was dissolved in 5 mL of water. A 0.1.times.
volume of piperidine was added to the solution and the mixture was
gently shaken for 45 minutes at room temperature. The product was
then purified by ethanol precipitation as described above and
isolated by centrifugation. The resulting pellet was washed twice
with cold EtOH and dried by lyophilization to give purified
Compound 3.
Cycle 1
[0227] To each well in a 96 well plate was added 12.5 of a 4 mM
solution of Compound 3 in water; 100 .mu.L of a 1 mM solution of
one of oligonucleotide tags 1.1 to 1.96, as shown in Table 3 (the
molar ratio of Compound 3 to tags was 1:2). The plates were heated
to 95.degree. C. for 1 minute and then cooled to 16.degree. C. over
10 minutes. To each well was added 10 .mu.L of 10.times. ligase
buffer, 30 units T4 DNA ligase (1 .mu.L of a 30 unit/.mu.L solution
(FermentasLife Science, Cat. No. EL0013)), 76.5 .mu.l of water and
the resulting solutions were incubated at 16.degree. C. for 16
hours.
[0228] After the ligation reaction, 20 .mu.L of 5 M aqueous NaCl
was added directly to each well, followed by 500 .mu.L cold
(-20.degree. C.) ethanol, and held at -20.degree. C. for 1 hour.
The plates were centrifugated for 1 hour at 3200 g in a Beckman
Coulter Allegra 6R centrifuge using Beckman Microplus Carriers. The
supernatant was carefully removed by inverting the plate and the
pellet was washed with 70% aqueous cold ethanol at -20.degree. C.
Each of the pellets was then dissolved in sodium borate buffer (50
.mu.L, 150 mM, pH 9.4) to a concentration of 1 mM and chilled to
4.degree. C.
[0229] To each solution was added 40 equivalents of one of the 96
building block precursors in DMF (13 .mu.L, 0.15 M) followed by 40
equivalents of DMT-MM in water (8 .mu.L, 0.25M), and the solutions
were gently shaken at 4.degree. C. After 2 hours, an additional 40
equivalents of one of each building block precursor and DMTMM were
added and the solutions were gently shaken for 16 hours at
4.degree. C. Following acylation, 10 equivalents of acetic
acid-N-hydroxy-succinimide ester in DMF (2 .mu.L, 0.25M) was added
to each solution and gently shaken for 10 minutes.
[0230] Following acylation, the 96 reaction mixtures were pooled
and 0.1 volume of 5M aqueous NaCl and 2.5 volumes of cold absolute
ethanol were added and the solution was allowed to stand at
-20.degree. C. for at least one hour. The mixture was then
centrifuged. Following centrifugation, as much supernatant as
possible was removed with a micropipette, the pellet was washed
with cold ethanol and centrifuged again. The supernatant was
removed with a 200 .mu.L pipet. Cold 70% ethanol was added to the
tube, and the resulting mixture was centrifuged for 5 min at
4.degree. C.
[0231] The supernatant was removed and the remaining ethanol was
removed by lyophilization at room temperature for 10 minutes. The
pellet was then dissolved in 2 mL of water and purified by Reverse
Phase HPLC with a Waters Xterra RP.sub.18 column. A binary mobile
phase gradient profile was used to elute the library using a 50 mM
aqueous triethylammonium acetate buffer at pH 7.5 and 99%
acetonitrile/1% water solution. The fractions containing the
library were collected, pooled, and lyophilized. The resulting
residue was dissolved in 2.5 mL of water and 250 .mu.L of
piperidine was added. The solution was shaken gently for 45 minutes
and then precipitated with ethanol as previously described. The
resulting pellet was dried by lyophilization and then dissolved in
sodium borate buffer (4.8 mL, 150 mM, pH 9.4) to a concentration of
1 mM.
[0232] The solution was chilled to 4.degree. C. and 40 equivalents
each of N-Fmoc-propargylglycine in DMF (1.2 mL, 0.15 M) and DMT-MM
in water (7.7 mL, 0.25 M) were added. The mixture was gently shaken
for 2 hours at 4.degree. C. before an additional 40 equivalents of
N-Fmoc-propargylglycine and DMT-MM were added and the solution was
shaken for a further 16 hours. The mixture was later purified by
EtOH precipitation and Reverse Phase HPLC as described above and
the N-Fmoc group was removed by treatment with piperidine as
previously described. Upon final purification by EtOH
precipitation, the resulting pellet was dried by lyophilization and
carried into the next cycle of synthesis
Cycles 2-4
[0233] For each of these cycles, the dried pellet from the previous
cycle was dissolved in water and the concentration of library was
determined by spectrophotometry based on the extinction coefficient
of the DNA component of the library, where the initial extinction
coefficient of Compound 2 is 131,500 L/(molecm). The concentration
of the library was adjusted with water such that the final
concentration in the subsequent ligation reactions was 0.25 mM. The
library was then divided into 96 equal aliquots in a 96 well plate.
To each well was added a solution comprising a different tag (molar
ratio of the library to tag was 1:2), and ligations were performed
as described for Cycle 1. Oligonucleotide tags used in Cycles 2, 3
and 4 are set forth in Tables 4, 5 and 6, respectively.
Correspondence between the tags and the building block precursors
for each of Cycles 1 to 4 is provided in Table 7. The library was
precipitated by the addition of ethanol as described above for
Cycle 1, and dissolved in sodium borate buffer (150 mM, pH 9.4) to
a concentration of 1 mM. Subsequent acylations and purifications
were performed as described for Cycle 1, except HPLC purification
was omitted during Cycle 3.
[0234] The products of Cycle 4 were ligated with the closing primer
shown below, using the method described above for ligation of
tags.
TABLE-US-00013 (SEQ ID NO: 889) 5'-PO.sub.3-CAG AAG ACA GAC AAG CTT
CAC CTG C (SEQ ID NO: 890) 5'-PO.sub.3-GCA GGT GAA GCT TGT CTG TCT
TCT GAA
Results:
[0235] The synthetic procedure described above has the capability
of producing a library comprising 96.sup.4 (about 10.sup.8)
different structures. The synthesis of the library was monitored
via gel electrophoresis and LC/MS of the product of each cycle.
Upon completion, the library was analyzed using several techniques.
FIG. 13a is a chromatogram of the library following Cycle 4, but
before ligation of the closing primer; FIG. 13b is a mass spectrum
of the library at the same synthetic stage. The average molecular
weight was determined by negative ion LC/MS analysis. The ion
signal was deconvoluted using ProMass software. This result is
consistent with the predicted average mass of the library.
[0236] The DNA component of the library was analyzed by agarose gel
electrophoresis, which showed that the majority of library material
corresponds to ligated product of the correct size. DNA sequence
analysis of molecular clones of PCR product derived from a sampling
of the library shows that DNA ligation occurred with high fidelity
and to near completion.
Library Cyclization
[0237] At the completion of Cycle 4, a portion of the library was
capped at the N-terminus using azidoacetic acid under the usual
acylation conditions. The product, after purification by EtOH
precipitation, was dissolved in sodium phosphate buffer (150 mM, pH
8) to a concentration of 1 mM and 4 equivalents each of CuSO.sub.4
in water (200 mM), ascorbic acid in water (200 mM), and a solution
of the compound shown below in DMF (200 mM) were added. The
reaction mixture was then gently shaken for 2 hours at room
temperature.
##STR00110##
[0238] To assay the extent of cyclization, 5 .mu.L aliquots from
the library cyclization reaction were removed and treated with a
fluorescently-labeled azide or alkyne (14 of 100 mM DMF stocks)
prepared as described in Example 4. After 16 hours, neither the
alkyne or azide labels had been incorporated into the library by
HPLC analysis at 500 nm. This result indicated that the library no
longer contained azide or alkyne groups capable of cycloaddition
and that the library must therefore have reacted with itself,
either through cyclization or intermolecular reactions. The
cyclized library was purified by Reverse Phase HPLC as previously
described. Control experiments using uncyclized library showed
complete incorporation of the fluorescent tags mentioned above.
Example 4
Preparation of Fluorescent Tags for Cyclization Assay
[0239] In separate tubes, propargyl glycine or
2-amino-3-phenylpropylazide (8 .mu.mol each) was combined with
FAM-OSu (Molecular Probes Inc.) (1.2 equiv.) in pH 9.4 borate
buffer (250 .mu.L). The reactions were allowed to proceed for 3 h
at room temperature, and were then lyophilized overnight.
Purification by HPLC afforded the desired fluorescent alkyne and
azide in quantitative yield.
##STR00111##
Example 5
Cyclization of Individual Compounds Using the Azide/Alkyne
Cycloaddition Reaction
Preparation of Azidoacetyl-Gly-Pro-Phe-Pra-NH.sub.2 (SEQ ID NO:
920):
[0240] Using 0.3 mmol of Rink-amide resin, the indicated sequence
was synthesized using standard solid phase synthesis techniques
with Fmoc-protected amino acids and HATU as activating agent
(Pra=C-propargylglycine). Azidoacetic acid was used to cap the
tetrapeptide. The peptide was cleaved from the resin with 20%
TFA/DCM for 4 h. Purification by RP HPLC afforded product as a
white solid (75 mg, 51%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz):
8.4-7.8 (m, 3H), 7.4-7.1 (m, 7H), 4.6-4.4 (m, 1H), 4.4-4.2 (m, 2H),
4.0-3.9 (m, 2H), 3.74 (dd, 1H, J=6 Hz, 17 Hz), 3.5-3.3 (m, 2H),
3.07 (dt, 1H, J=5 Hz, 14 Hz), 2.92 (dd, 1H, J=5 Hz, 16 Hz), 2.86
(t, 1H, J=2 Hz), 2.85-2.75 (m, 1H), 2.6-2.4 (m, 2H), 2.2-1.6 (m,
4H). IR (mull) 2900, 2100, 1450, 1300 cm.sup.-1. ESIMS 497.4
([M+H], 100%), 993.4 ([2M+H], 50%). ESIMS with ion-source
fragmentation: 519.3 ([M+Na], 100%), 491.3 (100%), 480.1
([M-NH.sub.2], 90%), 452.2 ([M-NH.sub.2--CO], 20%), 424.2 (20%),
385.1 ([M-Pra], 50%), 357.1 ([M-Pra-CO], 40%), 238.0 ([M-Pra-Phe],
100%).
Cyclization of Azidoacetyl-Gly-Pro-Phe-Pra-NH.sub.2 (SEQ ID NO:
920):
##STR00112##
[0242] The azidoacetyl peptide (31 mg, 0.62 mmol) was dissolved in
MeCN (30 mL). Diisopropylethylamine (DIEA, 1 mL) and
Cu(MeCN).sub.4PF.sub.6 (1 mg) were added. After stirring for 1.5 h,
the solution was evaporated and the resulting residue was taken up
in 20% MeCN/H.sub.2O. After centrifugation to remove insoluble
salts, the solution was subjected to preparative reverse phase
HPLC. The desired cyclic peptide was isolated as a white solid (10
mg, 32%). .sup.1H NMR (DMSO-d.sub.6, 400 MHz): 8.28 (t, 1H, J=5
Hz), 7.77 (s, 1H), 7.2-6.9 (m, 9H), 4.98 (m, 2H), 4.48 (m, 1H),
4.28 (m, 1H), 4.1-3.9 (m, 2H), 3.63 (dd, 1H, J=5 Hz, 16 Hz), 3.33
(m, 2H), 3.0 (m, 3H), 2.48 (dd, 1H, J=11 Hz, 14 Hz), 1.75 (m, 1H0,
1.55 (m, 1H), 1.32 (m, 1H), 1.05 (m, 1H). IR (mull) 2900, 1475,
1400 cm.sup.-1. ESIMS 497.2 ([M+H], 100%), 993.2 ([2M+H], 30%),
1015.2 ([2M+Na], 15%). ESIMS with ion-source fragmentation: 535.2
(70%), 519.3 ([M+Na], 100%), 497.2 ([M+H], 80%), 480.1
([M-NH.sub.2], 30%), 452.2 ([M-NH.sub.2--CO], 40%), 208.1
(60%).
Preparation of Azidoacetyl-Gly-Pro-Phe-Pra-Gly-OH (SEQ ID NO:
920):
[0243] Using 0.3 mmol of Glycine-Wang resin, the indicated sequence
was synthesized using Fmoc-protected amino acids and HATU as the
activating agent. Azidoacetic acid was used in the last coupling
step to cap the pentapeptide. Cleavage of the peptide was achieved
using 50% TFA/DCM for 2 h. Purification by RP HPLC afforded the
peptide as a white solid (83 mg; 50%). .sup.1H NMR (DMSO-d.sub.6,
400 MHz): 8.4-7.9 (m, 4H), 7.2 (m, 5H), 4.7-4.2 (m, 3H), 4.0-3.7
(m, 4H), 3.5-3.3 (m, 2H), 3.1 (m, 1H), 2.91 (dd, 1H, J=4 Hz, 16
Hz), 2.84 (t, 1H, J=2.5 Hz), 2.78 (m, 1H), 2.6-2.4 (m, 2H), 2.2-1.6
(m, 4H). IR (mull) 2900, 2100, 1450, 1350 cm.sup.-1. ESIMS 555.3
([M+H], 100%). ESIMS with ion-source fragmentation: 577.1 ([M+Na],
90%), 555.3 ([M+H], 80%), 480.1 ([M-Gly], 100%), 385.1
([M-Gly-Pra], 70%), 357.1 ([M-Gly-Pra-CO], 40%), 238.0
([M-Gly-Pra-Phe], 80%).
Cyclization of Azidoacetyl-Gly-Pro-Phe-Pra-Gly-OH (SEQ ID NO:
920):
[0244] The peptide (32 mg, 0.058 mmol) was dissolved in MeCN (60
mL). Diisopropylethylamine (1 mL) and Cu(MeCN).sub.4 PF.sub.6 (1
mg) were added and the solution was stirred for 2 h. The solvent
was evaporated and the crude product was subjected to RP HPLC to
remove dimers and trimers. The cyclic monomer was isolated as a
colorless glass (6 mg, 20%). ESIMS 555.6 ([M+H], 100%), 1109.3
([2M+H], 20%), 1131.2 ([2M+Na], 15%).
[0245] ESIMS with ion source fragmentation: 555.3 ([M+H], 100%),
480.4 ([M-Gly], 30%), 452.2 ([M-Gly-CO], 25%), 424.5 ([M-Gly-2CO],
10%, only possible in a cyclic structure).
Conjugation of Linear Peptide to DNA:
[0246] Compound 2 (45 nmol) was dissolved in 45 .mu.L sodium borate
buffer (pH 9.4; 150 mM). At 4.degree. C., linear peptide (18 .mu.L
of a 100 mM stock in DMF; 180 nmol; 40 equiv.) was added, followed
by DMT-MM (3.6 .mu.L of a 500 mM stock in water; 180 nmol; 40
equiv.). After agitating for 2 h, LCMS showed complete reaction,
and product was isolated by ethanol precipitation. ESIMS 1823.0
([M-3H]/3, 20%), 1367.2 ([M-4H]/4, 20%), 1093.7 ([M-5H]/5, 40%),
911.4 ([M-6H]/6, 100%).
Conjugation of Cyclic Peptide to DNA:
[0247] Compound 2 (20 nmol) was dissolved in 20 .mu.L sodium borate
buffer (pH 9.4, 150 mM). At 4.degree. C., linear peptide (8 .mu.L
of a 100 mM stock in DMF; 80 nmol; 40 equiv.) was added, followed
by DMT-MM (1.6 .mu.L of a 500 mM stock in water; 80 nmol; 40
equiv.). After agitating for 2 h, LCMS showed complete reaction,
and product was isolated by ethanol precipitation. ESIMS 1823.0
([M-3H]/3, 20%), 1367.2 ([M-4H]/4, 20%), 1093.7 ([M-5H]/5, 40%),
911.4 ([M-6H]/6, 100%).
Cyclization of DNA-Linked Peptide:
[0248] Linear peptide-DNA conjugate (10 nmol) was dissolved in pH 8
sodium phosphate buffer (10 .mu.L, 150 mm). At room temperature, 4
equivalents each of CuSO.sub.4, ascorbic acid, and the Sharpless
ligand were all added (0.2 .mu.L of 200 mM stocks). The reaction
was allowed to proceed overnight. RP HPLC showed that no linear
peptide-DNA was present, and that the product co-eluted with
authentic cyclic peptide-DNA. No traces of dimers or other
oligomers were observed.
##STR00113##
Example 6
Application of Aromatic Nucleophilic Substitution Reactions to
Functional Moiety Synthesis
[0249] General Procedure for Arylation of Compound 3 with Cyanuric
Chloride:
[0250] Compound 2 is dissolved in pH 9.4 sodium borate buffer at a
concentration of 1 mM. The solution is cooled to 4.degree. C. and
20 equivalents of cyanuric chloride is then added as a 500 mM
solution in MeCN. After 2 h, complete reaction is confirmed by LCMS
and the resulting dichlorotriazine-DNA conjugate is isolated by
ethanol precipitation.
Procedure for Amine Substitution of Dichlorotriazine-DNA:
[0251] The dichlorotriazine-DNA conjugate is dissolved in pH 9.5
borate buffer at a concentration of 1 mM. At room temperature, 40
equivalents of an aliphatic amine is added as a DMF solution. The
reaction is followed by LCMS and is usually complete after 2 h. The
resulting alkylamino-monochlorotriazine-DNA conjugate is isolated
by ethanol precipitation.
Procedure for Amine Substitution of Monochlorotriazine-DNA:
[0252] The alkylamino-monochlorotriazine-DNA conjugate is dissolved
in pH 9.5 borate buffer at a concentration of 1 mM. At 42.degree.
C., 40 equivalents of a second aliphatic amine is added as a DMF
solution. The reaction is followed by LCMS and is usually complete
after 2 h. The resulting diaminotriazine-DNA conjugate is isolated
by ethanol precipitation.
Example 7
Application of Reductive Amination Reactions to Functional Moiety
Synthesis
[0253] General Procedure for Reductive Amination of DNA-Linker
Containing a Secondary Amine with an Aldehyde Building Block:
[0254] Compound 2 was coupled to an N-terminal proline residue. The
resulting compound was dissolved in sodium phosphate buffer (50
.mu.L, 150 mM, pH 5.5) at a concentration of 1 mM. To this solution
was added 40 equivalents each of an aldehyde building block in DMF
(8 .mu.L, 0.25M) and sodium cyanoborohydride in DMF (8 .mu.L,
0.25M) and the solution was heated at 80.degree. C. for 2 hours.
Following alkylation, the solution was purified by ethanol
precipitation.
General Procedure for Reductive Aminations of DNA-Linker Containing
an Aldehyde with Amine Building Blocks:
[0255] Compound 2 coupled to a building block comprising an
aldehyde group was dissolved in sodium phosphate buffer (50 .mu.L,
250 mM, pH 5.5) at a concentration of 1 mM. To this solution was
added 40 equivalents each of an amine building block in DMF (8
.mu.L, 0.25M) and sodium cyanoborohydride in DMF (8 .mu.L, 0.25M)
and the solution was heated at 80.degree. C. for 2 hours. Following
alkylation, the solution was purified by ethanol precipitation.
Example 8
Application of Peptoid Building Reactions to Functional Moiety
Synthesis
General Procedure for Peptoid Synthesis on DNA-Linker:
##STR00114##
[0257] Compound 2 was dissolved in sodium borate buffer (50 .mu.L,
150 mM, pH 9.4) at a concentration of 1 mM and chilled to 4.degree.
C. To this solution was added 40 equivalents of
N-hydroxysuccinimidyl bromoacetate in DMF (13 .mu.L, 0.15 M) and
the solution was gently shaken at 4.degree. C. for 2 hours.
Following acylation, the DNA-Linker was purified by ethanol
precipitation and redissolved in sodium borate buffer (50 .mu.L,
150 mM, pH 9.4) at a concentration of 1 mM and chilled to 4.degree.
C. To this solution was added 40 equivalents of an amine building
block in DMF (13 .mu.L, 0.15 M) and the solution was gently shaken
at 4.degree. C. for 16 hours. Following alkylation, the DNA-linker
was purified by ethanol precipitation and redissolved in sodium
borate buffer (50 .mu.L, 150 mM, pH 9.4) at a concentration of 1 mM
and chilled to 4.degree. C. Peptoid synthesis is continued by the
stepwise addition of N-hydroxysuccinimidyl bromoacetate followed by
the addition of an amine building block.
Example 9
Application of the Azide-Alkyne Cycloaddition Reaction to
Functional Moiety Synthesis
General Procedure
[0258] An alkyne-containing DNA conjugate is dissolved in pH 8.0
phosphate buffer at a concentration of ca. 1 mM. To this mixture is
added 10 equivalents of an organic azide and 5 equivalents each of
copper (II) sulfate, ascorbic acid, and the ligand
(tris-((1-benzyltriazol-4-yl)methyl)amine all at room temperature.
The reaction is followed by LCMS, and is usually complete after 1-2
h. The resulting triazole-DNA conjugate can be isolated by ethanol
precipitation.
Example 10
Identification of a Ligand to Abl Kinase from Within an Encoded
Library
[0259] The ability to enrich molecules of interest in a DNA-encoded
library above undesirable library members is paramount to
identifying single compounds with defined properties against
therapeutic targets of interest. To demonstrate this enrichment
ability a known binding molecule (described by Shah et al., Science
305, 399-401 (2004), incorporated herein by reference) to rhAbl
kinase (GenBank U07563) was synthesized. This compound was attached
to a double stranded DNA oligonucleotide via the linker described
in the preceding examples using standard chemistry methods to
produce a molecule similar (functional moiety linked to an
oligonucleotide) to those produced via the methods described in
Examples 1 and 2. A library generally produced as described in
Example 2 and the DNA-linked Abl kinase binder were designed with
unique DNA sequences that allowed qPCR analysis of both species.
The DNA-linked Abl kinase binder was mixed with the library at a
ratio of 1:1000. This mixture was equilibrated with to rhAble
kinase, and the enzyme was captured on a solid phase, washed to
remove non-binding library members and binding molecules were
eluted. The ratio of library molecules to the DNA-linked Abl kinase
inhibitor in the eluate was 1:1, indicating a greater than 500-fold
enrichment of the DNA-linked Abl-kinase binder in a 1000-fold
excess of library molecules.
EQUIVALENTS
[0260] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
92919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gcaacgaag 929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2tcgttgcca 939DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 3gcgtacaag
949DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4tgtacgcca 959DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5gctctgtag 969DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 6acagagcca
979DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7gtgccatag 989DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8atggcacca 999DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 9gttgaccag
9109DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 10ggtcaacca 9119DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11cgacttgac 9129DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 12acgctgaac
9139DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 13cgtagtcag 9149DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14gactacgca 9159DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 15ccagcatag
9169DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 16atgctggca 9179DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17cctacagag 9189DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 18ctgtaggca
9199DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19ctgaacgag 9209DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20acgacttgc 9219DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 21ctccagtag
9229DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 22actggagca 9239DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23taggtccag 9249DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 24ggacctaca
9259DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 25gcgtgttgt 9269DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26aacacgcct 9279DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 27gcttggagt
9289DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 28tccaagcct 9299DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29gtcaagcgt 9309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 30gcttgacct
9319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 31caagagcgt 9329DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32gctcttgct 9339DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 33cagttcggt
9349DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 34cgaactgct 9359DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 35cgaaggagt 9369DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 36tccttcgct
9379DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 37cggtgttgt 9389DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 38aacaccgct 9399DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 39cgttgctgt
9409DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 40agcaacgct 9419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 41ccgatctgt 9429DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 42agatcggct
9439DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43ccttctcgt 9449DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 44gagaaggct 9459DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 45tgagtccgt
9469DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 46ggactcact 9479DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 47tgctacggt 9489DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 48cgttagact
9499DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49gtgcgttga 9509DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50aacgcacac 9519DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 51gttggcaga
9529DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 52tgccaacac 9539DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 53cctgtagga 9549DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 54ctacaggac
9559DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 55ctgcgtaga 9569DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 56tacgcagac 9579DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 57cttacgcga
9589DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 58gcgtaagac 9599DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 59tggtcacga 9609DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 60gtgaccaac
9619DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 61tcagagcga 9629DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 62gctctgaac 9639DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 63ttgctcgga
9649DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 64cgagcaaac 9659DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 65gcagttgga 9669DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 66caactgcac
9679DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 67gcctgaaga 9689DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 68ttcaggcac 9699DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 69gtagccaga
9709DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 70tggctacac 9719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 71gtcgcttga 9729DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 72aagcgacac
9739DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 73gcctaagtt 9749DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 74cttaggctc 9759DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 75gtagtgctt
9769DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 76gcactactc 9779DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 77gtcgaagtt 9789DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 78cttcgactc
9799DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 79gtttcggtt 9809DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 80ccgaaactc 9819DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 81cagcgtttt
9829DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 82aacgctgtc 9839DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 83catacgctt 9849DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 84gcgtatgtc
9859DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 85cgatctgtt 9869DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 86cagatcgtc 9879DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 87cgctttgtt
9889DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 88caaagcgtc 9899DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 89ccacagttt 9909DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 90actgtggtc
9919DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 91cctgaagtt 9929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 92cttcaggtc 9939DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 93ctgacgatt
9949DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 94tcgtcagtc 9959DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 95ctccacttt 9969DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 96agtggagtc
9979DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 97accagagcc 9989DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 98ctctggtaa 9999DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 99atccgcacc
91009DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 100tgcggataa 91019DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 101gacgacacc 91029DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 102tgtcgtcaa
91039DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 103ggatggacc 91049DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 104tccatccaa 91059DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 105gcagaagcc
91069DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 106cttctgcaa 91079DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 107gccatgtcc 91089DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide
108acatggcaa 91099DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 109gtctgctcc 91109DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 110agcagacaa 91119DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 111cgacagacc
91129DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 112tctgtcgaa 91139DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 113cgctactcc 91149DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 114agtagcgaa
91159DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 115ccacagacc 91169DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 116tctgtggaa 91179DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 117cctctctcc
91189DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 118agagaggaa 91199DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 119ctcgtagcc 91209DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 120ctacgagaa
912120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 121aaatcgatgt ggtcactcag
2012220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 122gagtgaccac atcgatttgg
2012320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 123aaatcgatgt ggactaggag
2012420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 124cctagtccac atcgatttgg
2012520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 125aaatcgatgt gccgtatgag
2012620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 126catacggcac atcgatttgg
2012720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 127aaatcgatgt gctgaaggag
2012820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 128ccttcagcac atcgatttgg
2012920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 129aaatcgatgt ggactagcag
2013020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 130gctagtccac atcgatttgg
2013120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 131aaatcgatgt gcgctaagag
2013220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 132cttagcgcac atcgatttgg
2013320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 133aaatcgatgt gagccgagag
2013420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 134ctcggctcac atcgatttgg
2013520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 135aaatcgatgt gccgtatcag
2013620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 136gatacggcac atcgatttgg
2013720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 137aaatcgatgt gctgaagcag
2013820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 138gcttcagcac atcgatttgg
2013920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 139aaatcgatgt gtgcgagtag
2014020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 140actcgcacac atcgatttgg
2014120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 141aaatcgatgt gtttggcgag
2014220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 142cgccaaacac atcgatttgg
2014320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 143aaatcgatgt gcgctaacag
2014420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 144gttagcgcac atcgatttgg
2014520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 145aaatcgatgt gagccgacag
2014620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 146gtcggctcac atcgatttgg
2014720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 147aaatcgatgt gagccgaaag
2014820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 148ttcggctcac atcgatttgg
2014920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 149aaatcgatgt gtcggtagag
2015020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 150ctaccgacac atcgatttgg
2015120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 151aaatcgatgt ggttgccgag
2015220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 152cggcaaccac atcgatttgg
2015320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 153aaatcgatgt gagtgcgtag
2015420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 154acgcactcac atcgatttgg
2015520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 155aaatcgatgt ggttgccaag
2015620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 156tggcaaccac atcgatttgg
2015720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 157aaatcgatgt gtgcgaggag
2015820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 158cctcgcacac atcgatttgg
2015920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 159aaatcgatgt ggaacacgag
2016020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 160cgtgttccac atcgatttgg
2016120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 161aaatcgatgt gcttgtcgag
2016220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 162cgacaagcac atcgatttgg
2016320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 163aaatcgatgt gttccggtag
2016420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 164accggaacac atcgatttgg
2016520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 165aaatcgatgt gtgcgagcag
2016620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 166gctcgcacac atcgatttgg
2016720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 167aaatcgatgt ggtcaggtag
2016820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 168acctgaccac atcgatttgg
2016920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 169aaatcgatgt ggcctgttag
2017020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 170aacaggccac atcgatttgg
2017120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 171aaatcgatgt ggaacaccag
2017220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 172ggtgttccac atcgatttgg
2017320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 173aaatcgatgt gcttgtccag
2017420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 174ggacaagcac atcgatttgg
2017520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 175aaatcgatgt gtgcgagaag
2017620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 176tctcgcacac atcgatttgg
2017720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 177aaatcgatgt gagtgcggag
2017820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 178ccgcactcac atcgatttgg
2017920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 179aaatcgatgt gttgtccgag
2018020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 180cggacaacac atcgatttgg
2018120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 181aaatcgatgt gtggaacgag
2018220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 182cgttccacac atcgatttgg
2018320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 183aaatcgatgt gagtgcgaag
2018420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 184tcgcactcac atcgatttgg
2018520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 185aaatcgatgt gtggaaccag
2018620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 186ggttccacac atcgatttgg
2018720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 187aaatcgatgt gttaggcgag
2018820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 188cgcctaacac atcgatttgg
2018920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 189aaatcgatgt ggcctgtgag
2019020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 190cacaggccac atcgatttgg
2019120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 191aaatcgatgt gctcctgtag
2019220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 192acaggagcac atcgatttgg
2019320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 193aaatcgatgt ggtcaggcag
2019420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 194gcctgaccac atcgatttgg
2019520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 195aaatcgatgt ggtcaggaag
2019620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 196tcctgaccac atcgatttgg
2019720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 197aaatcgatgt ggtagccgag
2019820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 198cggctaccac atcgatttgg
2019920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 199aaatcgatgt ggcctgtaag
2020020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 200tacaggccac atcgatttgg
2020120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 201aaatcgatgt gctttcggag
2020220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 202ccgaaagcac atcgatttgg
2020320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 203aaatcgatgt gcgtaaggag
2020420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 204ccttacgcac atcgatttgg
2020520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 205aaatcgatgt gagagcgtag
2020620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 206acgctctcac atcgatttgg
2020720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 207aaatcgatgt ggacggcaag
2020820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 208tgccgtccac atcgatttgg
2020920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 209aaatcgatgt gctttcgcag
2021020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 210gcgaaagcac atcgatttgg
2021120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 211aaatcgatgt gcgtaagcag
2021220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 212gcttacgcac atcgatttgg
2021320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 213aaatcgatgt ggctatggag
2021420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 214ccatagccac atcgatttgg
2021520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 215aaatcgatgt gactctggag
2021620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 216ccagagtcac atcgatttgg
2021719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 217aaatcgatgt gctggaaag
1921819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 218ttccagcaca tcgatttgg
1921920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 219aaatcgatgt gccgaagtag
2022020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 220acttcggcac atcgatttgg
2022120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 221aaatcgatgt gctcctgaag
2022220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 222tcaggagcac atcgatttgg
2022320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 223aaatcgatgt gtccagtcag
2022420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 224gactggacac atcgatttgg
2022520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 225aaatcgatgt gagagcggag
2022620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 226ccgctctcac atcgatttgg
2022720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 227aaatcgatgt gagagcgaag
2022820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 228tcgctctcac atcgatttgg
2022920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 229aaatcgatgt gccgaaggag
2023020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 230ccttcggcac atcgatttgg
2023120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 231aaatcgatgt gccgaagcag
2023220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 232gcttcggcac atcgatttgg
2023320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 233aaatcgatgt gtgttccgag
2023420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 234cggaacacac atcgatttgg
2023520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 235aaatcgatgt gtctggcgag
2023620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 236cgccagacac atcgatttgg
2023720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 237aaatcgatgt gctatcggag
2023820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 238ccgatagcac atcgatttgg
2023920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 239aaatcgatgt gcgaaaggag
2024020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 240cctttcgcac atcgatttgg
2024120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 241aaatcgatgt gccgaagaag
2024220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 242tcttcggcac atcgatttgg
2024320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 243aaatcgatgt ggttgcagag
2024420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 244ctgcaaccac atcgatttgg
2024520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 245aaatcgatgt ggatggtgag
2024620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 246caccatccac atcgatttgg
2024720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 247aaatcgatgt gctatcgcag
2024820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 248gcgatagcac atcgatttgg
2024920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 249aaatcgatgt gcgaaagcag
2025020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 250gctttcgcac atcgatttgg
2025120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 251aaatcgatgt gacactggag
2025220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 252ccagtgtcac atcgatttgg
2025320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 253aaatcgatgt gtctggcaag
2025420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 254tgccagacac atcgatttgg
2025520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 255aaatcgatgt ggatggtcag
2025620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 256gaccatccac atcgatttgg
2025720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 257aaatcgatgt ggttgcacag
2025820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 258gtgcaaccac atcgatttgg
2025920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 259aaatcgatgt gggcatcgag
2026020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 260cgatgcccca tccgatttgg
2026120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 261aaatcgatgt gtgcctccag
2026220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 262ggaggcacac atcgatttgg
2026320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 263aaatcgatgt gtgcctcaag
2026420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 264tgaggcacac atcgatttgg
2026520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 265aaatcgatgt gggcatccag
2026620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 266ggatgcccac atcgatttgg
2026720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 267aaatcgatgt gggcatcaag
2026820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 268tgatgcccac atcgatttgg
2026920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 269aaatcgatgt gcctgtcgag
2027020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 270cgacaggcac atcgatttgg
2027120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 271aaatcgatgt ggacggatag
2027220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 272atccgtccac atcgatttgg
2027320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 273aaatcgatgt gcctgtccag
2027420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 274ggacaggcac atcgatttgg
2027520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 275aaatcgatgt gaagcacgag
2027620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 276cgtgcttcac atcgatttgg
2027720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 277aaatcgatgt gcctgtcaag
2027820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 278tgacaggcac atcgatttgg
2027920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 279aaatcgatgt gaagcaccag
2028020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 280ggtgcttcac atcgatttgg
2028120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 281aaatcgatgt gccttcgtag
2028220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 282acgaaggcac atcgatttgg
2028320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 283aaatcgatgt gtcgtccgag
2028420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 284cggacgacac atcgatttgg
2028520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 285aaatcgatgt ggagtctgag
2028620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 286cagactccac atcgatttgg
2028720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 287aaatcgatgt gtgatccgag
2028820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 288cggatcacac atcgatttgg
2028920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 289aaatcgatgt gtcaggcgag
2029020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 290cgcctgacac atcgatttgg
2029120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 291aaatcgatgt gtcgtccaag
2029220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 292tggacgacac atcgatttgg
2029320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 293aaatcgatgt ggacggagag
2029420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 294ctccgtccac atcgatttgg
2029520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 295aaatcgatgt ggtagcagag
2029620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 296ctgctaccac atcgatttgg
2029720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 297aaatcgatgt ggctgtgtag
2029820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 298acacagccac atcgatttgg
2029920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 299aaatcgatgt ggacggacag
2030020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 300gtccgtccac atcgatttgg
2030120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 301aaatcgatgt gtcaggcaag
2030220DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 302tgcctgacac atcgatttgg
2030320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 303aaatcgatgt ggctcgaaag
2030420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 304ttcgagccac atcgatttgg
2030520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 305aaatcgatgt gccttcggag
2030620DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 306ccgaaggcac atcgatttgg
2030720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 307aaatcgatgt ggtagcacag
2030820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 308gtgctaccac atcgatttgg
2030920DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 309aaatcgatgt ggaaggtcag
2031020DNAArtificial SequenceDescription of Artificial Sequence
Synthetic
oligonucleotide 310gaccttccac atcgatttgg 2031120DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 311aaatcgatgt ggtgctgtag 2031220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 312acagcaccac atcgatttgg 203139DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 313gttgcctgt 93149DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 314aggcaacct
93159DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 315caggacggt 93169DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 316cgtcctgct 93179DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 317agacgtggt
93189DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 318cacgtctct 93199DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 319caggaccgt 93209DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 320ggtcctgct
93219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 321caggacagt 93229DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 322tgtcctgct 93239DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 323cactctggt
93249DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 324cagagtgct 93259DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 325gacggctgt 93269DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 326agccgtcct
93279DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 327cactctcgt 93289DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 328gagagtgct 93299DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 329gtagcctgt
93309DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 330aggctacct 93319DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 331gccacttgt 93329DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 332aagtggcct
93339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 333catcgctgt 93349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 334agcgatgct 93359DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 335cactggtgt
93369DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 336accagtgct 93379DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 337gccactggt 93389DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 338cagtggcct
93399DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 339tctggctgt 93409DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 340agccagact 93419DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 341gccactcgt
93429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 342gagtggcct 93439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 343tgcctctgt 93449DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 344agaggcact
93459DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 345catcgcagt 93469DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 346tgcgatgct 93479DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 347caggaaggt
93489DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 348cttcctgct 93499DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 349ggcatctgt 93509DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 350agatgccct
93519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 351cggtgctgt 93529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 352agcaccgct 93539DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 353cactggcgt
93549DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 354gccagtgct 93559DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 355tctcctcgt 93569DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 356gaggagact
93579DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 357cctgtctgt 93589DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 358agacaggct 93599DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 359caacgctgt
93609DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 360agcgttgct 93619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 361tgcctcggt 93629DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 362cgaggcact
93639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 363acactgcgt 93649DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 364gcagtgtct 93659DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 365tcgtcctgt
93669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 366aggacgact 93679DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 367gctgccagt 93689DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 368tggcagcct
93699DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 369tcaggctgt 93709DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 370agcctgact 93719DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 371gccaggtgt
93729DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 372acctggcct 93739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 373cggacctgt 93749DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 374aggtccgct
93759DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 375caacgcagt 93769DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 376tgcgttgct 93779DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 377cacacgagt
93789DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 378tcgtgtgct 93799DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 379atggcctgt 93809DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 380aggccatct
93819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 381ccagtctgt 93829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 382agactggct 93839DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 383gccaggagt
93849DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 384tcctggcct 93859DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 385cggaccagt 93869DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 386tggtccgct
93879DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 387ccttcgcgt 93889DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 388gcgaaggct 93899DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 389gcagccagt
93909DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 390tggctgcct 93919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 391ccagtcggt 93929DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 392cgactggct
93939DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 393actgagcgt 93949DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 394gctcagtct 93959DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 395ccagtccgt
93969DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 396ggactggct 93979DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 397ccagtcagt 93989DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 398tgactggct
93999DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 399catcgaggt 94009DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 400ctcgatgct 94019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 401ccatcgtgt
94029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 402acgatggct 94039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 403gtgctgcgt 94049DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 404gcagcacct
94059DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 405gactacggt 94069DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 406cgtagtcct 94079DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 407gtgctgagt
94089DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 408tcagcacct 94099DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 409gctgcatgt 94109DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 410atgcagcct
94119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 411gagtggtgt 94129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 412accactcct 94139DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 413gactaccgt
94149DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 414ggtagtcct 94159DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 415cggtgatgt 94169DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 416atcaccgct
94179DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 417tgcgactgt
94189DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 418agtcgcact 94199DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 419tctggaggt 94209DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 420ctccagact
94219DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 421agcactggt 94229DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 422cagtgctct 94239DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 423tcgcttggt
94249DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 424caagcgact 94259DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 425agcactcgt 94269DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 426gagtgctct
94279DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 427gcgattggt 94289DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 428caatcgcct 94299DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 429ccatcgcgt
94309DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 430gcgatggct 94319DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 431tcgcttcgt 94329DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 432gaagcgact
94339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 433agtgcctgt 94349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 434aggcactct 94359DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 435ggcataggt
94369DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 436ctatgccct 94379DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 437gcgattcgt 94389DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 438gaatcgcct
94399DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 439tgcgacggt 94409DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 440cgtcgcact 94419DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 441gagtggcgt
94429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 442gccactcct 94439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 443cggtgaggt 94449DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 444ctcaccgct
94459DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 445gctgcaagt 94469DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 446ttgcagcct 94479DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 447ttccgctgt
94489DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 448agcggaact 94499DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 449gagtggagt 94509DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 450tccactcct
94519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 451acagagcgt 94529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 452gctctgtct 94539DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 453tgcgaccgt
94549DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 454ggtcgcact 94559DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 455cctgtaggt 94569DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 456ctacaggct
94579DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 457tagccgtgt 94589DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 458acggctact 94599DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 459tgcgacagt
94609DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 460tgtcgcact 94619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 461ggtctgtgt 94629DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 462acagaccct
94639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 463cggtgaagt 94649DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 464ttcaccgct 94659DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 465caacgaggt
94669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 466ctcgttgct 94679DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 467gcagcatgt 94689DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 468atgctgcct
94699DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 469tcgtcaggt 94709DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 470ctgacgact 94719DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 471agtgccagt
94729DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 472tggcactct 94739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 473tagaggcgt 94749DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 474gcctctact
94759DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 475gtcagcggt 94769DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 476cgctgacct 94779DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 477tcaggaggt
94789DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 478ctcctgact 94799DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 479agcaggtgt 94809DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 480acctgctct
94819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 481ttccgcagt 94829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 482tgcggaact 94839DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 483gtcagccgt
94849DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 484ggctgacct 94859DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 485ggtctgcgt 94869DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 486gcagaccct
94879DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 487tagccgagt 94889DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 488tcggctact 94899DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 489gtcagcagt
94909DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 490tgctgacct 94919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 491ggtctgagt 94929DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 492tcagaccct
94939DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 493cggacaggt 94949DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 494ctgtccgct 94959DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 495ttagccggt
94969DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 496cggctaact 94979DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 497gagacgagt 94989DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 498tcgtctcct
94999DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 499cgtaaccgt 95009DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 500ggttacgct 95019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 501ttggcgtgt
95029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 502acgccaact 95039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 503atggcaggt 95049DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 504ctgccatct
95059DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 505cagctacga 95069DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 506gtagctgac 95079DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 507ctcctgcga
95089DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 508gcaggagac 95099DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 509gctgcctga 95109DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 510aggcagcac
95119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 511caggaacga 95129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 512gttcctgac 95139DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 513cacacgcga
95149DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 514gcgtgtgac 95159DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 515gcagcctga 95169DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 516aggctgcac
95179DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 517ctgaacgga 95189DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 518cgttcagac 95199DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 519ctgaaccga
95209DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 520ggttcagac 95219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 521tctggacga 95229DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 522gtccagaac
95239DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 523tgcctacga 95249DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 524gtaggcaac 95259DNAArtificial SequenceDescription
of Artificial Sequence Synthetic
oligonucleotide 525ggcatacga 95269DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 526gtatgccac
95279DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 527cggtgacga 95289DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 528gtcaccgac 95299DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 529caacgacga
95309DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 530gtcgttgac 95319DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 531ctcctctga 95329DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 532agaggagac
95339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 533tcaggacga 95349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 534gtcctgaac 95359DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 535aaaggcgga
95369DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 536cgcctttac 95379DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 537ctcctcgga 95389DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 538cgaggagac
95399DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 539cagatgcga 95409DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 540gcatctgac 95419DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 541gcagcaaga
95429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 542ttgctgcac 95439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 543gtggagtga 95449DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 544actccacac
95459DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 545ccagtagga 95469DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 546ctactggac 95479DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 547atggcacga
95489DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 548gtgccatac 95499DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 549ggactgtga 95509DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 550acagtccac
95519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 551ccgaactga 95529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 552agttcggac 95539DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 553ctcctcaga
95549DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 554tgaggagac 95559DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 555cactgctga 95569DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 556agcagtgac
95579DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 557agcaggcga 95589DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 558gcctgctac 95599DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 559agcaggaga
95609DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 560tcctgctac 95619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 561agagccaga 95629DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 562tggctctac
95639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 563gtcgttgga 95649DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 564caacgacac 95659DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 565ccgaacgga
95669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 566cgttcggac 95679DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 567cactgcgga 95689DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 568cgcagtgac
95699DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 569gtggagcga 95709DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 570gctccacac 95719DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 571gtggagaga
95729DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 572tctccacac 95739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 573ggactgcga 95749DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 574gcagtccac
95759DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 575ccgaaccga 95769DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 576ggttcggac 95779DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 577cactgccga
95789DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 578ggcagtgac 95799DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 579cgaaacgga 95809DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 580cgtttcgac
95819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 581ggactgaga 95829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 582tcagtccac 95839DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 583ccgaacaga
95849DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 584tgttcggac 95859DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 585cgaaaccga 95869DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 586ggtttcgac
95879DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 587ctggcttga 95889DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 588aagccagac 95899DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 589cacacctga
95909DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 590aggtgtgac 95919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 591aacgaccga 95929DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 592ggtcgttac
95939DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 593atccagcga 95949DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 594gctggatac 95959DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 595tgcgaagga
95969DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 596cttcgcaac 95979DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 597tgcgaacga 95989DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 598gttcgcaac
95999DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 599ctggctgga 96009DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 600cagccagac 96019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 601cacaccgga
96029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 602cggtgtgac 96039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 603agtgcagga 96049DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 604ctgcactac
96059DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 605gaccgttga 96069DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 606aacggtcac 96079DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 607ggtgagtga
96089DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 608actcaccac 96099DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 609ccttcctga 96109DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 610aggaaggac
96119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 611ctggctaga 96129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 612tagccagac 96139DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 613cacaccaga
96149DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 614tggtgtgac 96159DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 615agcggtaga 96169DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 616taccgctac
96179DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 617gtcagagga 96189DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 618ctctgacac 96199DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 619ttccgacga
96209DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 620gtcggaaac 96219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 621aggcgtaga 96229DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 622tacgcctac
96239DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 623ctcgactga 96249DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 624agtcgagac 96259DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 625tacgctgga
96269DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 626cagcgtaac 96279DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 627gttcggtga 96289DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 628accgaacac
96299DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 629gccagcaga 96309DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 630tgctggcac 96319DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 631gaccgtaga
96329DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 632tacggtcac
96339DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 633gtgctctga 96349DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 634agagcacac 96359DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 635ggtgagcga
96369DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 636gctcaccac 96379DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 637ggtgagaga 96389DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 638tctcaccac
96399DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 639ccttccaga 96409DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 640tggaaggac 96419DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 641ctcctacga
96429DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 642gtaggagac 96439DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 643ctcgacgga 96449DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 644cgtcgagac
96459DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 645gccgtttga 96469DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 646aaacggcac 96479DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 647gcggagtga
96489DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 648actccgcac 96499DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 649cgtgcttga 96509DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 650aagcacgac
96519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 651ctcgaccga 96529DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 652ggtcgagac 96539DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 653agagcagga
96549DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 654ctgctctac 96559DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 655gtgctcgga 96569DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 656cgagcacac
96579DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 657ctcgacaga 96589DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 658tgtcgagac 96599DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 659ggagagtga
96609DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 660actctccac 96619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 661aggctgtga 96629DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 662acagcctac
96639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 663agagcacga 96649DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 664gtgctctac 96659DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 665ccatcctga
96669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 666aggatggac 96679DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 667gttcggaga 96689DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 668tccgaacac
96699DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 669tggtagcga 96709DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 670gctaccaac 96719DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 671gtgctccga
96729DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 672ggagcacac 96739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 673gtgctcaga 96749DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 674tgagcacac
96759DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 675gccgttgga 96769DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 676caacggcac 96779DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 677gagtgctga
96789DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 678agcactcac 96799DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 679gctccttga 96809DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 680aaggagcac
96819DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 681ccgaaagga 96829DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 682ctttcggac 96839DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 683cactgagga
96849DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 684ctcagtgac 96859DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 685cgtgctgga 96869DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 686cagcacgac
96879DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 687ccgaaacga 96889DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 688gtttcggac 96899DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 689gcggagaga
96909DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 690tctccgcac 96919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 691gccgttaga 96929DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 692taacggcac
96939DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 693tctcgtgga 96949DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 694cacgagaac 96959DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 695cgtgctaga
96969DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 696tagcacgac 96979DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 697gcctgtctt 96989DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 698gacaggctc
96999DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 699ctcctggtt 97009DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 700ccaggagtc 97019DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 701actctgctt
97029DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 702gcagagttc 97039DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 703catcgcctt 97049DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 704ggcgatgtc
97059DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 705gccactatt 97069DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 706tagtggctc 97079DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 707cacacggtt
97089DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 708ccgtgtgtc 97099DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 709caacgcctt 97109DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 710ggcgttgtc
97119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 711actgaggtt 97129DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 712cctcagttc 97139DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 713gtgctggtt
97149DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 714ccagcactc 97159DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 715catcgactt 97169DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 716gtcgatgtc
97179DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 717ccatcggtt 97189DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 718ccgatggtc 97199DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 719gctgcactt
97209DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 720gtgcagctc 97219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 721acagaggtt 97229DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 722cctctgttc
97239DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 723agtgccgtt 97249DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 724cggcacttc 97259DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 725cggacattt
97269DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 726atgtccgtc 97279DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 727ggtctggtt 97289DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 728ccagacctc
97299DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 729gagacggtt 97309DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 730ccgtctctc 97319DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 731ctttccgtt
97329DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 732cggaaagtc 97339DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 733cagatggtt 97349DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 734ccatctgtc
97359DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 735cggacactt 97369DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 736gtgtccgtc 97379DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 737actctcgtt
97389DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 738cgagagttc 97399DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 739gcagcactt 97409DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide
740gtgctgctc 97419DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 741actctcctt 97429DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 742ggagagttc 97439DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 743accttggtt
97449DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 744ccaaggttc 97459DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 745agagccgtt 97469DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 746cggctcttc
97479DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 747accttgctt 97489DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 748gcaaggttc 97499DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 749aagtccgtt
97509DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 750cggactttc 97519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 751ggactggtt 97529DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 752ccagtcctc
97539DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 753gtcgttctt 97549DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 754gaacgactc 97559DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 755cagcatctt
97569DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 756gatgctgtc 97579DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 757ctatccgtt 97589DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 758cggatagtc
97599DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 759acactcgtt 97609DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 760cgagtgttc 97619DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 761atccaggtt
97629DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 762cctggattc 97639DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 763gttcctgtt 97649DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 764caggaactc
97659DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 765acactcctt 97669DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 766ggagtgttc 97679DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 767gttcctctt
97689DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 768gaggaactc 97699DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 769ctggctctt 97709DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 770gagccagtc
97719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 771acggcattt 97729DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 772atgccgttc 97739DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 773ggtgaggtt
97749DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 774cctcacctc 97759DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 775ccttccgtt 97769DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 776cggaaggtc
97779DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 777tacgctctt 97789DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 778gagcgtatc 97799DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 779acggcagtt
97809DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 780ctgccgttc 97819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 781actgacgtt 97829DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 782cgtcagttc
97839DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 783acggcactt 97849DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 784gtgccgttc 97859DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 785actgacctt
97869DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 786ggtcagttc 97879DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 787tttgcggtt 97889DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 788ccgcaaatc
97899DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 789tggtaggtt 97909DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 790cctaccatc 97919DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 791gttcggctt
97929DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 792gccgaactc 97939DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 793gccgttctt 97949DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 794gaacggctc
97959DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 795ggagaggtt 97969DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 796cctctcctc 97979DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 797cactgactt
97989DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 798gtcagtgtc 97999DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 799cgtgctctt 98009DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 800gagcacgtc
98019DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 801aatccgctt 98029DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 802gcggatttc 98039DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 803aggctggtt
98049DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 804ccagccttc 98059DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 805gctagtgtt 98069DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 806cactagctc
98079DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 807ggagagctt 98089DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 808gctctcctc 98099DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 809ggagagatt
98109DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 810tctctcctc 98119DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 811aggctgctt 98129DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 812gcagccttc
98139DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 813gagtgcgtt 98149DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 814cgcactctc 98159DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 815ccatccatt
98169DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 816tggatggtc 98179DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 817gctagtctt 98189DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 818gactagctc
98199DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 819aggctgatt 98209DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 820tcagccttc 98219DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 821acagacgtt
98229DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 822cgtctgttc 98239DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 823gagtgcctt 98249DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 824ggcactctc
98259DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 825acagacctt 98269DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 826ggtctgttc 98279DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 827cgagctttt
98289DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 828aagctcgtc 98299DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 829ttagcggtt 98309DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 830ccgctaatc
98319DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 831cctcttgtt 98329DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 832caagaggtc 98339DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 833ggtctcttt
98349DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 834agagacctc 98359DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 835gccagattt 98369DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 836atctggctc
98379DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 837gagaccttt 98389DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 838aggtctctc 98399DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 839cacacagtt
98409DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 840ctgtgtgtc 98419DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 841cctcttctt 98429DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 842gaagaggtc
98439DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 843tagagcgtt 98449DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 844cgctctatc 98459DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 845gcacctttt
98469DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 846aaggtgctc 98479DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 847ggcttgttt 98489DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 848acaagcctc 98499DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 849gacgcgatt
98509DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 850tcgcgtctc 98519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 851cgagctgtt 98529DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 852cagctcgtc
98539DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 853tagagcctt 98549DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 854ggctctatc 98559DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 855catccgttt
98569DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 856acggatgtc 98579DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 857ggtctcgtt 98589DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 858cgagacctc
98599DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 859gccagagtt 98609DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 860ctctggctc 98619DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 861gagaccgtt
98629DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 862cggtctctc 98639DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 863cgagctatt 98649DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 864tagctcgtc
98659DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 865gcaagtgtt 98669DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 866cacttgctc 98679DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 867ggtctcctt
98689DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 868ggagacctc 98699DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 869gccagactt 98709DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 870gtctggctc
98719DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 871ggtctcatt 98729DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 872tgagacctc 98739DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 873gagaccatt
98749DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 874tggtctctc 98759DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 875ccttcagtt 98769DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 876ctgaaggtc
98779DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 877gcacctgtt 98789DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 878caggtgctc 98799DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 879aaaggcgtt
98809DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 880cgccttttc 98819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 881cagatcgtt 98829DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 882cgatctgtc
98839DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 883cataggctt 98849DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 884gcctatgtc 98859DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 885ccttcactt
98869DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 886gtgaaggtc 98879DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 887gcacctctt 98889DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 888gaggtgctc
988925DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 889cagaagacag acaagcttca cctgc
2589027DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 890gcaggtgaag cttgtctgtc ttctgaa
2789119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 891gcctccctcg cgccatcag 1989219DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
892gccttgccag cccgctcag 1989310DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 893cagcgttcga
1089435DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 894gcaggtgaag cttgtctgnn nnntcgaacg ctgaa
3589533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 895cagcgttcga nnnnncagac aagcttcacc tgc
3389635DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 896gcaggtgaag cttgtctgnn nnntcgaacg ctgaa
3589747DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 897gcctccctcg cgccatcaga bbbabbbabb bagcatcgaa
cgctgaa 4789839DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 898gccttgccag cccgctcaga tgactcccaa
atcgatgtg 3989939DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 899gccttgccag cccgctcagc tgactcccaa
atcgatgtg 3990039DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 900gccttgccag cccgctcagg tgactcccaa
atcgatgtg 3990139DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 901gccttgccag cccgctcagt tgactcccaa
atcgatgtg 3990240DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 902gccttgccag cccgctcaga atgactccca
aatcgatgtg 4090340DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 903gccttgccag cccgctcaga ctgactccca
aatcgatgtg 4090440DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 904gccttgccag cccgctcaga gtgactccca
aatcgatgtg 4090540DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 905gccttgccag cccgctcaga ttgactccca
aatcgatgtg 4090640DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 906gccttgccag cccgctcagc atgactccca
aatcgatgtg 4090738DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 907gtctgttcga agtggacgag actaccgcgc
tccctccg 3890838DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 908gtctgttcga agtggacgcg actaccgcgc
tccctccg 3890938DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 909gtctgttcga agtggacggg actaccgcgc
tccctccg 3891038DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 910gtctgttcga agtggacgtg actaccgcgc
tccctccg 3891139DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 911gtctgttcga agtggacgaa gactaccgcg
ctccctccg 3991239DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 912gtctgttcga agtggacgca gactaccgcg
ctccctccg 3991339DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 913gtctgttcga agtggacgga gactaccgcg
ctccctccg 3991439DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 914gtctgttcga agtggacgta gactaccgcg
ctccctccg 3991539DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 915gtctgttcga agtggacgac gactaccgcg
ctccctccg 3991619DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 916tgactcccaa atcaatgtg
1991717DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 917cattgatttg ggagtca
1791821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 918ggcacattga tttgggagtc a 2191919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
919tgactcccaa atcaatgtg 199205PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 920Gly Pro Phe Xaa Gly1
592125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 921agtctggtac agggtgttct tttta
2592229DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 922accgtaaaaa gaacaccctg taccagact
2992312DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 923cggtggctgg ag 1292412DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 924aaggctccag cc 1292537DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 925agtctggtac agggtgttct ttttacggtg gctggag
3792641DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 926aaggctccag ccaccgtaaa aagaacaccc
tgtaccagac t 4192731DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 927cggaaacggg taccctaaaa
agaacaccct g 3192840DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 928agtctggtac agggtgttct
ttttagggta cccgtttccg 4092940DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 929cggaaacggg
taccctaaaa agaacaccct gtaccagact 40
* * * * *