U.S. patent application number 13/636668 was filed with the patent office on 2013-10-31 for method for making an enriched library.
This patent application is currently assigned to Vipergen. The applicant listed for this patent is Peter Blakskj.ae butted.r, Allan Beck Christensen, Nils Jakob Vest Hansen, Tara Heitner Hansen, Johan Holmkvist, Leif Kongskov Larsen, Lars Kolster Petersen, Judith Rasmussen-Dietvorst, Frank Abildgaard Slok. Invention is credited to Peter Blakskj.ae butted.r, Allan Beck Christensen, Nils Jakob Vest Hansen, Tara Heitner Hansen, Johan Holmkvist, Leif Kongskov Larsen, Lars Kolster Petersen, Judith Rasmussen-Dietvorst, Frank Abildgaard Slok.
Application Number | 20130288929 13/636668 |
Document ID | / |
Family ID | 43629282 |
Filed Date | 2013-10-31 |
United States Patent
Application |
20130288929 |
Kind Code |
A1 |
Hansen; Nils Jakob Vest ; et
al. |
October 31, 2013 |
Method for Making an Enriched Library
Abstract
A method for making an enriched library comprising specific
nucleic acid sequence information allowing to identifying at least
one binding entity that binds to at least one target wherein the
specific binding entity has been present in an in vitro display
library.
Inventors: |
Hansen; Nils Jakob Vest;
(Copenhagen, DK) ; Christensen; Allan Beck;
(Copenhagen, DK) ; Larsen; Leif Kongskov;
(Copenhagen, DK) ; Slok; Frank Abildgaard;
(Copenhagen, DK) ; Petersen; Lars Kolster;
(Copenhagen, DK) ; Rasmussen-Dietvorst; Judith;
(Copenhagen, DK) ; Blakskj.ae butted.r; Peter;
(Copenhagen, DK) ; Hansen; Tara Heitner;
(Copenhagen, DK) ; Holmkvist; Johan; (Copenhagen,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hansen; Nils Jakob Vest
Christensen; Allan Beck
Larsen; Leif Kongskov
Slok; Frank Abildgaard
Petersen; Lars Kolster
Rasmussen-Dietvorst; Judith
Blakskj.ae butted.r; Peter
Hansen; Tara Heitner
Holmkvist; Johan |
Copenhagen
Copenhagen
Copenhagen
Copenhagen
Copenhagen
Copenhagen
Copenhagen
Copenhagen
Copenhagen |
|
DK
DK
DK
DK
DK
DK
DK
DK
DK |
|
|
Assignee: |
Vipergen
Copenhagen
DK
|
Family ID: |
43629282 |
Appl. No.: |
13/636668 |
Filed: |
September 1, 2011 |
PCT Filed: |
September 1, 2011 |
PCT NO: |
PCT/EP2011/065117 |
371 Date: |
September 21, 2012 |
Current U.S.
Class: |
506/26 |
Current CPC
Class: |
C12N 15/1075 20130101;
C12N 15/11 20130101; C12N 15/1068 20130101 |
Class at
Publication: |
506/26 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 2010 |
EP |
10180435.9 |
Claims
1. A method for making an enriched library comprising specific
nucleic acid sequence information allowing to identifying at least
one binding entity that binds to at least one target wherein the
specific binding entity has been present in an in vitro display
library and wherein the method comprises the steps of: (i): making
an in vitro display library of at least 100 different binding
entities, wherein each binding entity is attached to a nucleic acid
molecule and the nucleic acid molecule comprises specific nucleic
acid sequence information allowing to identify the binding entity
once one knows the specific nucleic acid sequence information of
the nucleic acid molecule one directly knows the structure of the
specific binding entity attached to the nucleic acid molecule--the
structure of the binding entity, attached to the nucleic acid
molecule, is herein termed B-structure; (ii): making nucleic acid
molecules with at least one target T.sub.n attached to a nucleic
acid molecule and the nucleic acid molecule comprises specific
nucleic acid sequence information allowing to identify the specific
target, wherein the target is capable of binding to at least one of
the binding entities present in the library of step (i)--the
structure of the target attached to the nucleic acid molecule is
herein termed T-structure; and wherein the method is characterized
by that: (iii): mixing a solution comprising X, wherein X is a
number greater than 10.sup.4, numbers of B-structures of the
library of step (i) with a solution comprising Y, wherein Y is a
number greater than 10.sup.2, numbers of T-structures of step (ii)
under binding conditions, i.e. conditions where a B-structure
containing a binding entity capable of binding to a target
molecule, binds more efficiently to the corresponding T-structure,
than a B-structure containing a binding entity not capable of
binding to the same target do and wherein one gets binding of at
least one of the binding entities to at least one target thereby
creating a complex comprising a B-structure bound to a T-structure,
herein termed B.sub.BoundToT-structure; (iv): applying an in vitro
compartmentalization system--under binding conditions, i.e.
conditions where a B-structure containing a binding entity capable
of binding to a target molecule, binds more efficiently to the
corresponding T-structure, than a B-structure containing a binding
entity not capable of binding to the same target do--wherein the
compartmentalization system comprises at least 2 times more
individual compartments than the Y number of T-structures present
in step (iii) under conditions wherein the B-structures,
T-structures and B.sub.BoundToT-structures enter randomly into the
individual compartments; and (v): fusing the nucleic acid molecules
of a B-structure and a T-structure which are both present within
the same individual compartment--i.e. fusing the nucleic acid
molecule of the B-structure to the nucleic acid molecule of the
T-structure--this structure is herein termed BT.sub.Fused-structure
and the BT.sub.Fused-structure comprises the specific nucleic acid
sequence information allowing to identify the binding entity of
step (i) and the specific nucleic acid sequence information
allowing to identify the specific target of step (ii); and (vi):
combining the content of the individual compartments of step (v)
under conditions wherein there is no fusing of the nucleic acid
molecules of a B-structure and a T-structure--i.e. there is not
created any new BT.sub.Fused-structure not already created in step
(v)--in order to get a library of BT.sub.Fused-structures, wherein
the library is an enriched library of species of
BT.sub.Fused-structures originating from binding pairs of target
and binder entity when compared to BT.sub.Fused-structures
originating from nonbinding pairs of target and binder entity; and
wherein the method is further characterized by at least one of (a)
or (b) below: (a) B.sub.BoundToT-structures remain suspended in
solution in the individual compartments of step (iv) of the first
aspect; (b) the method does not rely on target immobilization on a
solid support.
2. The method of claim 1, wherein the binding entity of step (i) is
attached to the nucleic acid molecule by a covalent binding and
wherein the target of step (ii) is attached to the nucleic acid
molecule by a covalent binding and wherein the nucleic acid
molecule of the B-structure is DNA and the nucleic acid molecule of
the T-structure is DNA.
3. The method of claim 2, wherein the DNA nucleic acid molecule in
the B-structure is a double stranded nucleic acid molecule and
wherein the DNA nucleic acid molecule in the T-structure is a
double stranded nucleic acid molecule.
4. The method of claim 1, wherein the nucleic acid molecule
attached to the binding entity in the B-structure contains a PCR
priming site and wherein the nucleic acid molecule attached to the
target in the T-structure contains a PCR priming site.
5. The method of claim 1, wherein the in vitro library of step (i)
comprises at least 10.sup.5 different binding entities and wherein
the binding entities of step (i) are chemical compounds with an
average molecular weight MW below 5000 dalton.
6. The method of claim 1, wherein there is at least two different
targets in step (ii).
7. The method of claim 1, wherein at least one target is a
protein.
8. The method of claim 1, wherein there in step (iii) is at least
10.sup.5 copies of a T-structure of interest, "Y" is at least
10.sup.5 and wherein the concentration of T-structures in the
"mixing step (iii)" is at least 10.sup.-9 M.
9. The method of claim 1, wherein step (iii) is performed under
binding conditions, wherein a B-structure containing a binding
entity capable of binding to a target molecule, binds 100 fold more
efficiently to the corresponding T-structure, than a B-structure
containing a binding entity not capable of binding to the same
target do.
10. The method of claim 1, wherein said method comprises an
additional step (iii-b) that is performed before the step (iv),
comprising: (iii-b): diluting the solution of step (iii) at least
100 fold under binding conditions, i.e. conditions where a
B-structure containing a binding entity capable of binding to a
target molecule, binds more efficiently to the corresponding
T-structure, than a B-structure containing a binding entity not
capable of binding to the same target do.
11. The method of claim 1, wherein there in step (iv) is at least
100 times more individual compartments than the Y number of
T-structures present in step (iii) and wherein there in step (iv)
there is at least the square root of 10, 3.16, times more
individual compartments than the X number of B-structures in step
(iii).
12. The method of claim 1, wherein the in vitro
compartmentalization system of step (iv) is a water-in-oil emulsion
system and wherein the average compartments volume is less than
10.sup.-12 liter.
13. The method of claim 1, wherein the fusing of the nucleic acid
molecules of a B-structure and a T-structure which are both present
within the same individual compartment of step (v) is: (a): done by
a DNA ligase where a phosphodiester bond between a 3'-OH and a
5'-phosphate groups is formed; or (b): by using a DNA polymerase
using emulsion PCR.
14. The method of claim 1, wherein the in vitro
compartmentalization system of step (iv) is a water-in-oil emulsion
and wherein the content of the individual compartments of step (v)
is combined in step (vi) by a method, wherein the oil compartments
are disrupted.
15: The method of claim 1, wherein there is an extra step (vii),
wherein the BT.sub.Fused-structures present in the enriched library
of step (vi) is amplified by PCR and thereafter subjected to DNA
sequencing to identify at least one individual binding entity that
binds to at least one target of interest.
16: The method of claim 1, wherein there is an extra step (vii)
comprising use the enriched library of step (vi) to identify at
least one individual binding entity that binds to at least one
target of interest.
17-18. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for making an
enriched library comprising specific nucleic acid sequence
information allowing to identifying at least one binding entity
that binds to at least one target wherein the specific binding
entity has been present in an in vitro display library.
BACKGROUND
[0002] Display technologies have been developed to combine
information storage and amplification capabilities of nucleic acids
with the functional activities of other compound. Display
technologies rely on an association between a functional binding
entity (i.e. phenotype) and a nucleic acid sequence informative
(genotype) about the structure of the binding entity. Note: Nucleic
acid aptamer technology is considered a display technology although
a special case as the pheno- and genotype consist of the same
molecule (DNA or RNA).
[0003] An advantage of such methods is that very large libraries
can be constructed and probed for a desired activity of the
functional binding entities. Library members having the desired
activity can then be partitioned from library members not having
the desired activity, thus creating an enriched library with a
higher fraction of members having the desired activity. This
process is called selection or enrichment. Some display
technologies allows for rounds of selections, where the enriched
library from one round is amplified and used to prepare a new
enriched display library and used in a next round of selection and
so forth. The structures of the library members in the enriched
library can then be identified by their cognate nucleic acid
sequence, thus allowing identification even from minute amounts of
material.
[0004] Herein relevant libraries may according to the art be termed
"in vitro display libraries".
[0005] The term "in vitro display library" shall herein be
understood according to the art--i.e. as a library comprising
numerous different binding entities wherein each binding entity is
attached to a nucleic acid molecule and the nucleic acid molecule
comprises specific nucleic acid sequence information allowing to
identify the binding entity--i.e. once one knows the specific
nucleic acid sequence information of the nucleic acid molecule one
directly knows the structure of the specific binding entity
attached to the nucleic acid molecule--the structure of the binding
entity (i.e. phenotype) attached to the nucleic acid molecule
(genotype) is herein termed B-structure.
[0006] The prior art describes a number of different methods to
make such in vitro display libraries--herein suitable examples
include e.g. EP1809743B1 (Vipergen), EP1402024B1 (Nuevolution),
EP1423400B1 (David Liu), Nature Chem. Biol. (2009), 5:647-654
(Clark), WO 00/23458 (Harbury), Nature Methods (2006), 3(7),
561-570, 2006 (Miller), Nat. Biotechnol. 2004; 22, 568-574
(Melkko), Nature. (1990); 346(6287), 818-822 (Ellington), or Proc
Natl Acad Sci USA (1997). 94 (23): 12297-302 (Roberts),
WO06053571A2 (Rasmussen).
[0007] As described in e.g. above mentioned prior art--one can
today make in vitro display libraries comprising very many (e.g.
10.sup.15) specific binding entities (e.g. 10.sup.15 different
chemical compounds).
[0008] In view of this--it is evident that it would be very
interesting to be able to improve the selection/enrichment step of
such libraries to make an enriched library--e.g. to more efficient
be able to identify the structure of a specific binding entity
(e.g. a chemical compound) that binds to a target of interest (e.g.
a medical important receptor molecule).
[0009] In FIG. 3 herein is shown an example of the in vitro display
technology as described in EP1809743B1 (Vipergen)--as can be seen
in this FIG. 3--the selection step of this example is performed by
immobilizing the target (e.g. a receptor) to a solid surface (e.g.
a bead or a glass plate).
[0010] Without being limited to theory--to our knowledge, the
example in FIG. 3 herein may be seen as an example of herein
relevant in vitro display technology prior art (e.g. above
mentioned prior art)--i.e. to our knowledge the selection for
suitable binding entities present within in vitro display libraries
are in the prior art generally done by immobilizing the target
(e.g. a receptor) to a solid support (e.g. a glass plate, a column,
a bead, a nitrocellulose filter, a cell etc) before or after the
display library binding event. Non-binders and low affinity binders
are typically washed away, whereas the population enriched for
binders are recovered from the solid support.
[0011] In prior art in vitro compartmentalization (IVC) have been
described employed in technologies utilizing phenotype and genotype
linkage for interrogating libraries. These prior art technologies
can be divided into two groups: a) IVC utilized for facilitating
establishing correct phenotype and genotype linkage, which allows
for selection of function (e.g. specific target binding) later
(post compartment disruption), and b) IVC for facilitating
establishing correct phenotype and genotype linkage based on an
activity of the phenotype inside the compartment, i.e in a
compartment a gene is transcribed and translated and the resulting
protein's function inside the compartment is used directly or
indirectly for sorting, survival or amplification.
[0012] In other words herein relevant so-called IVC prior art
technologies--may be described as a:
group a)--wherein the phenotype activity is interrogated AFTER the
compartmentalized step; or group b)--wherein the phenotype activity
is interrogated DURING the compartmentalized step.
[0013] Examples of IVC prior art belonging to group a): [0014]
Bertschinger et al, (2007) Protein Engineering, Design &
Selection vol. 20 no. 2 pp. 57-68; [0015] Miller O J, Bernath K,
Agresti J J, Amitai G, Kelly B T, Mastrobattista E, Taly V,
Magdassi S, Tawfik D S, Griffiths A D. Directed evolution by in
vitro compartmentalization. Nat Methods. 2006 July; 3(7):561-70;
[0016] Doi, N. and Yanagawa, H. (1999) FEBS Lett., 457, 227-230;
and Yonezawa, M., Doi, N., Kawahashi, Y., Higashinakagawa, T. and
Yanagawa, H. (2003) Nucleic Acids Res., 31, e118.
[0017] Examples of IVC prior art belonging to group b): [0018]
Tawfik, D. S, and Griffiths, A. D. (1998) Man-made cell-like
compartments for molecular evolution. Nat. Biotechnol., 16,
652-656; [0019] Ghadessy, F. J., Ong, J. L. and Holliger, P. (2001)
Proc. Natl. Acad. Sci. USA, 98, 4552-4557; [0020] Tay Y, Ho C,
Droge P, Ghadessy F J. Selection of bacteriophage lambda integrases
with altered recombination specificity by in vitro
compartmentalization. Nucleic Acids Res. 2010 March; 38(4):e25.
Epub 2009 Dec. 4; [0021] Zheng Y, Roberts R J. Selection of
restriction endonucleases using artificial cells. Nucleic Acids
Res. 2007; 35(11):e83. Epub 2007; [0022] Mastrobattista E, Taly V,
Chanudet E, Treacy P, Kelly B T, Griffiths A D. High-throughput
screening of enzyme libraries: in vitro evolution of a
beta-galactosidase by fluorescence-activated sorting of double
emulsions. Chem. Biol. 2005 December; 12(12): 1291-300; [0023] Levy
M, Griswold K E, Ellington A D. Direct selection of trans-acting
ligase ribozymes by in vitro compartmentalization. RNA. 2005
October; 11(10):1555-62. Epub 2005 Aug. 30; [0024] Sepp A, Choo Y.
Cell-free selection of zinc finger DNA-binding proteins using in
vitro compartmentalization. J Mol Biol. 2005 Nov. 25; 354(2):212-9.
Epub 2005 Oct. 3; [0025] Bernath K, Magdassi S, Tawfik D S.
Directed evolution of protein inhibitors of DNA-nucleases by in
vitro compartmentalization (IVC) and nano-droplet delivery. J Mol
Biol. 2005 Feb. 4; 345(5):1015-26. Epub 2004 Dec. 7.
[0026] Examples of further IVC prior art may be found in: [0027]
Bertschinger et al, (2004) Protein Engineering, Design &
Selection vol. 20 no. 2 pp. 699-707; [0028] Chen Yu et al,
(November 2008) Nucleic Acid Research, Vol. 36, Nr. 19, Pages:
Article No. E128; [0029] Hansen et al. J. Am. Chem. Soc., 2009, 131
(3), pp 1322-1327.
SUMMARY OF THE INVENTION
[0030] The problem to be solved by the present invention may be
seen as to provide an improved in vitro display based method in
order to make an enriched library comprising at least one binding
entity (e.g. a chemical compound) that binds to a target of
interest (e.g. a medical relevant receptor).
[0031] In many cases, most notably in the development of
therapeutics, two parameters for a binding entity (drug) are
especially important, namely the potency (affinity) and the off
rate (dissociative half-life of drug:target complex). The present
invention provides an improved solution for in vitro display
methods to enrich for both these important binding parameters.
[0032] In other cases, the on-rate characteristic for a binding
identity is desired. The present invention provides an improved
solution for in vitro display methods to enrich for on-rate
characteristic for a binding identity.
[0033] The solution may be seen as based on that:
(i): making an in vitro display library of binding entities (i.e.
phenotype) attached to nucleic acid molecules (genotype)--this step
may be made according to known prior art techniques for making such
in vitro display libraries; (ii): making structures comprising
target (i.e. phenotype) attached to a nucleic acid molecule
(genotype)--this step may be made according to known prior art
techniques for making such structures; and wherein the method as
described herein may be seen as characterized by that: (iii): the
binding step is performed in solution (e.g. under aqueous
conditions); (iv): there is used a suitable in vitro
compartmentalization system (e.g. a water-in-oil emulsion system)
creating more individual compartments than target molecules; (v):
fuse co-compartmentalized target and binding entity genotypes;
(vi): de-compartmentalize; to get an enrichment of fused genotypes
(positive binders in the in vitro display library will have a
higher propensity for being fused than none-binders); and (vii):
optionally e.g. purify and/or preferential amplify the fused
genotypes.
[0034] Based on the detailed description herein and the common
general knowledge--the skilled person may perform the steps (iii)
to steps (vi) in a number of different ways.
[0035] Step (vii) is an optional step--as described herein once one
has obtained the enriched library of step (vi) one may use this
library in different ways according to art--e.g. the enriched
library may be considered as an enriched in vitro display library
that e.g. can be used in a second round of selection/enrichment or
one may identify the structure of a specific binding entity of
interest directly from the enriched library of step (vi).
[0036] As discussed above--herein relevant so-called IVC prior art
technologies--may be described as a:
group a)--wherein the phenotype activity is interrogated AFTER the
compartmentalized step; or group b)--wherein the phenotype activity
is interrogated DURING the compartmentalized step.
[0037] As evident from above and as further discussed herein--the
method as described herein is conceptionally different from such
so-called IVC prior art technologies--e.g. due to that the
phenotype activity is interrogated in step (iii) of first aspect,
which is BEFORE the compartmentalized step (iv) of first
aspect.
[0038] A simple way to explain the principle of the novel method as
described herein, is that non-binders in the display library is
randomly distributed in the compartments and therefore
co-compartmentalize with the target in a random fashion, with a
frequency depending on the ratio between the number of compartments
and the number of target molecules. In contrast, binders, due to
the binding activity, will co-compartmentalize together with target
molecules--independently of the ratio between the number of
compartments and the number of target molecules. Consequently,
enrichment of a binder is achieved when the ratio between the
number of compartments and the number of target molecules is larger
than 1--the higher ratio the higher enrichment.
[0039] In FIGS. 1 and 2 herein are provided illustrative examples
of the novel method as described herein.
[0040] In working examples 1 and 2 herein are provided an example
with herein relevant numbers of e.g. binding entities and target of
interest--as can be seen in the conclusion of the examples 1 and
2--by using the method as described herein one may get e.g. an 1000
times enrichment of binders in the library.
[0041] Accordingly, a first aspect of the invention relates to a
method a method for making an enriched library comprising specific
nucleic acid sequence information allowing to identifying at least
one binding entity that binds to at least one target wherein the
specific binding entity has been present in an in vitro display
library and wherein the method comprises the steps of:
(i): making an in vitro display library of at least 100 different
binding entities (B.sub.n (n=100 or more), wherein each binding
entity is attached to a nucleic acid molecule and the nucleic acid
molecule comprises specific nucleic acid sequence information
allowing to identify the binding entity--i.e. once one knows the
specific nucleic acid sequence information of the nucleic acid
molecule one directly knows the structure of the specific binding
entity attached to the nucleic acid molecule--the structure of the
binding entity (i.e. phenotype) attached to the nucleic acid
molecule (genotype) is herein termed B-structure; (ii): making
nucleic acid molecules with at least one target T.sub.n (n=1 or
more) attached to a nucleic acid molecule and the nucleic acid
molecule comprises specific nucleic acid sequence information
allowing to identify the specific target, wherein the target is
capable of binding to at least one of the binding entities present
in the library of step (i)--the structure of the target (i.e.
phenotype) attached to the nucleic acid molecule (genotype) is
herein termed T-structure; and wherein the method is characterized
by that: (iii): mixing a solution comprising X (X is a number
greater than 10.sup.4) numbers of B-structures of the library of
step (i) with a solution comprising Y (Y is a number greater than
10.sup.2) numbers of T-structures of step (ii) under binding
conditions, i.e. conditions where a B-structure containing a
binding entity capable of binding to a target molecule, binds more
efficiently to the corresponding T-structure, than a B-structure
containing a binding entity not capable of binding to the same
target do and wherein one gets binding of at least one of the
binding entities to at least one target thereby creating a complex
comprising a B-structure bound to a T-structure (herein termed
B.sub.BoundToT-structure); (iv): applying an in vitro
compartmentalization system--under binding conditions, i.e.
conditions where a B-structure containing a binding entity capable
of binding to a target molecule, binds more efficiently to the
corresponding T-structure, than a B-structure containing a binding
entity not capable of binding to the same target do--wherein the
compartmentalization system comprises at least 2 times more
individual compartments than the Y number of T-structures present
in step (iii) under conditions wherein the B-structures,
T-structures and B.sub.BoundToT-structures enter randomly into the
individual compartments; and (v): fusing the nucleic acid molecules
of a B-structure and a T-structure which are both present within
the same individual compartment--i.e. fusing the nucleic acid
molecule of the B-structure to the nucleic acid molecule of the
T-structure--this structure is herein termed BT.sub.Fused-structure
and the BT.sub.Fused-structure comprises the specific nucleic acid
sequence information allowing to identify the binding entity of
step (i) and the specific nucleic acid sequence information
allowing to identify the specific target of step (ii); and (vi):
combining the content of the individual compartments of step (v)
under conditions wherein there is no fusing of the nucleic acid
molecules of a B-structure and a T-structure--i.e. there is not
created any new BT.sub.Fused-structure not already created in step
(v)--in order to get a library of BT.sub.Fused-structures, wherein
the library is an enriched library of species of
BT.sub.Fused-structures originating from binding pairs of target
and binder entity when compared to BT.sub.Fused-structures
originating from nonbinding pairs of target and binder entity.
[0042] The method of the first aspect as described herein may be
termed Enrichment by Co-Compartmentalization (ECC).
[0043] Advantageous in ECC method as described herein is that
enrichment for important binding characteristics can be optimized
for in isolation--because ECC is a homogenous assay--target is not
immobilization to a solid support. Prior art methods are
heterogenous--rely on target immobilization to a solid support
(e.g. beads, columns, cells, plastic, filters etc). Heterogenous
assays are notoriously more difficult to control than homogenous
assay due e.g. avidity effects, density of coating, and
interference of the solid support itself with the assay.
[0044] As discussed above, in herein relevant in vitro display
technology prior art (e.g. above mentioned prior art)--selection
for suitable binding entities present within in vitro display
libraries are in the prior art generally done by immobilizing the
target (e.g. a receptor) to a solid support (e.g. a glass plate, a
column, a bead, a nitrocellulose filter, a cell etc) before or
after the display library binding event. Non-binders and low
affinity binders are typically washed away, whereas the population
enriched for binders are recovered from the solid support.
[0045] Accordingly, as understood by the skilled person, when there
above is said that herein relevant prior art methods "rely on
target immobilization to a solid support" is it understood by the
skilled person in the way that the selection for suitable binding
entities relies on this immobilization of target to a solid support
as an essential element to get the selection for suitable binding
entities.
[0046] As evident to the skilled person, the method of the first
aspect is not such a prior at method that rely on target
immobilization to a solid support, since the selection of the
binding entities is based on the separation of the
B.sub.BoundToT-structures into the individual compartments as
required in step (iv) of the first aspect.
[0047] In line of above and as understood by the skilled person--in
the method of the first aspect one could theoretically image a
situation, wherein the target T-structure of step (ii) would e.g.
comprise a bead. It could theoretically be a T-structure, wherein
the target is bound to a bead and the nucleic acid molecule that
comprises the specific nucleic acid sequence information allowing
identifying the specific target of the T-structure of step (ii) is
then also bound to the bead.
[0048] As evident to the skilled person--such a special T-structure
comprising a bead will not change the fact that the method of the
first aspect is not a method, wherein the selection for suitable
binding entities relies on this immobilization of target to a solid
support.
[0049] In line of above and as understood by the skilled person in
the present context, the method of the first aspect may be seen as
a method which implies that the B.sub.BoundToT-structures (i.e. the
target-binding entity complexes) remain suspended in solution in
the individual/separated compartments of step (iv) of the first
aspect.
[0050] ECC allows optimizing for major binding characteristic for
binding of binding entity to target in isolation. For example
potency (affinity), association rate (on rate) or dissociative
half-life of binding entity and target (off rate).
[0051] Affinity based selection is achieved by using equilibrium
conditions and controlled by the target concentration in the mixing
step (binding step), i.e. 90% of the molecules of a binding entity
in the display library having a K.sub.d equal to 10 times smaller
than the target concentration are target bound, whereas 50% of the
molecules of a binding entity having a K.sub.d equal to the target
concentration are, and 10% of the molecules of a binding entity
having a K.sub.d 10 times smaller than the target concentration
are. Consequently, enrichment for affinity is easily controlled by
the target concentration in the mixing step.
[0052] A separate aspect of the invention relates to an enriched
library of step (vi) of the first aspect and which is obtainable by
the method of the first aspect or herein related embodiments of the
first aspect.
[0053] Embodiments of the present invention are described below, by
way of examples only.
DRAWINGS
[0054] FIG. 1: Illustrative example of the principle of the
principle of the method as described herein.
[0055] FIG. 2: Illustrative example of the principle of the
principle of the method as described herein--it is an illustrative
example wherein emulsion PCR is used in the fusion step (v) of the
first aspect.
[0056] FIG. 3: Herein is shown an example of the in vitro display
technology as described in EP1809743B1 (Vipergen)--as can be seen
in this FIG. 3--the selection step of this example is performed by
immobilizing the target (e.g. a receptor) to a solid surface (e.g.
a bead or a glass plate).
[0057] FIGS. 4-7: These figures are further discussed in working
examples herein.
DETAILED DESCRIPTION OF THE INVENTION
In Vitro Display Library--Step (i) of First Aspect
[0058] The term "in vitro display library" shall be understood
according to the art--i.e. as a library comprising numerous
different binding entities wherein each binding entity is attached
to a nucleic acid molecule and the nucleic acid molecule comprises
specific nucleic acid sequence information allowing to identify the
binding entity--i.e. once one knows the specific nucleic acid
sequence information of the nucleic acid molecule one directly
knows the structure of the specific binding entity attached to the
nucleic acid molecule--the structure of the binding entity (i.e.
phenotype) attached to the nucleic acid molecule (genotype) is
herein termed B-structure.
[0059] As discussed herein--the prior art describes a number of
different methods to make such in vitro display libraries--i.e. an
in vitro display library of step (i).
[0060] Said in other words, it is today routine work for the
skilled person to properly make a structure of the binding entity
(i.e. phenotype) attached to the nucleic acid molecule
(genotype)--i.e. what is herein termed a "B-structure".
[0061] As known in the art--binding entity (i.e. phenotype) may be
attached to the nucleic acid molecule (genotype) by e.g. a covalent
binding or e.g. a high affinity non-covalent binding.
[0062] It may herein be preferred that the binding entity (i.e.
phenotype) is attached to the nucleic acid molecule (genotype) by a
covalent binding.
[0063] An in vitro display library of step (i) comprises a number
of different B-structures--i.e. in line of above it is routine work
for the skilled person to make an in vitro display library of step
(i).
[0064] Herein suitable examples include e.g. EP1809743B1
(Vipergen), EP1402024B1 (Nuevolution), EP1423400B1 (David Liu),
Nature Chem. Biol. (2009), 5:647-654 (Clark), WO 00/23458
(Harbury), Nature Methods (2006), 3(7), 561-570, 2006 (Miller),
Nat. Biotechnol. 2004; 22, 568-574 (Melkko), Nature. (1990);
346(6287), 818-822 (Ellington), or Proc Natl Acad Sci USA (1997).
94 (23): 12297-302 (Roberts).
[0065] Said in other words, the in vitro display library of step
(i) of first aspect may be made in a numbers of ways as described
in the prior art.
[0066] Without being limited to theory--herein suitable examples of
in vitro display library technologies include DNA Encoded Chemical
Library technologies, Aptamer technologies, RNA/DNA display
technologies such as CIS display, Ribosome display, mRNA display or
bead display system (using nucleic acids for encoding).
[0067] As described in the prior art (see e.g. EP1809743B1
(Vipergen))--the nucleic acid molecule of the B-structure may e.g.
be PNA, LNA, RNA, DNA or combinations thereof. Preferably, the
nucleic acid molecule of the B-structure is DNA.
[0068] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the binding entity
(phenotype) in the B-structure may be a double stranded nucleic
acid molecule.
[0069] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the binding entity
(phenotype) in the B-structure may be at least 0% double stranded
(i.e. single stranded), may be at least 10% double stranded, at
least 20% double stranded, at least 30% double stranded, at least
40% double stranded, at least 50% double stranded, at least 60%
double stranded, at least 70% double stranded, at least 80% double
stranded, at least 90% double stranded, or 100% double
stranded.
[0070] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the binding entity
(phenotype) in the B-structure may contain a PCR priming site or a
fraction hereof.
[0071] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the binding entity
(phenotype) in the B-structure may contain 2 PCR priming sites or
fractions hereof.
[0072] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the binding entity
(phenotype) in the B-structure may contain at least 3 PCR priming
sites or fractions hereof.
[0073] In some embodiments of the present invention a fraction of a
PCR priming site comprises at least 5 nucleotides, at least 6
nucleotides, at least 7 nucleotides, at least 8 nucleotides, at
least 9 nucleotides, at least 10 nucleotides, at least 11
nucleotides, at least 12 nucleotides, at least 13 nucleotides, at
least 14 nucleotides, at least 15 nucleotides, at least 16
nucleotides, at least 17 nucleotides, at least 18 nucleotides, at
least 19 nucleotides, or at least 20 nucleotides.
[0074] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the binding entity (phenotype)
in the B-structure may contain a single stranded overhang reverse
complement to a single stranded overhang of the genotype of the B
structure.
[0075] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the binding entity (phenotype)
in the B-structure may contain a single stranded overhang reverse
complement to a single stranded overhang of the genotype of the B
structure. The overhang may preferentially be 1 nucleotide, 2
nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6
nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10
nucleotides long.
Binding Entity
[0076] The Binding entity may any suitable binding entity of
interest.
[0077] Step (i) of first aspect reads "at least 100 different
binding entities (B.sub.n (n=100 or more)".
[0078] In practice, there may many times be many more different
binding entities present in the library of step (i)--such as e.g.
at least 10.sup.4, at least 10.sup.5 or at least 10.sup.6 different
binding entities--i.e. where n=at least 10.sup.4, n=at least
10.sup.5 or n=at least 10.sup.6.
[0079] Accordingly, in a theoretical situation, wherein the library
comprises exactly 10.sup.4 different binding entities--one may
herein express this as B.sub.n (n=10.sup.4) or B.sub.104.
[0080] Without being limited to theory it may be difficult to make
a library with more than 10.sup.20 different binding entities.
[0081] Suitable examples may be wherein the binding entity is at
least one binding entity selected from the group consisting of: a
protein, a polypeptide, a nucleic acid and a chemical compound
(preferably a small chemical compound with an average molecular
weight MW below 10000 dalton, more preferably an average molecular
weight MW below 5000 dalton, even more preferably an average
molecular weight MW below 1000 dalton.
[0082] Suitable examples of a herein relevant binding entity (such
as e.g. a chemical compound) may be found in the prior art--see
e.g. EP1809743B1 (Vipergen), EP1402024B1 (Nuevolution), EP1423400B1
(David Liu), Nature Chem. Biol. (2009), 5:647-654 (Clark), WO
00/23458 (Harbury), Nature Methods (2006), 3(7), 561-570, 2006
(Miller), Nat. Biotechnol. 2004; 22, 568-574 (Melkko), Nature.
(1990); 346(6287), 818-822 (Ellington), or Proc Natl Acad Sci USA
(1997). 94 (23): 12297-302 (Roberts).
[0083] In short, the skilled person is aware of numerous different
possible binding entities that could be of interest in the present
context.
Step (ii) of First Aspect
[0084] As discussed herein--the target shall be capable of binding
to at least one of the binding entities present in the library of
step (i)--otherwise it is not a suitable target that can be used to
identify a specific binding entity that binds to at least one
target.
[0085] In line of above--it is today routine work for the skilled
person to properly attach a target (i.e. phenotype) to a nucleic
acid molecule (genotype) and thereby make a structure of the target
(i.e. phenotype) attached to the nucleic acid molecule
(genotype)--i.e. what is herein termed "T-structure".
[0086] Said in other words, one may make herein relevant
"T-structure" based on e.g. the same prior art literature discussed
above for making the in vitro display library of step (i).
[0087] As known in the art--target (i.e. phenotype) may be attached
to the nucleic acid molecule (genotype) by e.g. a covalent binding
or e.g. a high affinity non-covalent binding.
[0088] It may herein be preferred that the target (i.e. phenotype)
is attached to the nucleic acid molecule (genotype) by a covalent
binding.
[0089] Step (i) of first aspect reads "at least one target Tn (n=1
or more)".
[0090] As discussed herein--an advantage of the method as described
herein is that one in an efficient and rapid way can simultaneous
screen for binding entities that could bind to e.g. two or more
targets.
[0091] For instance--the targets could be two different receptor
molecules and the method as described herein could then
simultaneous identify one binding entity that binds to one of the
receptors and another binding entity that binds to the other
receptor.
[0092] In the example above (with two different e.g. receptor
targets) we would have a situation, wherein the target Tn (n=2) or
alternatively expressed T.sub.2.
[0093] In line of above--it may be relevant to have at least two
different targets in step (ii) [i.e. Tn (n=2 or more], or to at
least three different targets in step (ii) [i.e. Tn (n=3 or more],
or to have at least ten different targets in step (ii) [i.e. Tn
(n=10 or more], or to at least hundred different targets in step
(ii) [i.e. Tn (n=100 or more].
[0094] Without being limited to theory it may be difficult to have
than 100.000 different targets in step (ii)--i.e. more than 100.000
different T-structures.
[0095] As described in the prior art (see e.g. EP1809743B1
(Vipergen))--the nucleic acid molecule of the T-structure may e.g.
be PNA, LNA, RNA, DNA or combinations thereof. Preferably, the
nucleic acid molecule of the T-structure is DNA.
[0096] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the target (phenotype)
in the T-structure may be at least 5 nucleotides long, at least 10
nucleotides long, at least 20 nucleotides long, at least 30
nucleotides long, at least 40 nucleotides long, at least 50
nucleotides long, at least 60 nucleotides long, at least 70
nucleotides long, at least 80 nucleotides long, at least 90
nucleotides long, at least 100 nucleotides long, at least 200
nucleotides long, at least 300 nucleotides long, at least 400
nucleotides long, or at least 500 nucleotides long.
[0097] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the target (phenotype)
in the T-structure may be a double stranded nucleic acid
molecule.
[0098] In a preferred embodiment of the present invention the
double stranded nucleic acid molecule (genotype) attached to the
target (phenotype) in the T-structure may be at least 5 base pairs
long, at least 10 base pairs long, at least 20 base pairs long, at
least 30 base pairs long, at least 40 base pairs long, at least 50
base pairs long, at least 60 base pairs long, at least 70 base
pairs long, at least 80 base pairs long, at least 90 base pairs
long, at least 100 base pairs long, at least 200 base pairs long,
at least 300 base pairs long, at least 400 base pairs long, or at
least 500 base pairs long.
[0099] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the target (phenotype)
in the T-structure may be at least 0% double stranded (i.e. single
stranded), may be at least 10% double stranded, at least 20% double
stranded, at least 30% double stranded, at least 40% double
stranded, at least 50% double stranded, at least 60% double
stranded, at least 70% double stranded, at least 80% double
stranded, at least 90% double stranded, or 100% double
stranded.
[0100] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the target (phenotype)
in the T-structure may contain a PCR priming site or a fraction
hereof.
[0101] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the target (phenotype)
in the T-structure may contain 2 PCR priming sites or fractions
hereof.
[0102] In a preferred embodiment of the present invention the
nucleic acid molecule (genotype) attached to the target (phenotype)
in the T-structure may contain at least 3 PCR priming sites or
fractions hereof.
[0103] In some embodiments of the present invention a fraction of a
PCR priming site comprises at least 5 nucleotides, at least 6
nucleotides, at least 7 nucleotides, at least 8 nucleotides, at
least 9 nucleotides, at least 10 nucleotides, at least 11
nucleotides, at least 12 nucleotides, at least 13 nucleotides, at
least 14 nucleotides, at least 15 nucleotides, at least 16
nucleotides, at least 17 nucleotides, at least 18 nucleotides, at
least 19 nucleotides, or at least 20 nucleotides.
[0104] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the target (phenotype) in the
T-structure may contain a single stranded overhang reverse
complement to a single stranded overhang of the genotype of the B
structure.
[0105] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the target (phenotype) in the
T-structure may contain a single stranded overhang reverse
complement to a single stranded overhang of the genotype of the B
structure. The overhang may preferentially be 1 nucleotide, 2
nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6
nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10
nucleotides long.
[0106] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the target (phenotype) in the
T-structure may contain a unique sequence specific for each target
molecule (Unique Molecule Identifier--UMI).
[0107] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the target (phenotype) in the
T-structure may contain a unique sequence specific for each target
molecule (Unique Molecule Identifier--UMI) consisting of at least
16 Ns (N=A, C, G, or T), at least 17 Ns, at least 18 Ns, at least
19 Ns, at least 20 Ns, at least 21 Ns, at least 22 Ns, at least 23
Ns, at least 24 Ns, at least 25 Ns, at least 26 Ns, at least 27 Ns,
at least 28 Ns, at least 29 Ns, or at least 30 Ns.
[0108] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the target (phenotype) in the
T-structure may contain a unique sequence specific for each target
molecule (Unique Molecule Identifier--UMI) consisting of a
continuous sequence.
[0109] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to the target (phenotype) in the
T-structure may contain a unique sequence specific for each target
molecule (Unique Molecule Identifier--UMI) consisting of a
discontinuous sequence.
[0110] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to a first target (phenotype) in
the T-structure may contain a first sequence different from a
second target's second genotype sequence (allowing
multiplexing).
[0111] In some embodiments of the present invention the nucleic
acid molecule (genotype) attached to a first target (phenotype) in
the T-structure may contain a first sequence different from a
second target's second genotype sequence (allowing multiplexing),
wherein the first and second target genotype comprise different PCR
priming sites.
Target
[0112] The target may be any suitable target of interest.
[0113] In a preferred embodiment of the present invention--specific
enriching methods for the enrichment facilitating identification of
binding entities with desired characteristics include but are not
limited to: enrichment on nucleic acid attached target molecules.
In this approach the target molecules is e.g. DNA, RNA, protein,
carbohydrate, organic or inorganic molecule.
[0114] As known in the art--a suitable target could e.g. be a
receptor molecule present in e.g. the human body and one would be
interested in identifying a binding entity (e.g. a chemical
compound) that can bind to the receptor.
[0115] In accordance with the prior art--suitable examples may be
wherein the target is DNA, RNA, protein, carbohydrate, organic or
inorganic molecule or fragments hereof.
[0116] In accordance with the prior art--suitable examples may be
wherein the target is an autoantigen, a bacterial protein, a blood
protein, a cell adhesion protein, a cytokine, a cytoskeleton
protein, a DNA-binding protein, a developmental protein, an
engineered protein, an enzyme, an extracellular matrix protein, a
GTP-binding protein regulator, a glycoprotein, a growth factor, a
heat shock protein, a lipoprotein, a membrane protein, a
metalloprotein, a motor protein, a phosphoprotein, a prion, a
protein complex, a protein domain, a RNA-binding protein, a
receptor, a recombinant protein, a seed storage protein, a
structural protein, a transcription coregulator protein, a
transport protein, a viral protein or fragments hereof.
[0117] In short, the skilled person is aware of numerous different
possible targets than could be of interest in the present
context.
Step (iii) of First Aspect:
[0118] In the illustrative example of FIG. 1 herein--this step
(iii) corresponds to the step "1 Binding".
[0119] As discussed above--step (iii) reads:
"mixing a solution comprising X (X is a number greater than
10.sup.4) numbers of B-structures of the library of step (i))"
[0120] The term "X" in relation to numbers of B-structures shall be
understood as the total numbers of B-structures of the library of
step (i).
[0121] For instance--if the library comprises 100 different binding
entities (B.sub.n (n=100)) and there are 100 copies of each of the
100 different B-structures then the number "X" is equal to
100.times.100=10.sup.4.
[0122] In practice the number X may many times be higher--for
instance, if the library comprises 10.sup.6 different binding
entities [B.sub.n (n=10.sup.6)] and there are 10.sup.4 copies of
each of the 10.sup.6 different B-structures then the number "X" is
equal to 10.sup.6.times.10.sup.4=10.sup.10.
[0123] As discussed above--step (iii) reads:
"a solution comprising Y (Y is a number greater than 10.sup.2)
numbers of T-structures of step (ii)"
[0124] The term "Y" in relation to numbers of T-structures shall be
understood as the total numbers of T-structures of the library of
step (ii).
[0125] For instance--if there is only one target in step (ii)
[T.sub.n (n=1)] and there are 10.sup.2 copies of each of the
T-structure then the number "Y" is equal to
1.times.10.sup.2=10.sup.2.
[0126] As discussed above--one could have e.g. 2 different targets
(e.g. two different receptor molecules)--in this case there would
be two targets in step (ii) [T.sub.n (n=2)] and if there would be
10.sup.2 copies of each of the two different T-structures then the
number "Y" would be equal to
2.times.10.sup.2=2.times.10.sup.2=200.
[0127] In practice one may many times have significant more copies
of a relevant T-structure--the reason for this is that one
preferably wants to have numerous copies of a relevant T-structure
in order to increase the probability for that the target on a
T-structure bind to the binding entity of a B-structure.
[0128] Accordingly, in a preferred embodiment there are at least
10.sup.0, at least 10.sup.1, at least 10.sup.2, at least 10.sup.3,
at least 10.sup.4, at least 10.sup.5, at least 10.sup.6, at least
10.sup.7 at least 10.sup.8, at least 10.sup.9, at least 10.sup.10,
at least 10.sup.11, at least 10.sup.12, at least 10.sup.13, at
least 10.sup.14, at least 10.sup.15 or at least 10.sup.16 copies of
a T-structure of interest.
[0129] Advantageous in ECC method as described herein is that
enrichment for important binding characteristics can be optimized
for in isolation--because ECC is a homogenous assay--target is not
immobilization to a solid support. Prior art methods are
heterogenous--rely on target immobilization to a solid support
(e.g. beads, columns, cells, plastic, filters etc). Heterogenous
assays are notoriously more difficult to control than homogenous
assay due e.g. avidity effects, density of coating, and
interference of the solid support itself with the assay.
[0130] ECC allows optimizing for major binding characteristic for
binding of binding entity to target in isolation. For example
potency (affinity), association rate (on rate) or dissociative
half-life of binding entity and target (off rate).
[0131] Affinity based selection is achieved in step (iii) e.g. by
using equilibrium conditions and controlled by the target
concentration in the mixing step (binding step), i.e. 90% of the
molecules of a binding entity in the display library having a
K.sub.d equal to 10 times smaller than the target concentration are
target bound, whereas 50% of the molecules of a binding entity
having a K.sub.d equal to the target concentration are, and 10% of
the molecules of a binding entity having a K.sub.d 10 times smaller
than the target concentration are. Consequently, enrichment for
affinity is easily controlled by the target concentration in the
mixing step.
[0132] In a preferred embodiment of the present invention--the
concentration of T-structures in the "mixing step (iii)" is at
least 10.sup.-15 M, at least 10.sup.-14 M, at least 10.sup.-13 M,
at least 10.sup.-12 M, at least 10.sup.-11 M, at least 10.sup.-19
M, at least 10.sup.-9 M, at least 10.sup.-8 M, at least 10.sup.-7
M, at least 10.sup.-6 M, at least 10.sup.-5 M, at least 10.sup.-4
M, or at least 10.sup.-3 M.
[0133] Alternatively, association rate based selection is achieved
by controlling the time allowed for the mixing step
(iii)--accordingly, the "mixing step" may be performed for a time
period shorter than the time needed to reach binding equilibrium
conditions.
[0134] Step (iii) further reads:
"under binding conditions, i.e. conditions where a B-structure
containing a binding entity capable of binding to a target
molecule, binds more efficiently to the corresponding T-structure,
than a B-structure containing a binding entity not capable of
binding to the same target do and wherein one gets binding of at
least one of the binding entities to at least one target thereby
creating a complex comprising a B-structure bound to a T-structure
(herein termed B.sub.BoundToT-structure)"
[0135] The term "binds more efficiently" shall be understood
according to common practice e.g. higher affinity, faster on rate,
or slower dissociation rate.
[0136] As known to the skilled person--in the present context it is
routine work for the skilled person to perform step (iii) under
conditions, wherein one get this "binds more efficiently"
effect.
[0137] For instance--one may easy obtain this "binds more
efficiently" effect by e.g. using B and T-structures genotypes that
essentially do not binds (e.g. by hybridization base pairing) under
the binding conditions of step (iii)--as evident to the skilled
person this could e.g. be obtained by using e.g. double stranded
DNA with none or very small single stranded base-pairing overlap as
genotypes for the B and T-structures.
[0138] It would be routine work for the skilled person to optimize
the binding conditions of step (iii) in order to get the required
"binds more efficiently" effect of step (iii).
[0139] As known to the skilled person--herein relevant optimization
parameters may e.g. be inonic strength, temperature etc.
[0140] Accordingly, under any practical herein relevant
circumstance--the skilled person would not be in any reasonable
doubt if he (after e.g. proper routine adjustment of the binding
conditions) would work under binding conditions of step (iii) or
not.
[0141] In a preferred embodiment, step (iii) is performed under
binding conditions, wherein a B-structure containing a binding
entity capable of binding to a target molecule, binds 10 fold (more
preferably 100 fold, even more preferably 1000 fold) more
efficiently to the corresponding T-structure, than a B-structure
containing a binding entity not capable of binding to the same
target do.
Step (iii-b)--Dilution Step--Preferred Embodiment:
[0142] In the illustrative example of FIG. 1 herein--this optional
step (iii-b) corresponds to the step "2 Dilution".
[0143] The mixing step (iii) may preferably be followed by a
dilution step--this is herein termed step (iii-b) and is performed
before the step (iv) of the first aspect.
[0144] Accordingly, in a preferred embodiment the method of the
first aspect comprises an additional step (iii-b) that is performed
before the step (iv) of the first aspect, comprising:
(iii-b): diluting the solution of step (iii) at least 2 fold under
binding conditions, i.e. conditions where a B-structure containing
a binding entity capable of binding to a target molecule, binds
more efficiently to the corresponding T-structure, than a
B-structure containing a binding entity not capable of binding to
the same target do.
[0145] The dilution solution introduced and the conditions (e.g.
temperature) in the dilution step (iii-b) may be different from the
binding conditions of the mixing step (iii)--but the above
described effects shall be maintained in dilution step (iii-b).
[0146] It may be preferred in step (iii-b) to have a diluting the
solution of step (iii) at least 10.sup.2 fold, or have a diluting
the solution of step (iii) at least 10.sup.3 fold, or have a
diluting the solution of step (iii) at least 10.sup.4 fold, or have
a diluting the solution of step (iii) at least 10.sup.5 fold, or
have a diluting the solution of step (iii) at least 10.sup.6 fold,
or have a diluting the solution of step (iii) at least 10.sup.7
fold, or have a diluting the solution of step (iii) at least
10.sup.8 fold or have a diluting the solution of step (iii) at
least 10.sup.9 fold.
[0147] An advantage of this diluting step is that enrichment can be
performed based on dissociative half-life of the BT-structures and
easily controlled by the degree of dilution and the incubation
time. When the mixing solution of step (iii) is diluted biding of
binding entity and target is a less likely event to happened
whereas the "un-binding event"--the off rate (the dissociative
half-life) is independent of the dilution. Consequently, in a very
dilute solution (T-structure concentration<<K.sub.d)
essentially only dissociation will take place. Therefore,
enrichment for dissociative half-life of the BT-structures is
conveniently controlled by the degree of dilution and the
incubation time.
[0148] The dissociative half-life together with the affinity is of
greatest importance in the usability of a binding entity. Most
notable, for development of effective new drugs where high affinity
and long dissociative half-life are critical parameters for
pharmacological effect (Nature Reviews Drug Discovery (2006) 5,
730-739, (Copeland). Hence, the method of the present new invention
permits enrichment for these two parameters in an unprecedented
effective and controllable manner. Moreover, the two parameters can
be controlled independently of each other.
Step (iv) of First Aspect:
[0149] A simple way to view this step is that the binding of target
with binding entity of step (iii) is "transformed" into
co-compartmentalization of B-structures and T-structures.
[0150] The conditions of this step (iv) shall be "under binding
conditions" that gives an effect corresponding to the effect in
step (iii)--see above.
[0151] In the illustrative example of FIG. 1 herein--this step (iv)
corresponds to the step "3 Emulsion w/o".
[0152] Step (iv) of first aspect further reads:
"wherein the compartmentalization system comprises at least 2 times
more individual compartments than the Y number of T-structures
present in step (iii)"
[0153] This may herein be seen as an essential step of the method
as described herein--i.e. it is essential to have "at least 2 times
more individual compartments than the Y number of T-structures
present in step (iii)".
[0154] In the FIG. 1 herein--the in vitro compartmentalization
system may be e.g. a water-in-oil emulsion system--as further
discussed below herein suitable water-in-oil emulsion systems are
well known in the art.
[0155] In the hypothetical theoretical illustrative example in FIG.
1 there is only one target in step (ii) [T.sub.n (n=1)] and there
are 3 copies of each of the T-structure--i.e. the number "Y" is
3.
[0156] Accordingly, in this theoretical illustrative example of
FIG. 1 there should be at least (2.times.3)=6 individual
compartments (e.g. oil droplets) in the in vitro
compartmentalization system--please note that in FIG. 1 herein are
there less than 30 individual compartments (i.e. FIG. 1 is just an
illustration of some of the elements of the method as described
herein).
[0157] As discussed above--in practice there may be for instance at
least 10.sup.4 copies of a T-structure of interest--i.e. Y could in
this case be 10.sup.4 and there should in this case be at least
(2.times.10.sup.4)=2.times.10.sup.4 individual compartments (e.g.
oil droplets) in the in vitro compartmentalization system.
[0158] An advantage of having this "at least 2 times more
individual compartments than the Y number of T-structures" is that
non-binders in the display library is randomly distributed in the
compartments and therefore co-compartmentalize with the target in a
random fashion, with a frequency depending on the ratio between the
number of compartments and the number of target molecules (in this
case 1 out of 10), whereas binders, due to the binding activity,
will co-compartmentalize together with target molecules
independently of the ratio between the number of compartments and
the number of target molecules (in the ideal case 1 out of 1).
Consequently, in this case binders will be enriched 2 fold when
compared to non-binders.
[0159] In line of above one may say one gets an even better
enrichment if there is relatively more individual compartments in
the in vitro compartmentalization system--accordingly, in a
preferred embodiment there is "at least 10 times more individual
compartments than the Y number of T-structures present in step
(iii)", more preferably there is "at least 100 times more
individual compartments than the Y number of T-structures present
in step (iii)", more preferably there is "at least 10 000 times
more individual compartments than the Y number of T-structures
present in step (iii)", more preferably there is "at least 100 000
times more individual compartments than the Y number of
T-structures present in step (iii)", more preferably there is "at
least 1 000 000 times more individual compartments than the Y
number of T-structures present in step (iii)".
[0160] In a preferred embodiment of the present invention the
number of compartments is larger than 2, 5, 10, 50, 100, 1000,
5000, 10 000, 50 000, 100 000, 500 000, 1 000 000, 5 000 000, or 10
000 000 times the Y number of T-structures of step (iii).
[0161] Step (iv) of first aspect further reads:
"under conditions wherein the B-structures, T-structures and
BT-structures enter randomly into the individual compartments"
[0162] This should understood as the skilled person would
understand it in the present context--relating to that in order to
get the herein advantageous enrichment one needs to have conditions
wherein the B-structures, T-structures and BT-structures enter
randomly into the individual compartments.
[0163] Said in other words--the propensity for any B-structures,
T-structures and BT-structures for being compartmentalized in any
given compartment is dependent on the volume of said compartment
and the total volume.
[0164] Said in other words--if virtually all the B-structures,
T-structures and BT-structures would only enter into one specific
individual compartment one will obviously not get the advantageous
enrichment as discussed herein.
[0165] As discussed below--if one e.g. uses a suitable water-in-oil
emulsion system as the in vitro compartmentalization system one can
easily identify conditions, wherein the B-structures, T-structures
and BT-structures enter randomly into the individual compartments
(e.g. an individual oil droplets)--in fact it would be quite
difficult to identify conditions, where it is not randomly--i.e.
wherein virtually all the B-structures, T-structures and
BT-structures would only enter into one specific individual
compartment (e.g. an individual oil droplets).
[0166] In a preferred embodiment of step (iv)--there is at least
square root 10 (3.16) times more individual compartments than the X
number of B-structures in step (iii).
[0167] A Poisson distribution is assumed to describe the
distribution of B-structures in the compartments. This implies that
all compartments are of equal volumes. The probability that a
compartment has n=0, 1, 2 or more B-structures molecules can be
calculated using the formula:
f ( n , .lamda. ) = - .lamda. .lamda. n n ! ##EQU00001##
where is the ratio between X number of B-structures and number of
compartments. When is square root 10 (3.16) less than 5% (4.1%) of
the compartments will have more than one B-structures. This means
that the effect of this is to lowering the enrichment of positive
binding entity with less than 5%, which is insignificant in most
cases.
[0168] As discussed above and as understood by the skilled person
in the present context, the method of the first aspect may be seen
as a method which implies that the B.sub.BoundToT-structures (i.e.
the target-binding entity complexes) remain suspended in solution
in the individual/separated compartments of step (iv) of the first
aspect.
[0169] Accordingly, just to make it 100% clear, one may express
this as that the method of the first aspect and herein relevant
embodiments of this method, is a method wherein the
B.sub.BoundToT-structures remain suspended in solution in the
individual compartments of step (iv) of the first aspect.
[0170] Said in other words, the method does not rely on target
immobilization on a solid support as for herein relevant prior art
methods as discussed above.
In Vitro Compartmentalization System
[0171] As discussed above, a herein suitable in vitro
compartmentalization system may e.g. be a water-in-oil emulsion
system.
[0172] In case wherein the in vitro compartmentalization system is
a water-in-oil emulsion system--one may say that the "applying an
in vitro compartmentalization system . . . to the solution of step
(iii)" of step (iv) may be expressed as "adding the solution of
step (iii) to an water-in-oil emulsion system".
[0173] Herein suitable water-in-oil emulsion systems are described
in the art e.g. Nat Methods. 2006 July; 3(7):545-50 (Williams et
al), Nat Methods. 2006 July; 3(7):551-9 (Diehl et al), Proc Natl
Acad Sci USA. 2003 Jul. 22; 100(15):8817-22 (Dressman et al), J
Biotechnol. 2003 Apr. 24; 102(2):117-24. (Nakano et al), or
Biomacromolecules. 6, 1824-1828 (2005) (Musyanovych et al).
[0174] As evident to the skilled person--in the case of using
emulsions as the compartmentalization system and in analogy with
similar size distributions, the compartment volume distribution is
modeled as a log-normal distribution, also called a Galton
distribution. By assuming a log-normal distribution and performing
measurements of the actual droplet sizes the expected value (mean)
and the standard deviation can be calculated for a specific
experiment. According to this distribution, 95% of the compartment
volumes will be within L logarithmic units from the mean (log)
volume, where L is 1.96 times the standard deviation of the
log-volumes.
[0175] In a preferred embodiment of the present invention the
average compartments size, the variation, and the standard
deviation is taken into account when analyzing the data.
[0176] In a preferred embodiment of the present invention
compartments with a volume, larger than 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 60, 70, 80, 90, or 100 times the average
compartment size are removed from the experiment.
[0177] In a preferred embodiment of the present invention
compartments with a volume, smaller than 1/100, 1/90, 1/80, 1/70,
1/60, 1/50, 1/40, 1/30, 1/20, 1/10, 1/9,1/8, 1/7, 1/6, 1/5, 1/4,
1/3, or 1/2, times the average compartment size are removed from
the experiment.
[0178] As evident to the skilled person--several technologies may
be employed to exclude compartments from the experiment based on
the volume of the compartments, for example by FACS sorting,
equilibrium centrifugation, filtration, or microfluridic systems
etc.
[0179] Alternatively, the in vitro compartmentalization system may
be agarose droplet microfluidics. (Lab Chip. 2010 Sep. 13. [Epub
ahead of print] Agarose droplet microfluidics for highly parallel
and efficient single molecule emulsion PCR. Leng X, Zhang W, Wang
C, Cui L, Yang C J).
[0180] Alternatively, the in vitro compartmentalization system may
simply be to disperse the solution into e.g. "a micro titer plate"
and of step (iii) is simply randomly putted into the individual
wells (i.e. the individual compartments) of the micro titer
plate--as known this may today be done rapidly and efficient by
e.g. a suitable robot machine or an open well system. An example of
such a system based on a high-density array of nanoliter PCR
assays, functionally equivalent to a microtiter plate, the
nanoplate system makes possible up to 3,072 simultaneous PCR
reactions in a device, the size of a standard microscope slide
(Methods Mol Biol. 2009; 496:161-74. (Brennan et al)).
[0181] Another example is a silicone device presenting a large
array of micrometer-sized cavities, which can be used it to tightly
enclose volumes of solution, as low as femtoliters, over long
periods of time. The microchip insures that the chambers are
uniform and precisely positioned (Nat Biotechnol. 2005 March;
23(3):361-5 (Rondelez et al)).
[0182] As evident to the skilled person--microfluridic devices can
be employed in the in vitro compartmentalization system (For review
see e.g. Angew Chem Int Ed Engl. 2010 Aug. 9; 49(34):5846-68
(Theberge et al)).
[0183] In a preferred embodiment of the present invention a
suitable average compartments volume is less than 10.sup.-6 liter,
less than 10.sup.-7 liter, less than 10.sup.-8 liter, less than
10.sup.-9 liter, less than 10.sup.-10 liter, less than 10.sup.-11
liter, less than 10.sup.-12 liter, less than 10.sup.-13 liter, less
than 10.sup.-14 liter, less than 10.sup.-16 liter, less than
10.sup.-16 liter, less than 10.sup.-17 liter, less than 10.sup.-18
liter, less than 10.sup.-19 liter, less than 10.sup.-20 liter, less
than 10.sup.-21 liter, or less than 10.sup.-22 liter.
[0184] As evident to the skilled person--the compartment volume
cannot be infinitely small as the compartment should be larger than
the molecules compartmentalized.
[0185] In short, the skilled person is aware of numerous different
in vitro compartmentalization systems than could be of interest in
the present context.
Step (v) of First Aspect
[0186] Step (v) of first aspect reads:
"fusing the nucleic acid molecules of a B-structure and a
T-structure which are both present within the same individual
compartment--i.e. fusing the nucleic acid molecule of the
B-structure to the nucleic acid molecule of the T-structure"
[0187] A simple way to view this step is that the
co-compartmentalization of B-structures and T-structures
origination of (iv) is "transformed" into fused cognate
genotypes.
[0188] In the present context "fusing the nucleic acid molecules of
a B-structure and a T-structure" shall be understood as joining the
genetic information carried by the two genotypes in the
compartment.
[0189] As known to the skilled person, this can be accomplished in
several ways such as e.g.:
a) information transfer by e.g. overlap PCR or overlap genome
extension (overlap PCR without outer primers)--one strand
originating from one genotype acts as a primer and uses a strand
originating from the other genotype as a template; b) information
joining catalyzed by an enzyme forming an amplifiable facilitating
bond--e.g. by a DNA ligase where a phosphordiester bond between at
least one of the strands from each genome is form; c) information
joining catalyzed by an enzyme forming an non-amplifiable
facilitating bond--e.g. having a moiety on each genome thus upon
induction an enzyme capable of linking the two moieties between at
least one of the strands from each genotype is form; d) information
joining not catalyzed by an enzyme--e.g. having a inducible
chemical reactive group on each genome thus upon induction a
chemical bond between at least one of the strands from each
genotype is form; and e) information joining transient--e.g. having
a affinity tag (these may be different or identical) on each genome
thus by providing an agent with affinity for both tags a ternary
complex containing both genotype is formed.
[0190] In the illustrative example of FIG. 1 herein--this step (v)
corresponds to fusing the nucleic acid molecules of the B-structure
and the T-structure present in the individual compartment number
three from the left.
[0191] A herein very important advantage is that during this step
one may say that the binding between the binding entity and target
is no longer relevant--i.e. when one here performs the fusion of
the nucleic acid molecules step one can do it under conditions,
wherein one does not have to worry about this binding entity to
target binding and spatial arrangements. A simple way to view this
step is that the binding of target with binding entity origination
from step (i) is now transformed into co-compartmentalization of
B-structures and T-structures.
[0192] This may be seen as a very big advantage of the method as
described herein.
[0193] For instance, if one wants to make the fusion of the nucleic
acid molecules of the B-structure and the T-structure by
hybridization of overlapping base pairing regions one can in this
step (v) adequately change e.g. the temperature to get the relevant
base pairing hybridization without being worried if the binding
between the binding entity and target could be destroyed.
[0194] Accordingly, the step (v) may be performed under conditions,
wherein there is essentially no binding of any of the binding
entities of step (i) to any of the target(s) of step (ii).
[0195] As already discussed above--the fusing of the nucleic acid
molecules of the B-structure and the T-structure may be done in
different ways than e.g. by hybridization of overlapping base
pairing regions.
[0196] For instance, if a e.g. a ligase enzyme is used in step (v)
to get the fusing nucleic acid molecules--then one does not need to
have any base pairing overlapping regions between the nucleic acid
molecules (genotype) of the B-structures step (i) and the nucleic
acid molecules (genotype) of the T-structures of step (ii).
[0197] As evident to the skilled person--if e.g. a ligase or a
polymerase enzyme is used--this ligase enzyme should preferably
have been added to the solution of step (iii) or during the
optional diluting step (iii-b) in order to properly be present in
the relevant individual compartments of step (v).
[0198] As evident to the skilled person--several different
enzymatic reactions may be employed to fuse the genome--a large
number enzymatic reactions have been reported in the literature
e.g. Kabanov et al., Biochimica et Biophysica Acta, 996 (1989)
147-152, Salon et al. Biochemistry 1992, 31, 8072-8079, Anarbaev et
al, Biochimica et Biophysica Acta 1384 1998. 315-324, Ong et al.,
(2006). J. Mol. Biol. 361: 537-50, Ghadessy, F. J. Ong, J. L. and
Holliger, P. (2001). Proc. Natl. Acad. Sci. USA 98: 4552-4557,
Protein Engineering, Design & Selection vol. 17 no. 3 pp.
201-204, 2004, Levy et al, RNA (2005), 11:1555-1562, Turner et al.,
Nucleic Acids Res. 2008 August; 36(13): e82.
[0199] In a preferred embodiment of the present invention the
co-compartmentalized genotypes are fused by an enzyme.
[0200] As evident to the skilled person--despite the concentration
of genotypes in the solution before compartmentalization may be
very low e.g. picomolar-micromolar range the concentration of
genotypes in a compartment with co-compartmentalized genotypes may
be high e.g. when the compartment volume is in the femtoliter
(10.sup.-15 liters) range the concentration of the genotypes are in
the nanomolar range (10.sup.-9 M), when the compartment volume is
in the attoliter (10.sup.-18 liters) range the concentration of the
genotypes are in the micromolar (10.sup.-6 M) range, or when the
compartment volume is in the zeptoliter (10.sup.-21 liters) range
the concentration of the genotypes are in the millimolar (10.sup.-3
M) range. Consequently, the genotype concentration in a compartment
may be controlled for facilitating enzymatic reactions and even
traditional chemical reactions.
[0201] As evident to the skilled person--if e.g. inducible chemical
cross-linking in emulsion is used--neither the inducer nor other
reagents may be mandatory before compartmentalization as these may
be "delivered" later to the formed compartments. For example
inducers may be light, temperature or a chemical activator
delivered though the continuous phase. Such embodiments may be
advantageous when small compartments are desired.
[0202] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00001##
[0203] The nucleophilic substitution reaction can essentially be
performed as described by: [0204] Z. J. Gartner, et al. J. Am.
Chem. Soc. 2001, 123, 6961.
[0205] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00002##
[0206] The nucleophilic aromatic substitution reaction can
essentially be performed as described by: Clark et al, Nature
Chemical Biology 5, 647-654 (2009)
[0207] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00003##
[0208] The nucleophilic substitution reaction can essentially be
performed as described by: [0209] Z. J. Gartner, et al. J. Am.
Chem. Soc. 2001, 123, 6961.
[0210] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00004##
[0211] The reductive amination can essentially be performed as
described by: [0212] Z. J. Gartner, et al. Angew. Chem. Int. Ed.
2002, 41, 1796.
[0213] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00005##
[0214] The Amine acylation can essentially be performed as
described by: [0215] Z. J. Gartner, et al. Angew. Chem. Int. Ed.
2002, 41, 1796.
[0216] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00006##
[0217] The Phosphoramidate formation can essentially be performed
as described by: [0218] Luther A, et al. Nature 1998,
396:245-248.
[0219] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00007##
[0220] The Aldol condensation reaction can essentially be performed
as described by: [0221] Zhuo Tang et al. Angew. Chem. Int. Ed.
2007, 46, 7297-7300
[0222] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00008##
[0223] The Cycloaddition reactions can essentially be performed as
described by: [0224] Buller et al. Bioorganic & Medicinal
Chemistry Letters 18 (2008) 5926-5931 [0225] Fujimoto K, J Am Chem
Soc 2000, 122:5646-5647. [0226] Gartner Z. J. et al. Angew Chem Int
Ed Engl 2002, 41:1796-1800. [0227] Gartner Z. J. et al. Angew Chem
Int Ed Engl 2003, 42:1370-1375. [0228] Poulin-Kerstien A. T. et al.
J Am Chem Soc 2003, 125:15811-15821.
[0229] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00009##
[0230] The Disulfide crosslink can essentially be performed as
described by: [0231] Mays, J. R. et al. Tetrahedron Lett. 2007, 48,
4579. [0232] Theodoropoulos, D. et al. Journal of Medicinal
Chemistry, 1985, vol. 28, 10, p. 1536-1539 [0233] Lorenz, Katrin B.
et al. Journal of Organic Chemistry, 2004, vol. 69, 11 p.
3917-3927
[0234] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00010##
[0235] The urea crosslink can essentially be performed as described
by: [0236] EP1809743B1 (Vipergen)
[0237] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00011##
[0238] The Wittig olefination reaction can be performed as
described by: [0239] Gartner Z. J. et al. Angew Chem Int Ed Engl
2002, 41:1796-1800
[0240] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00012##
[0241] The Wittig olefination reaction can essentially be performed
as described by: [0242] Gartner Z. J. et al. Angew Chem Int Ed Engl
2002, 41:1796-1800.
[0243] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00013## ##STR00014##
[0244] Transition metal catalysed reactions can essentially be
performed as described by: [0245] Czlapinski J. L. et al. J Am Chem
Soc 2001, 123: 8618-8619. [0246] Gartner Z. J. et al. Angew Chem
Int Ed Engl 2002, 41: 1796-1800 [0247] Calderone C. T. et al. Angew
Chem Int Ed Engl 2005, 44: 1-5 [0248] Kanan M. W. et al. Nature
431, 545-549, 2004
[0249] In a preferred embodiment of the present new invention a
chemical cross-linking is performed by
##STR00015##
[0250] Photo crosslinking can essentially be performed as described
by: [0251] Quamrul, A. et al. Bioorganic & Medicinal Chemistry
Letters 18 (2008) 5923-5925 [0252] Weber, T. et al. Journal of the
American Chemical Society; 117; 11; (1995); 3084-3095 [0253] Chee,
G. et al. Bioorganic and Medicinal Chemistry; 18; 2; (2010);
830-838 [0254] Nassal, M. Journal of the American Chemical Society;
106; (1984); 7540-7545 [0255] Pandurangi, R. S. et al. Bioorganic
Chemistry; 25; 2; (1997); 77-87 [0256] Patent; KENT STATE
UNIVERSITY; LELAND STANFORD JUNIOR UNIVERSITY; US2010/29952;
(2010); (A1)
[0257] In short, the skilled person is aware of numerous different
ways of fusing the nucleic acid molecules of a B-structure and a
T-structure which are both present within the same individual
compartment.
[0258] As understood by the skilled person in the present
context--it is preferred that there is no herein significant fusion
of the nucleic acid molecules of a B-structure and a T-structure in
the steps (iii) and (iv) of the method of the first aspect--said in
other words, there is preferably no significant creation of the
BT.sub.Fused-structures before step (v).
Step (Vi) of First Aspect
[0259] Step (vi) reads:
"combining the content of the individual compartments of step
(v)."
[0260] It is evident that the "combining the content of the
individual compartments of step (v)" is done in a suitable way
depending on the in vitro compartmentalization system used in step
(iv).
[0261] For instance, if the in vitro compartmentalization system
used in step (iv) is a micro titer plate like format (see above)
the content of the individual wells are simply combined (put
together).
[0262] For instance, if the in vitro compartmentalization system
used in step (iv) is a suitable water-in-oil emulsion--the
individual oil compartments may simply be disrupted by e.g.
centrifugation, increase the temperature or by adding a suitable
organic solvent.
[0263] In short, in the present context it is routine work for the
skilled person to combine the content of the individual
compartments of step (v).
[0264] Step (vi) further reads:
" . . . under conditions wherein there is no fusing of the nucleic
acid molecules of a B-structure and a T-structure--i.e. there is
not created any new BT.sub.Fused-structure not already created in
step (v)--in order to get a library of
BT.sub.Fused-structures."
[0265] As discussed above--one may say that the enrichment for the
herein wanted BT.sub.Fused-structures has already been obtained in
the steps above--accordingly, as evident to the skilled person one
is in this step not interested in creating more "new"
BT.sub.Fused-structures as such.
[0266] It is routine work for the skilled person to perform step
(vi) under such conditions--for instance, if a ligase has been used
in step (v) to obtain the wanted BT.sub.Fused-structures, this
ligase could be inactivated (e.g. by properly raising the
temperature) before step (vi) is performed.
[0267] In view of the discussions herein--it is evident for the
skilled person that this step (vi) will results in a library of
BT.sub.Fused-structures.
[0268] As evident to the skilled person--this library of
BT.sub.Fused-structures may be described as an enriched library of
species of BT.sub.Fused-structures, comprising nucleic acid
sequence information allowing to identify the binding entity and
the target--i.e. the sequence information of step (i) and (ii),
originating from binding pairs of target and binder entity when
compared to BT.sub.Fused-structures originating from nonbinding
pairs of target and binder entity.
Optional Step (Vii)--i.e. Subsequently Use of the Enriched Library
of Step (Iv) of First Aspect.
[0269] As discussed above--step (vii) is an optional step.
[0270] As described herein once one has obtained the enriched
library of step (vi) one may use this library in different ways
according to art--e.g. the enriched library may be considered as an
enriched in vitro display library that e.g. can be used in a second
round of selection/enrichment or one may identify the structure of
a specific binding entity of interest directly from the enriched
library of step (vi).
[0271] Accordingly, an embodiment of the invention relates to the
method as described herein, wherein there is an extra step (vii)
comprising use the enriched library of step (vi) to identify at
least one individual binding entity that binds to at least one
target of interest.
Purification of Fused Genotypes:
[0272] In a suitable embodiment of the present invention the fused
genotypes (i.e. the BT.sub.Fused-structures) may be purified.
[0273] The skilled person in the art can routinely identify
numerous different strategies in order to purify the fused
genotypes, without being limited for example by: agarose gel
electrophoresis, polyacrylamid gel electrophoresis, spun-columns,
enzymatic treatment, HPLC purified, affinity purified or capillary
electrophoresis (Molecular Cloning: A Laboratory Manual (3-Volume
Set), 3rd Edition, 2001-01 by Joseph Sambrook, David W. Russell,
Publisher: Cold Spring Harbor Laboratory Press)
[0274] In a preferred embodiment of the present invention the fused
genotypes are purified post compartmentalization e.g. by gel
purification or enzymatic degradation of undesired nucleic acid
species. For a skilled person in the art it is evident to design
such procedures. For example in the case of using overlap PCR for
genotype fusing the size of the genotypes may be chosen to
facilitate gel purification e.g the length of the display library
genotype could be chosen to around 250 bp and the length of the
target genotype could be around 100 bp and the overlap region to be
around 20 bases, the resulting fused genotypes will then be around
330 bp which are easily separated and purified from the original
un-fused species by standard agarose gels electrotrophoresis or
polyacrylamide gel electrotrophoresis. Furthermore, unused primers
and ssDNA originating from primer extension using un-fused
genotypes as templates may conveniently degraded enzymatically e.g.
by ExoSAP-IT (Amersham Biosciences). Another example in the case of
using ligase or chemical crosslinking or transient linking for
genotype fusing the size of the genotypes may be chosen to
facilitate gel purification e.g the length of the display library
genotype could be chosen to around 250 bp and the length of the
target genotype could be around 100 bp, the resulting fused
genotypes will then be around 350 bp which are easily separated and
purified from the original un-fused species by standard agarose
gels electrotrophoresis or polyacrylamide gel
electrotrophoresis
[0275] In a preferred embodiment of the present invention the fused
genotypes is gel purified.
[0276] In short, the skilled person is aware of numerous different
ways of purifying fused nucleic acid molecules of a B-structure and
a T-structure which was both present within the same individual
compartment.
Polish Fused Genotypes:
[0277] In a preferred embodiment of the present invention the fused
genotype may be polished (in cases where the genotypes are not
fused by an approach compatible with DNA amplification), i.e. to
form an amplifiable bond between the two genotypes in the fused
genomes--an amplifiable bond is a phosphordiester bond (or alike)
between a 3' end of one genotype with a 5' end of the other
genotype in the fused genotype.
[0278] The skilled person in the art can routinely identify
numerous different strategies in order to purify the fused
genotypes, for example without being limited: enzymatically (e.g.
E. coli DNA Ligase, Taq DNA Ligase, 9.degree. N.TM. DNA Ligase, T4
DNA Ligase, T4 RNA Ligase 1 (ssRNA Ligase), T4 RNA Ligase 2 (dsRNA
Ligase), T4 RNA Ligase 2, truncated) or chemically.
[0279] As evident to the skilled person--correct phosphordiester
bond (or alike) formation between cognate genotypes (genotypes
originating from the same compartment) post compartmentalization is
easily controlled because these are pseudo-intramolecular reaction,
thus, independent of concentration of the genotypes. In contrast,
incorrect phosphordiester bond (or alike) formation between
non-cognate genotypes is an intermolecular reaction, thus,
dependent on the concentration of the genotypes.
[0280] In a preferred embodiment of the present invention a DNA
ligase is used for polishing.
[0281] In short, the skilled person is aware of numerous different
ways of polishing fused nucleic acid molecules of a B-structure and
a T-structure which were both present within the same individual
compartment.
Removal or Inactivation of the Target Attached to the Nucleic
Acid
[0282] In a preferred embodiment of the present invention the
target attached to the fused genotypes may be removed or
inactivated.
[0283] The skilled person in the art can routinely identify
numerous different strategies in order to remove or inactivate the
target attached to the fused genotypes, for example without being
limited: heat, protease treatment, 6 M Guanidinium chloride, or
linker cleavage in case the target was attached by a cleavable
linker,
[0284] In a preferred embodiment of the present invention the
target attached to the fused genotypes is removed or inactivated by
heat protease treatment, or 6 M Guanidinium chloride.
[0285] In a preferred embodiment of the present invention the
target attached to the fused genotypes is removed proteinase K
treatment.
[0286] In a preferred embodiment of the present invention the
target attached to the fused genotypes is removed displacement by
primer extension.
[0287] In short, the skilled person is aware of numerous different
ways of removing or destroying the target attached to the fused
nucleic acid molecules of a B-structure and a T-structure which
were both present within the same individual compartment.
Next Round of ECC:
[0288] In a preferred embodiment of the present invention the fused
genotype may be subjected to a next round of ECC, i.e. in a next
round the fused genotypes will be fused with the new target's
genotype--the new target may be the same or a different type as the
previous target.
[0289] The skilled person in the art can appreciate that the
enriched library of fused genotypes of step (iv) is an in vitro
display library.
[0290] In a preferred embodiment of the present invention the
earlier round of ECC target attached to the nucleic acid is removed
or destroyed prior to a next round of ECC.
[0291] In a preferred embodiment of the present invention the
target in a next round of ECC is the same as in an earlier round of
ECC.
[0292] In a preferred embodiment of the present invention the
target in a next round of ECC is not the same as in an earlier
round of ECC.
[0293] In a preferred embodiment of the present invention the
genotype of the target in a next round of ECC is fused to a free
terminus end of the original genotype for the binding entity.
[0294] In a preferred embodiment of the present invention the
genotype of the target in a next round of ECC is fused to a free
terminus end of a target genotype from an earlier round of ECC.
Traditional Selection/Enrichment Methods:
[0295] In a preferred embodiment of the present invention the fused
genotype may be subjected to a round of prior art known traditional
selection/enrichment methods.
[0296] In a preferred embodiment of the present invention the
earlier round of ECC target attached to the nucleic acid is removed
or destroyed prior to a round of traditional selection/enrichment
methods.
[0297] The skilled person in the art can routinely identify
numerous different strategies in order to perform a round of a
round of traditional selection/enrichment methods, for example
without being limited: EP1809743B1 (Vipergen), EP1402024B1
(Nuevolution), EP1423400B1 (David Liu), Nature Chem. Biol. (2009),
5:647-654 (Clark), WO 00/23458 (Harbury), Nature Methods (2006),
3(7), 561-570, 2006 (Miller), Nat. Biotechnol. 2004; 22, 568-574
(Melkko), Nature. (1990); 346(6287), 0 818-822 (Ellington), or Proc
Natl Acad Sci USA (1997). 94 (23): 12297-302 (Roberts).
[0298] In short, the skilled person is aware of numerous different
ways to perform a round of traditional selection/enrichment methods
of the library of fused nucleic acid molecules of a B-structure and
a T-structure which was both present within the same individual
compartment.
Amplification of Fused Genotypes:
[0299] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes may be amplified--i.e. the
BT.sub.Fused-structures present in the enriched library of step
(vi) may be amplified.
[0300] The skilled person in the art can routinely identify
numerous different strategies in order to amplify the nucleic acid
in the fused genotypes, for example without being limited: PCR
(U.S. Pat. No. 4,683,202; Mullis), Emulsion PCR (Nakano et al., J
Biotechnol. 2003; 102(2):117-24), Digital PCR (Vogelstein, B;
Kinzler K W (1999). "Digital PCR". Proc Natl Acad Sci USA. 96 (16):
9236-41), NASBA (Compton J. Nucleic acid sequence-based
amplification. Nature. 1991; 350(6313):91-2), or Rolling Circle
Amplification (American Journal of Pathology. 2001; 159:63-69)
[0301] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the de-compartmentalization step.
[0302] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR.
[0303] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR, where the forward
PCR priming site is in the B-structure genotype and the backward
priming site is in the T-structure genotype.
[0304] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR where the forward
PCR priming site is in the B-structure genotypes and a part of the
backward priming site is in the first T-structure genotypes and the
remaining part is in the second T-structure genotype.
[0305] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR where the forward
PCR priming is in the first T-structure genotypes site and part of
the backward priming site is in the B-structure genotypes and the
remaining part is in the second T-structure genotype.
[0306] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR where part of the
forward PCR priming is in the first T-structure genotypes site and
the remaining part of the forward PCR priming is in the B-structure
genotypes and a part of the backward priming site is in the
B-structure genotypes and the remaining part is in the second
T-structure genotype.
[0307] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR where the forward
PCR priming site is in the B-structure genotypes and 30-70% of the
backward priming site is in the first T-structure genotypes and the
remaining 30-70% is in the second T-structure genotype.
[0308] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR where the forward
PCR priming is in the first T-structure genotypes site and 30-70%
of the backward priming site is in the B-structure genotypes and
the remaining 30-70% is in the second T-structure genotype.
[0309] In a preferred embodiment of the present invention the
nucleic acid in the fused genotypes is amplified subsequently to
the compartmentalization step performed by PCR where 30-70% of the
forward PCR priming is in the first T-structure genotypes site and
the remaining 30-70% of the forward PCR priming is in the
B-structure genotypes and 30-70% of the backward priming site is in
the B-structure genotypes and the remaining 30-70% is in the second
T-structure genotype.
[0310] In short, the skilled person is aware of numerous different
ways of amplifying the nucleic acid in fused nucleic acid molecules
of a B-structure and a T-structure which was both present within
the same individual compartment.
Translation:
[0311] In a preferred embodiment of the present invention the
nucleic acid of fused genotypes may be amplified and subjected to a
translation process where the library of enriched fused genotypes
is translated into a new enriched in vitro display library.
[0312] The skilled person in the art can routinely identify
numerous different strategies in order to amplify and subject the
fused genotypes to a translation process, for example without being
limited: EP1809743B1 (Vipergen), EP1423400B1 (David Liu), WO
00/23458 (Harbury), Nature Methods (2006), 3(7), 561-570, 2006
(Miller), Nature. (1990); 346(6287), 818-822 (Ellington), or Proc
Natl Acad Sci USA (1997). 94 (23): 12297-302 (Roberts).
[0313] In short, the skilled person is aware of numerous different
ways to amplify and perform a translation process of nucleic acid
molecules of a B-structure and a T-structure which was both present
within the same individual compartment.
Analysis for Identities and Composition:
[0314] In a preferred embodiment of the present invention the
nucleic acid of the fused genotype may be analyzed for identities
and composition.
[0315] The skilled person in the art can routinely identify
numerous different strategies in order to analyzed for identities
of the nucleic acid of the fused genotypes, for example without
being limited: sequencing (for review: Metzker, Michael L. (2010).
"Sequencing technologies--the next generation". Nat Rev Genet 11
(1): 31-46.), DNA hybridization technologies (Science 270 (5235):
467-470), restriction enzyme digest, PCR, methods in EP1809743B1
(Vipergen), EP1402024B1 (Nuevolution), EP1423400B1 (David Liu),
Nature Chem. Biol. (2009), 5:647-654 (Clark), WO 00/23458
(Harbury), Nature Methods (2006), 3(7), 561-570, 2006 (Miller),
Nat. Biotechnol. 2004; 22,568-574 (Melkko), Nature. (1990);
346(6287), 818-822 (Ellington), or Proc Natl Acad Sci USA (1997).
94 (23): 12297-302 (Roberts), WO06053571A2 (Rasmussen).
[0316] In a preferred embodiment of the present invention the
nucleic acid of the fused genotype may be analyzed for identities
and composition by DNA sequencing.
[0317] In a preferred embodiment of the present invention the
nucleic acid of the fused genotype may be analyzed for identities
and composition by DNA sequencing using the 454 technology
(Margulies M, Egholm M, Altman W E, et al (September 2005). "Genome
sequencing in microfabricated high-density picolitre reactors".
Nature 437 (7057): 376-80).
[0318] In short, the skilled person is aware of numerous different
ways for analysis for identities and composition of the nucleic
acid of the fused genotype of a B-structure and a T-structure which
was both present within the same individual compartment.
A Separate Independent Aspect of the Invention
[0319] A separate independent aspect of the invention is described
below.
[0320] As understood by the skilled person--the method of this
separate independent aspect of the invention uses the same basic
technical principles as described above for the first aspect of the
invention and thereto related embodiments.
[0321] In line of this and as understood by the skilled
person--specific preferred embodiments of the first aspect of the
invention (such as e.g. that the in vitro compartmentalization
system of step (iv) is a water-in-oil emulsion system) may also be
corresponding preferred embodiments of this separate independent
aspect of the invention.
[0322] Accordingly, a separate independent aspect of the invention
relates to a method for making an enriched library comprising
specific nucleic acid sequence information allowing to identifying
at least one binding entity that binds to at least one target
wherein the specific binding entity has been present in an in vitro
display library and wherein the method comprises the steps of:
(i): making an in vitro display library of at least 100 different
binding entities (B.sub.n (n=100 or more), wherein each binding
entity is attached to a nucleic acid molecule and the nucleic acid
molecule comprises specific nucleic acid sequence information
allowing to identify the binding entity--i.e. once one knows the
specific nucleic acid sequence information of the nucleic acid
molecule one directly knows the structure of the specific binding
entity attached to the nucleic acid molecule--the structure of the
binding entity (i.e. phenotype) attached to the nucleic acid
molecule (genotype) is herein termed B-structure; (ii): making
structures with one target T attached to an enzyme capable of
fusing two DNA molecules, wherein the target is capable of binding
to at least one of the binding entities present in the library of
step (i)--the structure of the target attached to the enzyme
capable of fusing two DNA molecules is herein termed T-structure;
and wherein the method is characterized by that: (iiia): mixing a
solution comprising X (X is a number greater than 10.sup.4) numbers
of B-structures of the library of step (i) with a solution
comprising Y (Y is a number greater than 10.sup.2) numbers of
T-structures of step (ii) under binding conditions, i.e. conditions
where a B-structure containing a binding entity capable of binding
to a target molecule, binds more efficiently to the corresponding
T-structure, than a B-structure containing a binding entity not
capable of binding to the same target do and wherein one gets
binding of at least one of the binding entities to at least one
target thereby creating a complex comprising a B-structure bound to
a T-structure (herein termed B.sub.BoundToT-structure); (iiib):
mixing to the solution of step (iiia) a solution comprising at
least 2 times more nucleic acid molecules than the Y number of
T-structures present in step (iiia), wherein the nucleic acid
molecules comprise specific nucleic acid sequence information
allowing to identify the specific target (herein termed
Target-DNA); (iv): applying an in vitro compartmentalization
system--under binding conditions, i.e. conditions where a
B-structure containing a binding entity capable of binding to a
target molecule, binds more efficiently to the corresponding
T-structure, than a B-structure containing a binding entity not
capable of binding to the same target do--wherein the
compartmentalization system comprises at least 2 times more
individual compartments than the Y number of T-structures present
in step (iii) under conditions wherein the B-structures,
T-structures, B.sub.BoundToT-structures and Target-DNA enter
randomly into the individual compartments; and (v): fusing the
nucleic acid molecules of a B-structure and a Target-DNA which are
both present within the same individual compartment--this structure
is herein termed BT.sub.Fused-structure and the
BT.sub.Fused-structure comprises the specific nucleic acid sequence
information allowing to identify the binding entity of step (i) and
the specific nucleic acid sequence information allowing to identify
the specific target of step (ii); and (vi): combining the content
of the individual compartments of step (v) under conditions wherein
there is no fusing of the nucleic acid molecules of a B-structure
and a T-structure--i.e. there is not created any new
BT.sub.Fused-structure not already created in step (v)--in order to
get a library of BT.sub.Fused-structures, wherein the library is an
enriched library of species of BT.sub.Fused-structures originating
from binding pairs of target and binder entity when compared to
BT.sub.Fused-structures originating from nonbinding pairs of target
and binder entity.
[0323] As known to the skilled person--suitable examples of an
enzyme capable of fusing two DNA molecules are e.g. a ligase or a
polymerase.
[0324] In line of above discussion of the first aspect of the
invention and herein relates embodiment to this--it may be
preferred that the herein relevant nucleic acid molecules are DNA
molecules and in line of this it may be preferred that the ligase
or polymerase is a DNA ligase or a DNA polymerase.
[0325] As understood by the skilled person in the present
context--the fusing of the nucleic acid molecules of a B-structure
and a Target-DNA of step (v) of this separate independent aspect of
the invention is done by the enzyme capable of fusing two DNA
molecules (e.g. a ligase or a polymerase) as present in the
T-structure of step (ii) of this separate independent aspect of the
invention.
[0326] Contrary to the method of the first aspect as discussed
herein (wherein there may be more than one different target T
present)--there is only one target T present in this separate
independent aspect of the invention.
[0327] Accordingly, the specific nucleic acid sequence information
allowing identifying the specific target of the nucleic acid
molecules of step (iiib) of this separate independent aspect of the
invention may simply be a herein relevant characterizing single
sequence.
[0328] In line of above discussion of the first aspect of the
invention it is preferred that this specific nucleic acid sequence
information allowing to identify the specific target is a PCR
amplifiable sequence, since the BT.sub.Fused-structure of step (v)
can then be PCR amplified.
EXAMPLES
Example 1
Enrichment by Co-Compartmentalization Using Overlap ePCR for
Genetype-Genotype Fusion
Spiking Experiment
[0329] For overview see FIG. 2.
Methods
DNA Oligonucleotides Used
[0330] Continuous strand of DNA analogue to the yoctoreactor
[Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp 1322-13271:
TABLE-US-00001
CGCTAAtggtccctggcagtctccTTAGCGgaccGACTCcTgctcGAAGACAACGGTgttttacAC
CGTTGTCTTCgagcTgtACCTGCgcAAGTGCgttttacGCACTTgcGCAGGTacTgtGCAT
CgacAAGACCgttttacGGTCTTgtcGATGCacTgGAGTCggtcCTGTTCGATCTTGGGCG TAT
vip1461: ATACGCCCAAGATCGAACAG vip2501: x-TGGTCCCTGGCAGTCTCC (x =
5'-biotin-TEG) vip2504:
CTGTTCGATCTTGGGCGTATGAGAAGAGCCAGAAACGTGGCTTCAGGCACCAA GGAAGAC
vip2512: GCCTTGCCAGCCCGCTCAGGCAAGTCTTACAGCCGATCAGTCTTCCTTGGTGCC
TGAAG vip2502: CTGTTCGATCTTGGGCGTAT vip2500: x-GCCTTGCCAGCCCGCTCAG
(x = 5' carboxyl) vip157: GCCTTGCCAGCCCGCTCAG vip660:
TGGTCCCTGGCAGTCT vip1481: GAACAGGACCGA vip1471:
CTGTTCGATCTTGGGCGTAT
Preparation of Yoctoreactor Library
[0331] The library is constructed according to Hansen et al. J. Am.
Chem. Soc., 2009, 131 (3), pp 1322-1327) with the following
modification: The splint oligonucleotide vip1481 and the
oligonucleotide vip1471 are used for introducing the backward
priming site.
Preparation of Known Target Binder (Biotin) Attached to Encoding
DNA
[0332] A continuous stranded DNA analogue to the yoctoreactor
library sequences is used for PCR using the vip1461 primer and the
vip2501 primer, which has a 5'-biotin. Thus, a 5'-biotin is
introduced in the yoctoreactor DNA analogue.
Protocol
[0333] PCR mixture:
[0334] 50 .mu.L 2.times.PCR mastermix (40 mM Tris-HCl, 20 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM KCl, 16 mM MgSO.sub.4, 0.2% Triton
X-100, 0.2 mg/mL BSA, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, pH
8.8 @ 25.degree. C.)
10 .mu.L 5M Betaine (final conc. 0.5M) 1 .mu.L 50 .mu.M vip2501
(final conc 0.5 .mu.M) 1 .mu.L 50 .mu.M vip1461 (final conc. 0.5
.mu.M) 1 .mu.L (10.sup.7 molecules) of continuos stranded DNA
analogue to the yoctoreactor 1 .mu.L (2 u/.mu.L) Vent (exo-)
polymerase 36 .mu.L water
[0335] The mixture is subjected to thermal cycling by applying the
following program in a PCR machine:
92 degrees 2 min, 25 cycles of (92 degrees 30 seconds, 72 degrees 1
min), 72 degrees 2 min), 72 degrees for 2 min.
[0336] The 185 bp DNA fragment is purified by PAGE purification
according to standard procedure (Molecular Cloning: A Laboratory
Manual (3-Volume Set), 3rd Edition, 2001-01 by Joseph Sambrook,
David W. Russell, Publisher: Cold Spring Harbor Laboratory Press)
and ethanol precipitated
Preparation of Target DNA (TD)
[0337] The 99-mer target DNA is prepared in a one-step overlapping
PCR protocol and subsequently purified on a 10% TBE-PAGE native
gel
Protocol
PCR Mixture:
[0338] 50 .mu.L 2.times.PCR mastermix (40 mM Tris-HCl, 20 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM KCl, 16 mM MgSO.sub.4, 0.2% Triton
X-100, 0.2 mg/mL BSA, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, pH
8.8 @ 25.degree. C.) 10 .mu.L 5M Betaine (final conc. 0.5M) 1 .mu.L
50 .mu.M vip2500 (final conc 0.5 .mu.M) 1 .mu.L 50 .mu.M vip2502
(final conc. 0.5 .mu.M) 1 .mu.L 20 .mu.M vip2504 (final
concentration 0.2 .mu.M) 1 .mu.L 20 .mu.M vip2512 (final
concentration 0.2 .mu.M) 1 .mu.L (2 u/.mu.L) Vent (exo-) polymerase
35 .mu.L water
[0339] The mixture is subjected to thermal cycling by applying the
following program in a PCR machine:
92 degrees 2 min, 25 cycles of (92 degrees 30 seconds, 72 degrees 1
min), 72 degrees for 2 min.
[0340] The 99 bp DNA fragment is purified by PAGE purification
according to standard procedure (Molecular Cloning: A Laboratory
Manual (3-Volume Set), 3rd Edition, 2001-01 by Joseph Sambrook,
David W. Russell, Publisher: Cold Spring Harbor Laboratory Press).
and ethanol precipitated
DNA--Target Conjugation
Materials
[0341] 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC, 100 mM stock freshly prepared, Aldrich E6383)
N-Hydroxysulfosuccinimide (s-NHS, 200 mM stock, Aldrich 56485)
Morpholinoethanesulfonic acid, MES buffer pH 6.0, 500 mM
beta-Mercaptoethanol, 500 mM stock PCR product with 5' carboxyl
group on lower strand (see above) Streptavidin protein (AbCam
78833) (Target protein)
Protocol
[0342] The carboxyl modified oligonucleotide (ssDNA) or PCR product
with terminal carboxyl can be pre-activated using EDC/s-NHS system
prior to reaction with target protein (see refs for examples of
activation of various types of carboxyls). Exposure of target
protein to EDC may render it inactive, e.g. by chemically modifying
tyrosine or cysteine residues. Thus, before mixing the activated
DNA-carboxyl with target protein, residual EDC optionally may be
quenched by addition of e.g. beta-mercaptoethanol to a final
concentration of 20 mM.
Example Pre-Activation Mixture
Final Conc
TABLE-US-00002 [0343] 10 .mu.L DNA (ssDNA or dsDNA) 10 .mu.L 500 mM
MES, pH 6 100 mM 2.5 .mu.L 100 mM EDC 5 mM 2.5 .mu.L 200 mM s-NHS
10 mM 25 .mu.L water Total 50 .mu.L
[0344] Carboxylic acid activation is allowed to incubate at 20 C
for 15-30 min.
[0345] Optional: To quench residual EDC, 2 .mu.L of 500 mM
beta-mercaptoethanol in water was added (final conc. 20 mM).
[0346] Subsequently, the s-NHS-activated ester should be used
immediately.
Test of Pre-Activation
[0347] An aliquot of the preactivation mixture (25 pmoles ssDNA)
can be diluted with water and a 1% phenethylamine in MeCN (primary
amine that quenches the activated ester). This mixture can be
allowed to react for 15 min, followed by precipitated using EtOH.
After dissolution in 100 mM triethylammonium acetate (TEAA, pH 7),
the DNA product can be analyzed by RP-HPLC using a gradient of MeCN
in 100 mM TEAA. Shift from initial retention time to higher
retention time indicates 1) transformation of carboxyl->NHS
ester and 2) subsequent reaction with amine.
Reaction with Target Protein
[0348] Check enzyme shipment buffer composition for primary amines.
This protocol should tolerate the presence of e.g. DTT or EDTA in
the protein stock solution, but primary amines must be removed e.g.
by dialysis. Primary amines will quench the activated ester thus
abolishing DNA--protein cross-link.
[0349] Otherwise, mix pre-activation mixture and enzyme as
concentrated as possible to drive chemical reactions.
[0350] This should be allowed to incubate for 1-2 h at 20 C
(possibly overnight in cases of slow reaction) then purify
DNA--protein complex by e.g. electrophoresis.
Association Mixture
[0351] A diverse YoctoReactor library consisting of 10.sup.6
different molecules and a total of 10.sup.9 molecules i.e.
potential ligands coupled to double stranded (ds) DNA, is spiked
with 10.sup.6 biotin molecules coupled to ds DNA (known target
binder). The spiked library is mixed with 10.sup.7 molecules
streptavadin coupled to dsDNA (target attached to DNA). The
molecules in the mixtures are allowed to associate in a total
volume of 3 .mu.l Binding Buffer (PBS, 0.05% tween20, 0.2% BSA for
1 hour at room temperature to reach equilibrium. The concentration
of streptavidin (the target) is around 6 .mu.M which is more that
100 fold more than the reported K.sub.d of the biotin-streptavidin
complex of .about.10.sup.-14 mol/L, which means that practical all
biotin will be streptavidin bound at equilibrium.
PCR Mixture
[0352] 67 mM Tris-HCl (pH 8.8), 16.6 mM NH.sub.4SO.sub.4, 6.7 mM
MgCl.sub.2, 10 mM 2-mercaptoethanol, 1 mM of each dNTP, 7.5 .mu.M
of each primer (vip157 and vip660), 45 units of Taq polymerase in a
total volume of 610 .mu.l
Emulsion PCR
[0353] Two mL of an emulsion consisting of approx. 5.times.10.sup.9
compartments per ml is prepared by a method similar to the method
described by Dressman et al., 2003.
[0354] The DNA fragments coupled to the target (streptavadin) or
ligands (non-binding or binding), respectively, have overlapping
regions resulting in the potential assembly of the three fragments
to combined fragments i.e. two types of combined fragment may be
generated through assembly PCR per mixture; fragment (A) signifies
that Streptavadin and Biotin have been present in the same
compartment and fragment (B) signifies that Streptavadin and a
random library molecule (not biotin linked) have been present in
the same compartment. The two types of fragments can be
differentiated through sequencing or restriction site
digestion.
[0355] One mL and 500 .mu.L (1.5 mL) continuous phase is prepared
by dissolving 4.5% (vol/vol) Span80 in mineral oil, followed by
0.40% (vol/vol) Tween80 and 0.05% (vol/vol) Triton X-100 under
constant stirring (1,400 rpm) in a 5 ml round bottom Cryo vial,
using a magnetic stirring bar with a pivot ring. The continuous
phase is split into two times 600 .mu.L in separate 5 ml round
bottom Cryo vials.
[0356] The aqueous phase is made by adding 597 .mu.l PCR mixture to
the 3 .mu.L association mixture. Three hundred (300) .mu.L of the
aqueous phases is gradually added (10 .mu.L every 15 s) to each of
the two continuous phases under constant stirring (1400 rpm) using
a magnetic stirring bar with a pivot ring. After addition of the
aqueous phases, the stirring is continued for 30 min.
[0357] The emulsions are aliquoted into approx. twenty wells of a
96-well PCR plate, each containing 100 .mu.L. The amplification
program comprises of 30 cycles with the following steps: initial
denaturation at 92.degree. C. for 2 min; 20 cycles consisting of
dsDNA denaturation at 92.degree. C. for 30 s, primer annealing and
extension at 72.degree. C. for 2 min and 30 s; and final elongation
at 72.degree. C. for 2 min.
Breaking the Emulsion
[0358] The DNA fragments from the emulsion PCR are rescued by
pooling the emulsions and centrifuging at 13,000 g for 5 min at
25.degree. C. The oil phase is discarded. Residual mineral oil and
surfactants are removed from the emulsion by performing the
following extraction twice: add 1 ml of water-saturated diethyl
ether, vortex the tube, and dispose of the upper (solvent)
phase.
Anticipated Results
[0359] Assuming; equal size spherical compartments, random
distribution of molecules and complex in compartments, 100%
association of Streptavadin-Biotin complex, no dissociation of
Streptavadin-Biotin complex, assuming none or few binders with
adequate binding affinity for streptavidin in the yR library, no
bias of the PCR reaction, and 100% reaction efficiency in "primer
extension" (only one priming site present--no
co-compartmentalization) during the PCR cycling and 10 000 fold
amplification when both priming sites present
(co-compartmentalization).
[0360] After emulsion PCR the theoretical amounts of the different
DNA species are:
Streptavadin fragment (100 bp): 10.sup.7 molecules Streptavadin
fragment (100 nt): 20 cycles times 10.sup.7
molecules=2.times.10.sup.8 molecules YoctoReactor fragment (250
bp): 10.sup.9 molecules YoctoReactor fragment (250 nt): 20 cycles
times 10.sup.9 molecules=2.times.10.sup.16 molecules (all original
Biotin fragment assumed converted to fused species see below)
[0361] Fused genotype--known binder: Fragment A
(Streptavadin-Biotin DNA fragment) (330 bp): 10 000.times.10.sup.6
molecules=10.sup.10 (PCR amplification times number of biotin
fragments)--the probability under the above mentioned assumption
for co-compartmentalize the Biotin fragment with target DNA is
1.
[0362] Fused genotype--non-binder: Fragment B (random
co-compartmentalized fragments) (330 bp): 10
000.times.10.sup.-3.times.10.sup.9 molecules=10.sup.10 (PCR
amplification times probability for random co-compartmentalization
times number of initial yR library molecules)--the probability
under the above mentioned assumption for co-compartmentalize a
non-binder is #target molecules/#
compartments=10.sup.7/10.sup.10=10.sup.-3
[0363] Consequently, after this process 50% of the 330 bp fragments
contain DNA origination from biotin. Moreover, the 330 bp fragment
constitutes about 15 ng and constitutes most of the total double
stranded DNA. The single stranded DNA is conveniently removed by
ExoSAP-IT (Amersham Biosciences) according to manufactures
instructions and the 330 bp fragment is conveniently PAGE purified
by standard procedure.
Analysis of Enrichment by DNA Sequencing Using 454 Sequencing
Technology
[0364] The 454 sequencing priming sites is introduced by PCR using
primers with terminal A and B sequences. The resulting fragment is
PAGE purified and submitted for 454 DNA sequencing using
manufactures protocol.
[0365] The DNA sequences are analyzed and the frequency of the
biotin genotype calculated.
Conclusions
[0366] As can be understood from above--by using the method as
described herein one gets an 1000 times enrichment--It is expected
from the above calculation that the biotin genotype will be
observed with a high frequency .about.1 out 2 whereas each of the
assumed non-binding yoctoreactor library members will be observed
.about.1 out of 2 million on average. Consequently, the binder has
been enriched 1000 fold over each of the non-binders. This will
demonstrate the feasibility of the present new invention.
Example 2
Enrichment by Co-Compartmentalization Using eLigation for
Genetype-Genotype fusion
Spiking Experiment
[0367] For overview see FIG. 1.
Methods
DNA Oligonucleotides Used
TABLE-US-00003 [0368] vip1481: GAACAGGACCGA vip1471:
CTGTTCGATCTTGGGCGTAT vip2513: ACGCCCAAGATCGAACAG
[0369] Biotin-modified continuos one-stranded DNA analogue to the
yoctoreactor:
TABLE-US-00004 X-
CGCTAAtggtccctggcagtctccTTAGCGgaccGACTCcTgctcGAAGACAACGGTgttttacAC
CGTTGTCTTCgagcTgtACCTGCgcAAGTGCgttttacGCACTTgcGCAGGTacTgtGCAT
CgacAAGACCgttttacGGTCTTgtcGATGCacTgGAGTCggtcCTGTTCGATCTTGGGCG TAT
(x = 5'-biotin-TEG)
[0370] The biotin-modified continuous one-stranded DNA analogue to
the yoctoreactor may be assembled by smaller oligonucleotides
essentially as described in Hansen et al. J. Am. Chem. Soc., 2009,
131 (3), pp 1322-1327) using a 5'-biotin TEG-modified
oligonucleotide in the 5'-position
TABLE-US-00005 vip2514: x-
GCCTTGCCAGCCCGCTCAGGGGAAGGACGTTGGTGTAGAAGCGTTCACTT GGTGGAAGTAT (x =
5' carboxyl) vip2515: ACTTCCACCAAGTGAACGCT vip157:
GCCTTGCCAGCCCGCTCAG vip660: TGGTCCCTGGCAGTCT
Preparation of Yoctoreactor Library
[0371] The library is constructed according to Hansen et al. J. Am.
Chem. Soc., 2009, 131 (3), pp 1322-1327) with the following
modifications: [0372] 1) The splint oligonucleotide vip1481 and the
oligonucleotide vip1471 are used for introducing the backward
priming site [0373] 2) The oligonucleotide vip2513 is used for the
dismantling of the yoctoreactor by primer extension
Preparation of Known Target Binder (Biotin) Attached to Encoding
DNA
[0374] The known target binder (biotin) attached to double-stranded
encoding DNA is made by dismantling the biotin-modified continuos
one-stranded DNA analogue to the yoctoreactor by primer extension
using vip2513. (Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp
1322-1327). The primer is chosen, so a 3'-overhang of 2 nt is made.
The 3'-overhang will facilitate the subsequent ligation.
[0375] The double stranded DNA fragment is purified by PAGE
purification according to standard procedure (Molecular Cloning: A
Laboratory Manual (3-Volume Set), 3rd Edition, 2001-01 by Joseph
Sambrook, David W. Russell, Publisher: Cold Spring Harbor
Laboratory Press) and ethanol precipitated.
Preparation of Target DNA (TD)
Protocol
[0376] The 61-mer target DNA is prepared by primer extension and
subsequently purified on a 10% TBE-PAGE native gel. The primer is
chosen, so a 2 nucleotide 3'-overhang, complementary to the
3'-overhang of the yoctoreactor 3'-overhang, is made. Furthermore,
ligation is enabled by phosphorylating the primer.
Example:
Phosphorylation of Primer
[0377] 2 .mu.L (200 pmol) vip2515 20 .mu.L 10.times. buffer 0 (50
mM Tris-HCl (pH 7.5 at 37.degree. C.), 10 mM MgCl 2, 100 mM NaCl,
0.1 mg/ml BSA) 2 .mu.l 100 mM ATP (final concentration 2 mM)
10 .mu.L T4 Polynucleotide Kinase (100 u)
[0378] 66 .mu.L Water, nuclease-free
[0379] The phosphorylation reaction is incubated @37 degrees C. for
30 minutes, and the kinase is inactivated by incubation @75 degrees
C. for 10 minutes
[0380] The DNA is precipitated by ethanol precipitation, washed in
70% ethanol, and resuspended in 10 .mu.L TE buffer.
Primer Extension:
[0381] 10 .mu.L phosphorylated vip2515 (200 pmoles) 10 .mu.L
10.times. buffer 0 (New England Biolabs) 2 .mu.L 10 mM dNTP mix
(final concentration 0.2 mM of dATP, dCTP, dGTP, and dTTP) 77 .mu.L
water 1 .mu.L (5 u) Klenow (exo-, 5 u/.mu.L)
[0382] The reaction is allowed to proceed for 15 minutes, and the
double stranded DNA purified by extraction from a 15% acrylamide
gel according to standard procedure (Molecular Cloning: A
Laboratory Manual (3-Volume Set), 3rd Edition, 2001-01 by Joseph
Sambrook, David W. Russell, Publisher: Cold Spring Harbor
Laboratory Press). and ethanol precipitated
DNA--Target Conjugation
Materials
[0383] 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC, 100 mM stock freshly prepared, Aldrich E6383)
N-Hydroxysulfosuccinimide (s-NHS, 200 mM stock, Aldrich 56485)
Morpholinoethanesulfonic acid, MES buffer pH 6.0, 500 mM
beta-Mercaptoethanol, 500 mM stock PCR product with 5' carboxyl
group on lower strand (see above) Streptavidin protein (AbCam
78833) (Target protein)
Protocol
[0384] The carboxyl modified oligonucleotide (ssDNA) or PCR product
with terminal carboxyl can be pre-activated using EDC/s-NHS system
prior to reaction with target protein (see refs for examples of
activation of various types of carboxyls). Exposure of target
protein to EDC may render it inactive, e.g. by chemically modifying
tyrosine or cysteine residues. Thus, before mixing the activated
DNA-carboxyl with target protein, residual EDC optionally may be
quenched by addition of e.g. beta-mercaptoethanol to a final
concentration of 20 mM.
Example Pre-Activation Mixture
Final Conc
TABLE-US-00006 [0385] 10 .mu.L DNA (ssDNA or dsDNA) 10 .mu.L 500 mM
MES, pH 6 100 mM 2.5 .mu.L 100 mM EDC 5 mM 2.5 .mu.L 200 mM s-NHS
10 mM 25 .mu.L water Total 50 .mu.L
[0386] Carboxylic acid activation is allowed to incubate at 20 C
for 15-30 min.
[0387] Optional: To quench residual EDC, 2 .mu.L of 500 mM
beta-mercaptoethanol in water was added (final conc. 20 mM).
[0388] Subsequently, the s-NHS-activated ester should be used
immediately.
Test of Pre-Activation
[0389] An aliquot of the preactivation mixture (25 pmoles ssDNA)
can be diluted with water and a 1% phenethylamine in MeCN (primary
amine that quenches the activated ester). This mixture can be
allowed to react for 15 min, followed by precipitated using EtOH.
After dissolution in 100 mM triethylammonium acetate (TEAA, pH 7),
the DNA product can be analyzed by RP-HPLC using a gradient of MeCN
in 100 mM TEAA. Shift from initial retention time to higher
retention time indicates 1) transformation of carboxyl->NHS
ester and 2) subsequent reaction with amine.
Reaction with Target Protein
[0390] Check enzyme shipment buffer composition for primary amines.
This protocol should tolerate the presence of e.g. DTT or EDTA in
the protein stock solution, but primary amines must be removed e.g.
by dialysis. Primary amines will quench the activated ester thus
abolishing DNA--protein cross-link.
[0391] Otherwise, mix pre-activation mixture and enzyme as
concentrated as possible to drive chemical reactions.
[0392] This should be allowed to incubate for 1-2 h at 20 C
(possibly overnight in cases of slow reaction) then purify
DNA--protein complex by e.g. electrophoresis.
Association Mixture
[0393] A diverse YoctoReactor library consisting of 10.sup.6
different molecules and a total of 10.sup.9 molecules i.e.
potential ligands coupled to double stranded (ds) DNA, is spiked
with 10.sup.6 biotin molecules coupled to ds DNA (known target
binder). The spiked library is mixed with 10.sup.7 molecules
streptavadin coupled to dsDNA (target attached to DNA). The
molecules in the mixtures are allowed to associate in a total
volume of 3 .mu.l Binding Buffer (PBS, 0.05% tween20, 0.2% BSA for
1 hour at room temperature to reach equilibrium. The concentration
of streptavidin (the target) is around 6 .mu.M which is more that
100 fold more than the reported K.sub.d of the biotin-streptavidin
complex of 10.sup.-14 mol/L, which means that practically all
biotin will be streptavidin bound at equilibrium.
Ligation Mixture
[0394] 1.times.Tag ligation buffer (20 mM Tris-HCl, 25 mM potassium
acetate, 10 mM Magnesium Acetate, 1 mM NAD, 10 mM Dithiothreitol
0.1% Triton X-100 pH 7.6 @ 25.degree. C.) is added 2 .mu.L (40
u/.mu.L) Taq DNA ligase in a total volume of 610 .mu.L.
[0395] 67 mM Tris-HCl (pH 8.8), 16.6 mM NH.sub.4SO.sub.4, 6.7 mM
MgCl.sub.2, 10 mM 2-mercaptoethanol, 1 mM of each dNTP, 7.5 .mu.M
of each primer (vip157 and vip660), 45 units of Taq polymerase in a
total volume of 610 .mu.l
Ligation in Emulsion
[0396] Two mL of an emulsion consisting of approx. 5.times.10.sup.9
compartments per ml is prepared by a method similar to the method
described by Dressman et al., 2003.
[0397] The DNA fragments coupled to the target (streptavadin) or
ligands (non-binding or binding), respectively, are able to be
ligated on one strand. i.e. two types of combined fragment may be
generated through ligation; fragment (A) signifies that
Streptavadin and Biotin have been present in the same compartment
and fragment (B) signifies that Streptavadin and a random library
molecule (not biotin linked) have been present in the same
compartment. The two types of fragments can be differentiated
through sequencing or restriction site digestion.
[0398] One mL and 500 .mu.L (1.5 mL) continuous phase is prepared
by dissolving 4.5% (vol/vol) Span80 in mineral oil, followed by
0.40% (vol/vol) Tween80 and 0.05% (vol/vol) Triton X-100 under
constant stirring (1,400 rpm) in a 5 ml round bottom Cryo vial,
using a magnetic stirring bar with a pivot ring. The continuous
phase is split into two times 600 .mu.L in separate 5 ml round
bottom Cryo vials and is kept ice-cold. The aqueous phase is made
by adding 597 .mu.l ice-cold ligation mixture to the 3 .mu.L
association mixture. Three hundred (300) .mu.L of the aqueous phase
is gradually added (10 .mu.L every 15 s) to each of the two
continuous phases under constant stirring (1400 rpm) using a
magnetic stirring bar with a pivot ring. After addition of the
aqueous phase, the stirring is continued for 30 min under ice-cold
conditions.
[0399] The emulsions are heated to 45 degrees C. and allowed to
ligate for one hour.
Breaking the Emulsion
[0400] The ligation mixtures are added 30 .mu.L 500 mM EDTA each
and vortexed briefly. The DNA fragments are rescued by pooling the
emulsions and centrifuging at 13,000 g for 5 min at 25.degree. C.
The oil phase is discarded. Residual mineral oil and surfactants
are removed from the emulsion by performing the following
extraction twice: add 1 ml of water-saturated diethyl ether, vortex
the tube, and dispose of the upper (solvent) phase.
[0401] The DNA is concentrated by precipitation, is fractionated by
size on denaturing 10% polyacrylamide gels and the ligated
fragments isolated by PAGE purification according to standard
procedure (Molecular Cloning: A Laboratory Manual (3-Volume Set),
3rd Edition, 2001-01 by Joseph Sambrook, David W. Russell,
Publisher: Cold Spring Harbor Laboratory Press), ethanol
precipitated and resuspended in 10 .mu.L TE buffer.
Amplification of Ligated DNA
[0402] Finally, the ligated fragments are amplified by PCR
Example:
PCR Mixture:
[0403] 10 .mu.L purified ligated DNA 50 .mu.L 2.times.PCR mastermix
(40 mM Tris-HCl, 20 mM (NH.sub.4).sub.2SO.sub.4, 20 mM KCl, 16 mM
MgSO.sub.4, 0.2% Triton X-100, 0.2 mg/mL BSA, 0.4 mM each of dATP,
dTTP, dGTP, and dCTP, pH 8.8 @ 25.degree. C.) 10 .mu.L 5M Betaine
(final conc. 0.5M) 1 .mu.L 50 .mu.M vip167 (final conc 0.5 .mu.M) 1
.mu.L 50 .mu.M vip660 (final conc. 0.5 .mu.M) 1 .mu.L (2 u/.mu.L)
Vent (exo-) polymerase 27 .mu.L water
[0404] The mixture is subjected to thermal cycling by applying the
following program in a PCR machine:
92 degrees C., 2 min, 25 cycles of (92 degrees C. 30 seconds, 72
degrees C. 1 min), 72 degrees C. for 2 min.
[0405] The resulting library of DNA fragments is sequenced, and the
enrichment for the binding fragment calculated.
Anticipated Results
[0406] Assuming; equal size spherical compartments, random
distribution of molecules and complex in compartments, 100%
association of Streptavadin-Biotin complex, no dissociation of
Streptavadin-Biotin complex, assuming none or few binders with
adequate binding affinity for streptavidin in the yR library, no
bias of the ligation reaction or the following PCR reaction, --no
co-compartmentalization) during the ligation and 100 reaction
efficiency when the library and target DNA fragments are
co-compartmentalized.
[0407] After emulsion ligation, theoretical amounts of the ligated
DNA species are: (all original Biotin fragment assumed converted to
fused species see below)
[0408] Ligated genotype--known binder: Fragment A
(Streptavadin-Biotin DNA fragment) (250 bp): 10.sup.6
molecules--the probability under the above mentioned assumption for
co-compartmentalize the Biotin fragment with target DNA is 1.
[0409] Fused genotype--non-binder: Fragment B (random
co-compartmentalized fragments) (250 bp): 10.sup.-3.times.10.sup.9
molecules=10.sup.6 (Probability for random co-compartmentalization
times number initial yR library molecules)--the probability under
the above mentioned assumption for co-compartmentalize a non-binder
is #target molecules/#
compartments=10.sup.7/10.sup.10=10.sup.-3
[0410] The final PCR amplification is expected to be of same
efficiency for the two types of molecules, and Consequently, after
this process 50% of the 330 by fragments contain DNA originating
from the biotin-streptavidin binding.
Analysis of Enrichment by DNA Sequencing Using 454 Sequencing
Technology
[0411] The 454 sequencing priming sites is introduced by PCR using
primers with terminal A and B sequences. The resulting fragment is
PAGE purified and submitted for 454 DNA sequencing using
manufactures protocol.
[0412] The DNA sequences are analyzed and the frequency of the
biotin genotype calculated.
Conclusions
[0413] As can be understood from above--by using the method as
described herein one gets an 1000 times enrichment--It is expected
from the above calculation that the biotin genotype will be
observed with a high frequency .about.1 out 2 whereas each of the
assumed non-binding yoctoreactor library members will be observed
.about.1 out of 2 million on average. Consequently, the binder has
been enriched 1000 fold over each of the non-binders. This will
demonstrate the feasibility of the present new invention.
Examples Below
[0414] All the Examples below were made based on the technical
information disclosed above (e.g. in the working examples above)
plus based on the common general knowledge of the skilled
person.
Example 3
Enrichment by Co-Compartmentalization Using Overlap ePCR for
Genotype-Genotype Fusion
[0415] The fundamental principle of ECC, co-compartmentalization of
binding partners and fusion of their attached DNA as a result
hereof, was demonstrated, using biotin and streptavidin (SA) as the
binding partners. Biotin DNA conjugate (yR_biotin) was subjected to
ECC using SA conjugated to DNA (SA_TD001) as the target. As a
negative control ECC was run in parallel using SA_TD001
preincubated with biotin as the targets. For overview see FIG.
2.
Methods
DNA Oligonucleotides Applied:
[0416] Applied for continuous strand of DNA analogue to the
yoctoreactor (Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp
1322-1327):
TABLE-US-00007
CGCTAAtggtccctggcagtctccTTAGCGgaccGACTCcTgctcGAAGACAACGGTgttttacAC
CGTTGTCTTCgagcTgtACCTGCgcAAGTGCgttttacGCACTTgcGCAGGTacTgtGCAT
CgacAAGACCgttttacGGTCTTgtcGATGCacTgGAGTCggtcCTGTTCGATCTTGGGCG TAT
vip1481: GAACAGGACCGA vip1471: CTGTTCGATCTTGGGCGTAT Applied for
yR_biotin vip1461: ATACGCCCAAGATCGAACAG vip2501:
x-TGGTCCCTGGCAGTCTCC (x = biotin-TEG) Applied for e PCR vip157:
GCCTTGCCAGCCCGCTCAG vip660: TGGTCCCTGGCAGTCT Applied for TD001
vip2500: x-GCCTTGCCAGCCCGCTCAG (x = carboxyl modification) vip2502:
CTGTTCGATCTTGGGCGTAT vip2512:
GCCTTGCCAGCCCGCTCAGGCAAGTCTTACAGCCGATCAGTCTTCCTTGGTGCC TGAAG
vip2507: CTGTTCGATCTTGGGCGTATTGTTTTAGCTGCCCCAACTCCTTCAGGCACCAAG
GAAGAC Applied for Rescue PCR vip2549: GCAAGTCTTACAGCCGATCA vip660:
TGGTCCCTGGCAGTCT
Preparation of yR
[0417] Continuous strand of DNA analogue to the yoctoreactor was
constructed as described in example 1
Preparation of yR_Biotin
[0418] Preparation of known target binder (biotin) attached to yR
was constructed as described in example 1
Preparation of Target DNA (TD001)
[0419] The TD001 was prepared as described in example 1 except the
oligo vip2507 being applied instead of Vip2504.
Materials
[0420] MOPS 3-(N-Morpholino) propanesulfonic acid (Sigma-Aldrich)
Silicone polyether/cyclopentasiloxane (Dow Corning, DC5225C)
Cyclopentasiloxane/trimethylsiloxysilicate (Dow Corning, DC749)
[0421] AR20 silicone oil (Sigma-Aldrich)
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC,
100 mM stock freshly prepared, Aldrich E6383)
N-Hydroxysulfosuccinimide (s-NHS, 200 mM stock, Aldrich 56485)
Morpholinoethanesulfonic acid, MES buffer pH 6.0, 500 mM
beta-Mercaptoethanol, 500 mM stock PCR product with 5' carboxyl
group on lower strand (see above) Streptavidin protein (AbCam
78833) (Target protein) Slide-A-lyzer mini (Pierce)
TissueLyzer II (Qiagen)
DNA--Target Conjugation
Pre-Activation
[0422] Pre-activation was done by mixing 5.4 .mu.l TD001 [9.3
.mu.M] with 1 .mu.l MOPS pH 6 [1 M], 1 .mu.l EDC [50 mM], 1 .mu.l
s-NHS [100 mM] and 1.6 .mu.l water.
[0423] Carboxylic acid activation is allowed to incubate at
20.degree. C. for 30 min.
[0424] To quench residual EDC, 1 .mu.l of 250 mM
beta-mercaptoethanol in water was added.
Reaction with Target Protein
[0425] Prior to conjugation, SA was dialyzed 2 times 30 min against
Dialysis Buffer (10 mM MOPS (pH 8), 50 mM NaCl) using Slide-A-Lyzer
mini dialysis device according to manufactures instructions
(Pierce).
[0426] 5 .mu.l of dialyzed SA [58 .mu.M] was added to 1.6 .mu.l
MOPS pH 6 [1 M], 1.6 .mu.l NaCl in water [1 M] and 11 .mu.l TD001
[4.6 .mu.M]. The reaction was allowed for 2 hours at 20.degree.
C.
[0427] To quench the reaction, 2 .mu.l Tris pH 8 [1 M] was added.
The SA_TD001 conjugate was isolated from reactants by PAGE from a
6% TBE gel that was run for 40 min at 200V.
[0428] The bands are extracted 3 times in 500 .mu.l Extraction
Buffer (50 mM Tris pH8, 150 mM NaCl, 0.1% Tween20) at 4.degree. C.
(30 min/o.n./30 min).
[0429] Residual gel was removed by filtration, and the samples
concentrated in a Microcon YM30 device according to manufactures
instructions (Millipore). The concentration of the conjugate was
estimated to be 0.38 .mu.M by measuring the DNA concentration using
Picogreen according to manufactures instructions (Molecular
Probes).
Association Reactions (Binding Reaction)
[0430] Prior to yR_biotin and SA_TD001 binding, 6e8 molecules
SA_TD001 molecules/.mu.l in a total volume of 50 .mu.l Association
Buffer (10 mM Tris-HCl (pH 7.8), 0.05% Triton-X100.) was incubated
with or without 1 .mu.M biotin (6e11 molecules biotin/.mu.l) for 30
min at 20.degree. C.
[0431] The following binding reactions were made in Association
Buffer: [0432] 1) 3e8 yR_biotin molecules/.mu.l and 3e8 SA_TD001
molecules/.mu.l in a total volume of 50 .mu.l, using SA_TD001 that
had not been pre-incubated with biotin [0433] 2) 3e8 yR_biotin
molecules/.mu.l and 3e8 SA_TD001 molecules/.mu.l in a total volume
of 50 .mu.l, using SA_TD001 that had been pre-incubated with
biotin.
[0434] The binding reaction was incubated for 1 h at 20.degree. C.
and hereafter diluted to a concentration of 3e6 molecules/.mu.l of
yR_biotin and 3e6 molecules/.mu.l SA_TD001 in Association
Buffer.
Emulsion PCR (ePCR)
[0435] Assembly of yR and TD001 and amplification of yR_TD001
fusion molecule in emulsion using PCR.
Continuous Phase
[0436] Continuous phase was prepared as described by (Turner and
Hurles, Nat Protoc. 2009; 4(12): 1771-1783).
1200 .mu.l continuous phase was made per reaction 480 .mu.l
Silicone polyether/cyclopentasiloxane (DC5225C)
360 .mu.l Cyclopentasiloxane/trimethylsiloxysilicate (DC749)
[0437] 360 .mu.l AR20 silicone oil
PCR Aqueous Phase
[0438] 600 .mu.l PCR aq. was made per reaction: 60 .mu.l Pfu buffer
(10.times.) 12 .mu.l BSA (50 mg/ml) 12 .mu.l dNTP (10 mM)
3 .mu.l Vip157 (100 .mu.M)
3 .mu.l Vip660 (100 .mu.M)
[0439] 4 .mu.l Pfu-turbo (2.5 u/.mu.l) 446 .mu.l water 60 .mu.l
template the resulting concentration of yR_biotin and SA_TD001 are
3e5 molecules/.mu.l of each
Emulsification
[0440] In a 2 ml Eppendorf tube 1000 .mu.l continuous phase and 500
.mu.l PCR phase, and a 5 mm steel bead were added per reaction
[0441] The reaction was emulsified by mixing for 8 min at 30 Hz in
a Tissuelyser II at 20.degree. C. 100 .mu.l emulsion was added per
PCR tube and the mixture was subjected to thermal cycling by
applying the following program in a PCR machine:
92.degree. C. for 2 min, 30 cycles of (92.degree. C. 30 seconds,
55.degree. C. for 1 min and 72.degree. C. for 1.5 min), 72.degree.
C. for 5 min.
Recovery of DNA
[0442] The emulsion was broken by adding 100 .mu.l 1-butanol per
PCR tube. The contents of 8 PCR tubes per condition were pooled and
600 .mu.l NaCl in water [4 M] was added. The content was mixed by
vortexing for 10 sec at max speed, and the organic phase was
removed after centrifugation at 14000 g for 1 min. Another 800
.mu.l 1-butanol was added to the pooled PCR product and the
vortexing and centrifugation step was repeated. The extraction with
1-butanol was repeated one more time.
[0443] The DNA was further purified by PCR purification columns
(Macherey-Nagel) according to manufactures instructions. Elute with
50 .mu.l elution buffer per condition
[0444] The eluted DNA was diluted 20 fold in Dilution Buffer (10 mM
Tris (pH 7.8), 20 mM NaCl, 0.1% Triton-X100) prior to the rescue
PCR.
Rescue PCR
[0445] Amplification of yR_TD001 fusion molecule
PCR Mixture Per Reaction:
[0446] 50 .mu.L 2.times.PCR mastermix (40 mM Tris-HCl, 20 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM KCl, 16 mM MgSO.sub.4, 0.2% Triton
X-100, 0.2 mg/mL BSA, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, pH
8.8 at 25.degree. C.) 1 .mu.L 50 .mu.M vip2549 (final conc 0.5
.mu.M) 1 .mu.L 50 .mu.M vip660 (final conc. 0.5 .mu.M) 1 .mu.L (2
u/.mu.L) Vent (exo-) polymerase 10 .mu.l template 37 .mu.L
water
[0447] The mixture was subjected to thermal cycling by applying the
following program in a PCR machine:
92.degree. C. for 2 min, 20 cycles of (92.degree. C. 30 seconds,
55.degree. C. for 1 min and 72.degree. C. for 1.5 min), 72.degree.
C. for 5 min.
Results
[0448] The fusion product yR_TD001 has a predicted length of 245
bp. The DNA products were visualized on a 10% TBE PAGE run for 40
min. at 200V. The results showed the presence of a band with the
expected size, if the association reaction was performed without
pre-incubation with biotin (lane 1) and the absence of a band if
association reaction was performed on molecules that had been
pre-incubated with biotin (lane 2), see FIG. 4.
Conclusion
[0449] This result demonstrated co-compartmentalization of binding
partners and fusion of their attached DNA as a result hereof.
Example 4
Enrichment by Co-Compartmentalization Using Overlap ePCR for
Genotype-Genotype Fusion
Spiking Experiment
[0450] ECC was demonstrated by enriching for yR_biotin that was
spiked into a diverse yR library using SA_TD001 as the target. As a
negative control ECC was run in parallel using SA_TD001
preincubated with biotin as the targets.
Methods
DNA Oligonucleotides Applied
[0451] Applied for continuous strand of DNA analogue to the
yoctoreactor (Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp
1322-1327):
TABLE-US-00008
CGCTAAtggtccctggcagtctccTTAGCGgaccGACTCcTgctcGAAGACAACGGTgttttacAC
CGTTGTCTTCgagcTgtACCTGCgcAAGTGCgttttacGCACTTgcGCAGGTacTgtGCAT
CgacAAGACCgttttacGGTCTTgtcGATGCacTgGAGTCggtcCTGTTCGATCTTGGGCG TAT
vip1481: GAACAGGACCGA vip1471: CTGTTCGATCTTGGGCGTAT Applied for
yR_biotin vip1461: ATACGCCCAAGATCGAACAG vip2501:
x-TGGTCCCTGGCAGTCTCC (x = biotin-TEG) Applied for e PCR vip157:
GCCTTGCCAGCCCGCTCAG vip660: TGGTCCCTGGCAGTCT Applied for TD001
vip2500: x-GCCTTGCCAGCCCGCTCAG (x = carboxyl modification) vip2502:
CTGTTCGATCTTGGGCGTAT vip2512:
GCCTTGCCAGCCCGCTCAGGCAAGTCTTACAGCCGATCAGTCTTCCTTGGTGCC TGAAG
vip2507: CTGTTCGATCTTGGGCGTATTGTTTTAGCTGCCCCAACTCCTTCAGGCACCAAG
GAAGAC Applied for Rescue PCR vip2549: GCAAGTCTTACAGCCGATCA vip660:
TGGTCCCTGGCAGTCT Applied for PCR of a yR diverse library vip341:
TGGTCCCTGGCAGTCTCC vip1461: ATACGCCCAAGATCGAACAG Applied for 454
PCR vip2593: CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGTCTTCCTTGGTGCCTGAAG
vip2465: CCATCTCATCCCTGCGTGTCTCCGACTCAGAGGTTGGTCCCTGGCAGTCTCC
vip2467: CCATCTCATCCCTGCGTGTCTCCGACTCAGATCGTGGTCCCTGGCAGTCTCC
vip2468: CCATCTCATCCCTGCGTGTCTCCGACTCAGATGCTGGTCCCTGGCAGTCTCC
vip2469: CCATCTCATCCCTGCGTGTCTCCGACTCAGCACTTGGTCCCTGGCAGTCTCC
vip2470: CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGATGGTCCCTGGCAGTCTCC
vip2471: CCATCTCATCCCTGCGTGTCTCCGACTCAGCCATTGGTCCCTGGCAGTCTCC
Preparation of yR_Biotin
[0452] yR_biotin was prepared as described in example 1
Preparation of a Diverse Yoctoreactor Library
[0453] The yR library was essentially constructed as described by
(Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp 1322-1327). The
diverse yR library was PCR amplified by the following method;
PCR Mixture Per Reaction:
[0454] 50 .mu.L 2.times.PCR mastermix (40 mM Tris-HCl, 20 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM KCl, 16 mM MgSO.sub.4, 0.2% Triton
X-100, 0.2 mg/mL BSA, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, pH
8.8 at 25.degree. C.) 10 .mu.L 5M Betaine (final conc. 0.5M) 1
.mu.L 50 .mu.M vip341 (final conc 0.5 .mu.M) 1 .mu.L 50 .mu.M
vip1461 (final conc. 0.5 .mu.M) 1 .mu.L (10.sup.8 molecules) of
continuous stranded DNA analogue to the yoctoreactor 1 .mu.L (2
u/.mu.L) Vent (exo-) polymerase 36 .mu.L water
[0455] The mixture was subjected to thermal cycling by applying the
following program in a PCR machine:
92.degree. C. for 2 min, 15 cycles of (92.degree. C. for 30
seconds, 72.degree. C. for 1 min), 72.degree. C. for 2 min.
[0456] The 185 bp DNA fragment was purified by PAGE purification
according to standard procedure (Molecular Cloning: A Laboratory
Manual (3-Volume Set), 3rd Edition, 2001-01 by Joseph Sambrook,
David W. Russell, Publisher: Cold Spring Harbor Laboratory Press)
and ethanol precipitated
Association Reactions (Binding Reactions)
[0457] Binding reactions was performed as described in example 3,
with the following changes.
[0458] Prior to yR_biotin and SA_TD001 binding, 6e8 molecules
SA_TD001 molecules/.mu.l in a total volume of 50 .mu.l association
buffer was incubated with or without 1 .mu.M biotin (6e11 molecules
biotin/.mu.l) for 30 min at 20.degree. C.
[0459] The following association reactions were made in Association
Buffer: [0460] 1) 3e7 yR_biotin molecules/.mu.l and 3e8 SA_TD001
molecules/.mu.l in a total volume of 50 .mu.l, using SA_TD001 that
had not been pre-incubated with biotin [0461] 2) 3e7 yR_biotin
molecules/.mu.l and 3e8 SA_TD001 molecules/.mu.l in a total volume
of 50 .mu.l, using SA_TD001 that had been pre-incubated with
biotin.
[0462] Binding reactions were incubated for 1 h at 20.degree.
C.
[0463] Hereafter the following conditions were setup: [0464] A)
Without biotin pre-incubation: A 1000 fold dilution of binding
reaction (1) to a concentration of 3e4 molecules/.mu.l of yR_biotin
and 3e5 molecules/.mu.l SA_TD001 in Association Buffer containing
3e7 molecules yR library/.mu.l (final concentration). [0465] B)
With biotin pre-incubation: A 1000 fold dilution of association
reaction (2) to a concentration of 3e4 molecules/.mu.l of yR_biotin
and 3e5 molecules/.mu.l SA_TD001 in Association Buffer containing
3e7 molecules yR library/.mu.l (final concentration). [0466] C)
Without yR-biotin: Association Buffer containing a 3e7 yR library
molecules/.mu.l and 3e5 molecules/.mu.l SA_TD001 in Association
Buffer was made
Emulsion PCR
[0467] ePCR was performed as described in example 3, but performed
in duplicate and with 40 PCR cycles
Recovery of DNA
[0468] Breaking of emulsions was performed as described in example
3, but performed by pooling the emulsions from 16 PCR tubes and
eluting with 100 .mu.l elution buffer per condition
Rescue PCR
[0469] Rescue PCR was performed as described in example 3, but with
the following thermal profile during PCR amplification; 92.degree.
C. for 2 min, 20 cycles of (92.degree. C. for 30 seconds,
72.degree. C. for 1.5 min), 72.degree. C. for 5 min.
Preparation for 454-Sequencing
[0470] PCR protocol for amplification of yR_TD001 fusion molecules
hereby including 454 sequence tags into the sequences leading to a
predicted size of 309 bp. For each condition and duplicate a unique
forward primer was applied.
PCR Mixture Per Reaction:
[0471] 50 .mu.L 2.times.PCR mastermix (40 mM Tris-HCl, 20 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM KCl, 16 mM MgSO.sub.4, 0.2% Triton
X-100, 0.2 mg/mL BSA, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, pH
8.8 at 25.degree. C.)
10 .mu.L 5M Betaine (final conc. 0.5M) 1 .mu.L 50 .mu.M vip2593
(final conc 0.5 .mu.M) 2 .mu.L 25 .mu.M vip2465, vip2467, vip2468,
vip2469, vip2470 or vip2471 (final conc. 0.5 .mu.M) 5 .mu.L
Template from each condition and duplicate 1 .mu.L (2 u/.mu.L) Vent
(exo-) polymerase 31 .mu.L water
[0472] The DNA was purified on PCR purification columns
(Macherey-Nagel) according to manufactures instructions and the DNA
concentrations were determined using a spectrophotometer
(Eppendorf). The concentrations were adjusted upon comparative
visual inspection of the products on 10% TBE gels that were run for
40 min at 200V. The DNA products were pooled so that all DNA
products were represented by similar amounts of DNA. The pooled DNA
was run on a 10% TBE PAGE gel and a DNA fragment of approx. 309 bp
was purified by PAGE purification according to standard procedure
(Molecular Cloning: A Laboratory Manual (3-Volume Set), 3rd
Edition, 2001-01 by Joseph Sambrook, David W. Russell, Publisher:
Cold Spring Harbor Laboratory Press), and ethanol precipitated.
454-Sequencing
[0473] 454-sequencing was performed as described by (Hansen et al.
J. Am. Chem. Soc., 2009, 131 (3), pp 1322-1327).
Results
[0474] The sequencing results showed, see FIG. 5, that although the
yR_biotin was spiked into the yR library in a 1000 fold lower
concentration than the yR library the percentages of yR_biotin
counts were:
37.6% and 32% for the duplicates in condition (A) i.e. enrichment
without pre-incubation of SA_TD001 with biotin. 0.30% and 0.72% for
the duplicates in condition (B) i.e. enrichment with pre-incubation
of SA_TD001 with biotin. 0.02% and 0.05% for the duplicates in
condition (C) i.e. enrichment without yR_biotin included in the
sample.
[0475] Consequently, more than 300 fold enrichment of yR_biotin was
observed using SA_TD001 as the target. In contrast, the negative
control target, SA_TD001 preincubated with biotin, provided a 3-7
fold enrichment.
CONCLUSION
[0476] ECC was demonstrated by enriching for yR_biotin that was
spiked into a diverse yR library using SA_TD001 as the target.
Example 5
Enrichment by Co-Compartmentalization Using eLigation for
Genotype-Genotype Fusion
[0477] The fundamental principle of ECC, co-compartmentalization of
binding partners and fusion of their attached DNA as a result
hereof, was demonstrated, using desthiobiotin (desBio) and
streptavidin (SA) as the binding partners. Desthiobiotin DNA
conjugate (yR_desBio) was subjected to ECC using SA conjugated to
DNA (SA_TD002) as the target. As a negative control ECC was run in
parallel using SA_TD002 preincubated with biotin as the
targets.
[0478] For overview see FIG. 1.
Methods
[0479] DNA oligonucleotides used are described in example 2. In
addition, the following were applied:
TABLE-US-00009 Applied for yR labeled with desthiobiotin
(desBio_yR): vip2815: x-TGGTCCCTGGCAGTCTCC (x = desthiobiotin)
vip2535: CACCACGATGGCAATGCATTCTTCGCTGCCATTCTG Applied for rescue
PCR: vip660: TGGTCCCTGGCAGTCT vip2824: CGATGTCCTGAGGTGGAAGT Applied
for `Scavenger DNA`: vip2554:
GGCAAGTGATTGTCCATGTGCATGAGAAGAGGCCCACATT vip2555:
CACATGGACAATCACTTGCC vip2556: AATGTGGGCCTCTTCTCATG Applied for
TD002 vip2528: TCCACATCCTCCAGTTCA vip2529:
ACTTCCACCTCAGGACATCGAGCTGGAGCTTGCTGTTAGC vip2530:
AGGTTCGCTCCCTCCTTAAGTCAGGAGGATGTGACACCAA vip2531:
CGATGTCCTGAGGTGGAAGTTGAACTGGAGGATGTGGACA vip2532:
CTTAAGGAGGGAGCGAACCTGCTAACAGCAAGCTCCAGCT vip2558:
x-TTGGTGTCACATCCTCCTGA (x = C6-amino modification)
Preparation of yR Labeled with Desthiobiotin (desBio_yR)
[0480] A 5'-desthiobiotin was introduced in the yR analogue by
using primers vip2815 and vip2535 in PCR with a continuous stranded
DNA analogue to the yoctoreactor library sequences as template DNA
(Hansen et al. J. Am. Chem. Soc., 2009, 131 (3), pp 1322-1327). To
create a 2 bp overhang suitable for ligation to target DNA
conjugated with streptavidin, the PCR product was digested with
BseMI.
Preparation of Target DNA (TD002)
Protocol
[0481] TD002 (98 bp double stranded DNA with a GA nucleotide
overhang and 5' carboxyl group on the lower strand) was assembled
by ligation of phosphorylated oligonucleotides vip2528, vip2529,
vip2530, vip2531 and vip2532 and the non-phosphorylated
oligonucleotide vip2558. Phosphorylation with T4 Polynucleotide
Kinase and ligation with T4 DNA ligase was performed according to
manufactures instructions (Fermentas). The double stranded DNA
fragment was purified by PAGE purification according to standard
procedure (Molecular Cloning: A Laboratory Manual (3-Volume Set),
3rd Edition, 2001-01 by Joseph Sambrook, David W. Russell,
Publisher: Cold Spring Harbor Laboratory Press) and precipitated
with ethanol. Prior to ligation vip2559 was modified to have a 5'
carboxyl group. Thus, simple C6-amino modification was interchanged
to a carboxylic acid by treatment with disuccinimidylsuberate (DSS,
C8-di-NHS ester, Pierce #21580). The oligonucleotide was treated
with 40 mM DSS in HEPBS buffer pH 9 in a water--NMP 1:1 mixture
over night followed by treatment with LiOH to hydrolyse the
remaining NHS ester. After neutralization and precipitation, the
crude carboxy modified oligonucleotide was used without further
modification.
Preparation of Target DNA Conjugated with Streptavidin
(SA_TD002)
[0482] The SA_TD002 was prepared as described in example 3.
Association Reactions (Binding Reaction)
Materials
1 M Tris-HCl, pH 7.5
4 M NaCl
[0483] 10% triton X-100 10 .mu.M biotin
SA_TD002
[0484] desBio_yR
Protocol
[0485] In a total volume of 0.5 .mu.l Binding Buffer (10 mM
Tris-HCl (pH7.5), 50 mM NaCl, 0.1% triton), 3E8 desBio_yR molecules
were mixed with 1.4E9 molecules SA_TD002 in the presence or absence
of 1 .mu.M biotin (inhibitor). Association of the molecules was
allowed by incubating the binding mixtures for 1 hour on ice.
Dissociation Reactions (Dilution)
Materials
Standard Ligation Buffer:
50 mM Tris-HCl, pH 7.5
50 mM NaCl
0.1% Triton X-100
0.75 .mu.M BSA
9 mM KCL
4.5% Glycerol
0.2 mM EDTA
1 mM DTT
2 mM ATP
[0486] 1 .mu.M T4 DNA ligase (Fermentas) 0.01 .mu.M `Scavenger DNA`
(40-mer nicked dsDNA fragment): 40-mer dsDNA fragment containing a
single nick was prepared by assembly of oligonucleotides vip2554,
phosphorylated vip2555 and vip2556.
[0487] Continuous phase was prepared as described in example 3
[0488] 2 mL micro tubes with screw cap
Protocol
[0489] A volume of 0.12 .mu.L was transferred from the binding
mixture to the lid of a 2 mL Eppendorf tube containing 600 .mu.L
aqueous phase containing 1 .mu.M T4 DNA ligase (standard ligation
buffer). The dissociation reaction was initiated by mixing the
binding mixture with the aqueous phase by inverting the tubes twice
followed by vortexing the tubes for 10 seconds. After a short spin
in the microcentrifuge, 500 .mu.L of the mixture was transferred to
an ice-cold 2 mL micro tube containing 1 mL continuous phase and
left on ice for the remaining time to finally obtain a dissociation
time of 2 minutes.
Emulsification
Materials
Induction Buffer:
50 mM Tris-HCl, pH 7.5
50 mM NaCl
0.1% Triton X-100
1.5 .mu.M BSA
10 mM KCL
5% Glycerol
0.2 mM EDTA
1 mM DTT
2 mM ATP
135 mM MgCl2
Protocol
[0490] The dissociation reactions were terminated exactly 2 min.
after initiation by mixing the continuous phase (1 mL) and the
aqueous phase (0.5 mL) by emulsification for 3.times.20 seconds at
5500 rpm (with 10 seconds pause in between the 20 seconds runs) on
the Precellys24 (Bertin Technologies). In parallel,
induction-emulsions containing magnesium but no ligase for the
activation of T4 DNA ligase were prepared by emulsification for
3.times.20 seconds at 5500 rpm of 1 mL continuous phase and 0.5 mL
aqueous phase containing 135 mM MgCl.sub.2 (Induction Buffer).
Ligation in Emulsion
Protocol
[0491] A volume of 150 .mu.L induction-emulsion containing
MgCl.sub.2 was added per emulsion and mixed by rotation for one
hour at RT to activate T4 DNA ligase. Ligation of desBio_yR and
SA_TD002 in emulsion was allowed by incubating the emulsions (1650
.mu.L) for 16 hours in a thermo block at 16.degree. C. and 300
rpm.
Emulsion Breaking and DNA Recovery
Materials
[0492] 1-butanol
Isopropanol
[0493] 100% ethanol 100 bp no-limits DNA PCR clean-up kit
(NucleoSpin ExtractII, Macherey-Nagel) 10% triton X-100
Protocol
[0494] The ligation reaction was stopped by incubating the tubes
for 30 minutes at 65.degree. C. followed by a short spin in the
microcentrifuge. For breaking of the emulsions, half of the volume
of each emulsion was transferred to a clean 2 mL eppendorf tube. To
each tube 850 .mu.L 1-butanol plus 15 ng 100 bp no-limits DNA [10
ng/.mu.L] was added and mixed by thoroughly vortexing for 10
seconds. The tubes were centrifuged for 1 min at 14,000.times.g and
the supernatant was discarded. Residual silicone oil and
surfactants were removed from the emulsion by repeating the
1-butanol extraction once more with the addition of 1 volume of
1-butanol. The recovered water-phases of the previously splitted
emulsions were pooled into one tube and the DNA fragments
(desBio_yR_TD002_SA fusion molecules) were rescued by purification
using a PCR clean-up kit (NucleoSpin ExtractII, Macherey-Nagel)
according to the supplier's recommendations. The DNA was eluted
into EB buffer (5 mM Tris/HCl, pH 8.5) containing 0.1% triton
X-100. Prior to qPCR analysis, the eluted DNA was diluted 10 fold
in Dilution Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.05%
Triton-X100).
Amplification and Analysis of Ligated DNA (Rescue PCR)
Materials
2.times.PCR Mastermix (pH 8.8 at 25.degree. C.):
40 mM Tris-HCl
20 mM (NH.sub.4).sub.2SO.sub.4
20 mM KCl
16 mM MgSO.sub.4,
0.2% Triton X-100,
[0495] 0.2 mg/mL BSA 0.4 mM of each nucleotide suitable for
hot-start PCR dATP, dTTP, dGTP, and dCTP (CleanAmp, TriLink)
Protocol
[0496] To analyze the number of desBio_yR_TD002_SA fusion molecules
present in the different emulsions, qPCR with primers vip660 and
vip2824 and using 5 .mu.L of the 10 fold diluted purified recovered
DNA samples as a template was run. For the standard curve 3E8, 3E7,
3E6, 3E5 or 3E4 copies of pre-ligated yR_TD002 (control template)
were added per qPCR reaction.
qPCR Mixture Per Reaction: 5 .mu.L template 10 .mu.L 2.times.PCR
mastermix 2 .mu.L 5M Betaine (final conc. 0.5 M) 0.4 .mu.L SyBR
Green (final conc. 2.5E-5%) 0.2 .mu.L 100 .mu.M vip660 (final conc.
1 .mu.M) 0.2 .mu.L 100 .mu.M vip2824 (final conc 1 .mu.M) 0.2 .mu.L
(2 u/.mu.L) Vent (exo-) polymerase (Fermentas) 2 .mu.L water
[0497] The mixture was subjected to thermal cycling by applying the
following program in the qPCR machine:
92.degree. C. for 10 min
[0498] 30 cycles of 95.degree. C. 30 seconds and 72.degree. C. for
2 min 30 seconds
72.degree. C. for 5 min
Results
[0499] In order to calculate the number of fusion molecules present
in the different qPCR reactions, the standard curve was defined.
The calculated numbers of desBio_yR_TD002_SA fusion molecules were
translated into a column chart to visualize the difference between
the signal obtained in association reactions incubated in presence
or absence of 1 .mu.M biotin (FIG. 6). The results show the
presence of a significantly higher number of fusion molecules if
the association reaction was performed in the absence of biotin
compared to if the association reaction was performed in the
presence of the inhibitor biotin.
Conclusion
[0500] This result demonstrated co-compartmentalization of
desBio_yR and SA_TD002 molecules originating from the
desthiobiotin-streptavidin binding and ligation of their attached
DNA as a result hereof.
Example 6
Enrichment by Co-Compartmentalization Using eLigation for
Genotype-Genotype Fusion
Spiking Experiment
[0501] ECC was demonstrated by enriching for
N-benzyl-4-sulfamoyl-benzamide conjugated to yR DNA (BSB_yR) that
was spiked into a diverse yR library using human Carbonic anhydrase
II as the target. As a negative control ECC was run in parallel
using target preincubated with BSB as the target. Furthermore,
dissociation time dependent enrichment was demonstrated in the same
system.
[0502] For overview see FIG. 1.
Methods
[0503] DNA oligonucleotides used are described in example 2 and 5.
In addition, the following were applied:
TABLE-US-00010 Used for yR labeled with BSB (BSB_yR): vip2260:
ATGAAAGACGTGGCCATTGC vip2724_vip2607:
CTGACATGGTCCCTGGCAGTCTCCTGTCAGGACCGACTCCXGCTCGAAGA C (x =
dT-C6-amino modification) vip2970:
CTATCGGTTTTACCGATAGGTCTTCGAGCTGTACCTGCGC vip2973:
AGCTAGGTTTTACCTAGCTGCGCAGGTACTGTGCATCGAC vip2980:
CTATCGGTTTTACCGATAGGTCGATGCACTGGAGTCGGTC Used for TD003: vip2536:
CTTATGCTGGCAGTTTCA vip2529:
ACTTCCACCTCAGGACATCGAGCTGGAGCTTGCTGTTAGC vip2538:
AGGTTCGCTCCCTCCTTAAGCCAGCAGTGGTAATTCGACA vip2996:
CGATGTCCTGAGGTGGAAGTTGAAACTGCCAGCATAAGGA vip2532:
CTTAAGGAGGGAGCGAACCTGCTAACAGCAAGCTCCAGCT vip2559:
x-TGTCGAATTACCACTGCTGG (x = C6-amino modification) Used for
454-sequencing: Vip3018:
CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCGATGTCCTGAGGTGGAAGT vip2459:
CCATCTCATCCCTGCGTGTCTCCGACTCAGAACCTGGTCCCTGGCAGTCT CC vip2460:
CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGGTGGTCCCTGGCAGTCT CC vip2461:
CCATCTCATCCCTGCGTGTCTCCGACTCAGACACTGGTCCCTGGCAGTCT CC vip2462:
CCATCTCATCCCTGCGTGTCTCCGACTCAGACTGTGGTCCCTGGCAGTCT CC vip2463:
CCATCTCATCCCTGCGTGTCTCCGACTCAGAGAGTGGTCCCTGGCAGTCT CC vip2464:
CCATCTCATCCCTGCGTGTCTCCGACTCAGAGCATGGTCCCTGGCAGTCT CC vip2465:
CCATCTCATCCCTGCGTGTCTCCGACTCAGAGGTTGGTCCCTGGCAGTCT CC vip2466:
CCATCTCATCCCTGCGTGTCTCCGACTCAGAGTCTGGTCCCTGGCAGTCT CC vip2467:
CCATCTCATCCCTGCGTGTCTCCGACTCAGATCGTGGTCCCTGGCAGTCT CC vip2468:
CCATCTCATCCCTGCGTGTCTCCGACTCAGATGCTGGTCCCTGGCAGTCT CC vip2469:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCACTTGGTCCCTGGCAGTCT CC vip2470:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGATGGTCCCTGGCAGTCT CC vip2471:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCCATTGGTCCCTGGCAGTCT CC vip2472:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCCTATGGTCCCTGGCAGTCT CC vip2473:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCGAATGGTCCCTGGCAGTCT CC vip2474:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCTACTGGTCCCTGGCAGTCT CC vip2475:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCTCATGGTCCCTGGCAGTCT CC vip2476:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCTGTTGGTCCCTGGCAGTCT CC vip2477:
CCATCTCATCCCTGCGTGTCTCCGACTCAGCTTGTGGTCCCTGGCAGTCT CC vip2478:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGAACTGGTCCCTGGCAGTCT CC vip2479:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGACATGGTCCCTGGCAGTCT CC vip2480:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGAGTTGGTCCCTGGCAGTCT CC vip2481:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGATGTGGTCCCTGGCAGTCT CC vip2482:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGCAATGGTCCCTGGCAGTCT CC vip2483:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGTCTTGGTCCCTGGCAGTCT CC vip2484:
CCATCTCATCCCTGCGTGTCTCCGACTCAGGTGATGGTCCCTGGCAGTCT CC vip2485:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTACGTGGTCCCTGGCAGTCT CC vip2486:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTAGCTGGTCCCTGGCAGTCT CC vip2487:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTCAGTGGTCCCTGGCAGTCT CC vip2488:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTCCATGGTCCCTGGCAGTCT CC vip2489:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTCGTTGGTCCCTGGCAGTCT CC vip2490:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCTGGTCCCTGGCAGTCT CC vip2491:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTGACTGGTCCCTGGCAGTCT CC vip2492:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTGTGTGGTCCCTGGCAGTCT CC vip2493:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCCTGGTCCCTGGCAGTCT CC vip2494:
CCATCTCATCCCTGCGTGTCTCCGACTCAGTTGGTGGTCCCTGGCAGTCT CC
Preparation of Yoctoreactor Library
[0504] The library was constructed according to (Hansen et al. J.
Am. Chem. Soc., 2009, 131 (3), pp 1322-1327) but in the tetramer
format instead of the trimer format.
Preparation of yR Labeled with BSB (BSB_yR)
Materials
[0505] 10 mM Fmoc-NH-PEG(12)-CO.sub.2H
100 mM 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMT-MM) 200 mM
N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) pH 9
(HEPBS) N-Methyl-2-pyrrolidone (NMP) 0.5 M piperidine in NMP
10 mM Fmoc-L-Phenylglycine (Fmoc-L-Phg)
[0506] 10 mM 4-carboxybenzenesulfonamide 0.1 M
acetonitrile/triethylammonium acetate (pH 7) BSB: BSB was
synthesized according to (Drabovich et al., Anal. Chem. 2009, 81,
490-494)
Protocol
[0507] BSB_yR was prepared by ligation. Position 2 oligonucleotide
vip2970, position 3 oligonucleotide vip2973 and position 4
oligonucleotide vip2980 were prepared for ligation by
phosphorylation with T4 Polynucleotide Kinase performed according
to manufactures instructions (Fermentas). BSB labeling of position
1 oligonucleotide vip2724_vip2607 was synthesized according to the
following reaction scheme:
##STR00016##
[0508] Amino-PEG12 derived oligonucleotide was synthesized from a
51 mer oligonucleotide (vip2724_vip2607) [5 nmol] with internally
modified dT (amino-C6-dT) that was coupled with
Fmoc-NH-PEG(12)-CO.sub.2H [10 mM] in a solution of 100 mM DMT-MM,
200 mM HEPBS pH 9, in 200 L NMP:water 1:1. After 1 hour the mixture
was ethanol precipitated and dissolved in 100 L water. A volume of
100 L 0.5 M piperidine in NMP was added and incubated at 25.degree.
C. for 2 hr. The amino-PEG12-oligonucleotide was isolated by
ethanol precipitation and used without further purification in the
next coupling.
[0509] BSB-PEG12 labeled position 1 conjugate was synthesized by
conjugating Fmoc-L-Phg to a 51 mer amino modified oligonucleotide
in which the primary amine was linked through a PEG12 linker on an
internally modified dT. The amino-PEG12 derived oligonucleotide was
coupled with Fmoc-L-Phg [10 mM] in a solution of 100 mM DMT-MM and
200 mM HEPBS pH 9 in 200 L NMP:water 1:1. After 1 hour the mixture
was ethanol precipitated and dissolved in 100 L water. 100 L 0.5 M
piperidine in NMP was added and incubated at 25.degree. C. for 2
hr. Final coupling of 4-carboxybenzenesulfonamide was made by
treatment of a solution of the L-Phg-PEG12 derived oligonucleotide
with 4-carboxybenzenesulfonamide [10 mM] in a solution of 100 mM
DMT-MM and 200 mM HEPBS pH 9 in 200 L NMP:water 1:1. After
incubation at 25.degree. C. for 1 hour the crude oligonucleotide
conjugate was isolated by ethanol precipitation and purified by
reverse phase HPLC on a C-18 Waters XBridge column with
acetonitrile/triethylammonium acetate (pH 7, 0.1 M) gradient 6-50%
acetonitrile over 20 min. Appropriate fractions were collected and
evaporated in vacuo and resulted into 1680 pmol of vip2724_vip2607
labeled with BSB. Equivalent amounts of the stem complimentary
position 1 oligonucleotide vip2724_vip2607 conjugated with BSB,
position 2, position 3 and position 4 oligonucleotides were mixed
and ligated to form the yR (Hansen et al. J. Am. Chem. Soc., 2009,
131 (3), pp 1322-1327). Ligation with T4 DNA ligase was performed
according to manufactures instructions (Fermentas). The BSB_yR was
un-folded and made double stranded via primer extension using
phosphorylated vip2260 in a Klenow (exo-) driven reaction performed
according to manufactures instructions (Fermentas).
Preparation of Target DNA (TD003)
[0510] TD003 was prepared similar to TD002 as described in example
5.
Conjugation of Carbonic Anhydrase II to TD003 (CAII_TD003)
[0511] Recombinant human CAII (RnD systems; 2184CA)
Protocol
[0512] Conjugation of TD003 to CAII was done similarly as described
in example 3. Pre-activation mixture with target DNA for
conjugation with CAII. Pre-activation was done by mixing 21 .mu.L
TD003 [4.7 .mu.M] with 3 .mu.L MOPS pH 6 [1 M], 3 .mu.L EDC [50
mM], and 3 .mu.L s-NHS [100 mM].
[0513] Carboxylic acid activation was allowed to incubate at
20.degree. C. for 30 min. The buffer was removed by using a G25
Illustra column according to manufactures instructions (GE
Healthcare).
[0514] Prior to conjugation, the protein was dialyzed 2.times.30
min against a Dialysis Buffer at 4.degree. C. using Slide-A-Lyzer
mini dialysis device according to manufactures instructions
(Pierce).
[0515] For the conjugation reaction 1 .mu.L MOPS [1M] pH 8.0, 1
.mu.L NaCl [1 M] and 1 .mu.L water was added to 9 .mu.L of dialyzed
CAII protein. Approx. 35 .mu.L activated DNA was added to this
mixture. The reaction was incubated at 4.degree. C. for 20 h.
[0516] The conjugation reaction was quenched by adding Tris (pH 8)
to a final concentration of 50 mM. The CAII_TD003 conjugate was
isolated from reactants by PAGE from a 6% TBE gel as described in
example 3. The concentration of the conjugate was estimated to be
0.21 .mu.M by measuring the DNA concentration using Picogreen
according to manufactures instructions (Molecular Probes).
Association Reactions (Binding Reaction)
Materials
1 M Tris-HCl, pH 7.5
4 M NaCl
[0517] 10% triton
10 .mu.M BSB
Protocol
[0518] In a total volume of 1.5 .mu.l Binding Buffer (10 mM
Tris-HCl (pH7.5), 50 mM NaCl, 0.1% triton X-100), 6.5E10 library
molecules and 3.3E5 BSB_yR molecules (YoctoReactor library
consisting of 1E12 molecules spiked 1 to 200 000 with 5E6 BSB_yR
molecules) were mixed with 9E9 molecules CAII_TD003 in the presence
or absence of 1 .mu.M BSB (inhibitor). Association of the molecules
was allowed by incubating the binding mixtures for 1 hour on
ice.
Dissociation Reactions (Dilution)
Materials
[0519] Standard ligation buffer:
50 mM Tris-HCl, pH 7.5
50 mM NaCl
0.1% Triton X-100
0.75 .mu.M BSA
9 mM KCL
4.5% Glycerol
0.2 mM EDTA
1 mM DTT
2 mM ATP
[0520] 1 .mu.M T4 DNA ligase (Fermentas)
0.01 .mu.M `Scavenger` DNA
[0521] Continuous phase was prepared as described in example 3 2 mL
micro tubes with screw cap
Protocol
[0522] A volume of 0.12 .mu.l was transferred from the binding
mixture to the lid of a 2 mL Eppendorf tube containing 600 .mu.L
aqueous phase containing 1 .mu.M T4 DNA ligase (standard ligation
buffer). The dissociation reaction was initiated by mixing the
binding mixture with the aqueous phase by inverting the tubes twice
followed by vortexing the tubes thoroughly for 10 seconds. After a
short spin on the micro centrifuge, 500 .mu.L of the mixture was
transferred to an ice-cold 2 mL tube containing 1 mL continuous
phase and left on ice for the remaining time to finally obtain
dissociation times of 2 or 30 minutes.
Emulsification
Materials
[0523] Induction buffer:
50 mM Tris-HCl, pH 7.5
50 mM NaCl
0.1% Triton X-100
1.5 .mu.M BSA
10 mM KCL
5% Glycerol
0.2 mM EDTA
1 mM DTT
2 mM ATP
135 mM MgCl.sub.2
Protocol
[0524] The dissociation reaction was terminated exactly 2 min or 30
min after initiation by mixing the continuous phase (1 mL) and the
aqueous phase (0.5 mL) by emulsification for 3.times.20 seconds at
5500 rpm (with 10 seconds pause in between the seconds runs) on the
Precellys 24 (Bertin Technologies). In parallel,
induction-emulsions containing magnesium but no ligase for the
activation of T4 DNA ligase were prepared by emulsification for
3.times.20 seconds at 5500 rpm of 1 mL continuous phase and 0.5 mL
aqueous phase containing 135 mM MgCl.sub.2 (induction buffer).
Ligation in Emulsion
Protocol
[0525] A volume of 150 .mu.L induction-emulsion containing
MgCl.sub.2 was added per emulsion and mixed by rotation for one
hour at RT to activate T4 DNA ligase. Ligation was allowed by
incubating the emulsions (1650 .mu.L) for 16 hours in a thermo
block at 16.degree. C. and 300 rpm.
Emulsion Breaking and DNA Recovery
Materials
[0526] 1-butanol
Isopropanol
[0527] 100% ethanol 100 bp no-limits DNA
TissueLyser II (Qiagen)
[0528] PCR clean-up kit (NucleoSpin ExtractII, Macherey-Nagel) 10%
triton X-100
Protocol
[0529] The ligation reaction was stopped by incubating the tubes
for 30 minutes at 65.degree. C. followed by a short spin on the
micro centrifuge. For breaking of the emulsions, 300
.mu.L1-butanol, 150 .mu.L isopropanol, 50 .mu.L ethanol and 20 ng
100 bp no-limits DNA [10 ng/.mu.L] was added per emulsion and mixed
on the TissueLyser II (Qiagen) for 1 min at 15 Hz. Subsequently the
tubes were rotated for 1 hour at RT, centrifuged for 2 min at
14,000.times.g and the supernatant was discarded. Residual silicone
oil and surfactants were removed from the emulsion by performing
the following extraction twice: addition of 1 volume of 1-butanol,
mixing for 1 min at 15 Hz on the TissueLyser, and discarding the
upper phase. The DNA fragments were rescued by purification using a
PCR clean-up kit (NucleoSpin ExtractII, Macherey-Nagel) according
to the supplier's recommendations. The DNA was eluted into EB
buffer (5 mM Tris/HCl, pH 8.5) containing 0.1% triton.
Amplification of Ligated DNA (Rescue PCR)
Materials
2.times.PCR Mastermix (pH 8.8 at 25.degree. C.):
40 mM Tris-HCl
20 mM (NH.sub.4).sub.2SO.sub.4
20 mM KCl
16 mM MgSO.sub.4,
0.2% Triton X-100,
[0530] 0.2 mg/mL BSA 0.4 mM of each nucleotide suitable for
hot-start PCR dATP, dTTP, dGTP, and dCTP (CleanAmp, TriLink)
Protocol
[0531] The ligated fragments were amplified by PCR using 10 .mu.L
of the purified recovered DNA samples as a template in a total
volume of 100 .mu.L.
PCR mixture: 10 .mu.L template DNA
50 .mu.L 2.times.PCR Mastermix
[0532] 10 .mu.L 5 M Betaine (final conc. 0.5 M) 1 .mu.L 100 .mu.M
vip660 (final conc. 1 .mu.M) 1 .mu.L 100 .mu.M vip2824 (final conc.
1 .mu.M) 1 .mu.L (2 u/.mu.L) Vent (exo-) polymerase (Fermentas) 27
.mu.L water
[0533] The mixture was subjected to thermal cycling by applying the
following program in a PCR machine:
10 min at 95.degree. C.
[0534] 32 cycles of 30 sec at 95.degree. C. and 2 min 30 sec at
72.degree. C.
2 min at 72.degree. C.
Preparation for 454-Sequencing
Protocol
[0535] Samples for 454-sequencing were prepared as described for
the yR_TD001 fusion molecules in example 4. 454-Sequencing tags
were included in the PCR for amplification of yR_TD003 fusion
molecules using unique forward primers vip2459 to vip2494.
DNA Sequencing
Protocol
[0536] 454-Sequencing was performed as described by (Hansen et al.
J. Am. Chem. Soc., 2009, 131 (3), pp 1322-1327). The DNA sequences
were analyzed and the frequency of the BSB genotype calculated.
Results
[0537] The sequencing results (FIG. 7) showed that the BSB_yR was
successfully enriched for (about 1300 fold) by CA II using a
dissociation time of two minutes. In contrast, no enrichment of
BSB_DNA was observed when using a dissociation time of 30 minutes
or when CA II was preincubated with BSB prior to the binding
step.
Conclusion
[0538] ECC was demonstrated by enriching in dissociation time
dependent fashion for BSB_yR that was spiked into a diverse yR
library using CAII as the target.
REFERENCE LIST
[0539] 1: EP1809743B1 (Vipergen) [0540] 2: EP1402024B1
(Nuevolution) [0541] 3: EP1423400B1 (David Liu) [0542] 4: Nature
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(Harbury) [0544] 6: Nature Methods (2006), 3(7), 561-570 7: 2006
(Miller) [0545] 7: Nat. Biotechnol. 2004; 22,568-574 (Melkko)
[0546] 8: Nature. (1990); 346(6287), 818-822 (Ellington) [0547] 9:
Proc Natl Acad Sci USA (1997). 94 (23): 12297-302 (Roberts) [0548]
10: WO06053571A2 (Rasmussen) [0549] 11: Bertschinger et al, (2007)
Protein Engineering, Design & Selection vol. 20 no. 2 pp. 57-68
[0550] 12: Miller O J, Bernath K, Agresti J J, Amitai G, Kelly B T,
Mastrobattista E, Taly V, Magdassi S, Tawfik D S, Griffiths A D.
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(1998) Man-made cell-like compartments for molecular evolution.
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and Holliger, P. (2001) Proc. Natl. Acad. Sci. USA, 98, 4552-4557;
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R J. Selection of restriction endonucleases using artificial cells.
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Mastrobattista E, Taly V, Chanudet E, Treacy P, Kelly B T,
Griffiths A D. High-throughput screening of enzyme libraries: in
vitro evolution of a beta-galactosidase by fluorescence-activated
sorting of double emulsions. Chem Biol. 2005 December;
12(12):1291-300 [0558] 20: Levy M, Griswold K E, Ellington A D.
Direct selection of trans-acting ligase ribozymes by in vitro
compartmentalization. RNA. 2005 October; 11(10):1555-62. Epub 2005
Aug. 30; [0559] 21: Sepp A, Choo Y. Cell-free selection of zinc
finger DNA-binding proteins using in vitro compartmentalization. J
Mol Biol. 2005 Nov. 25; 354(2):212-9. Epub 2005 Oct. 3; [0560] 22:
Bernath K, Magdassi S, Tawfik D S. Directed evolution of protein
inhibitors of DNA-nucleases by in vitro compartmentalization (IVC)
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Nat Protoc. 2009; 4(12): 1771-1783
Sequence CWU 1
1
781191DNAArtificial Sequencesource1..191/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 1cgctaatggt
ccctggcagt ctccttagcg gaccgactcc tgctcgaaga caacggtgtt 60ttacaccgtt
gtcttcgagc tgtacctgcg caagtgcgtt ttacgcactt gcgcaggtac
120tgtgcatcga caagaccgtt ttacggtctt gtcgatgcac tggagtcggt
cctgttcgat 180cttgggcgta t 191220DNAArtificial
Sequencesource1..20/mol_type="DNA" /note="Primer"
/organism="Artificial Sequence" 2atacgcccaa gatcgaacag
20318DNAArtificial Sequencesource1..18/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 3tggtccctgg cagtctcc
18460DNAArtificial Sequencesource1..60/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 4ctgttcgatc
ttgggcgtat gagaagagcc agaaacgtgg cttcaggcac caaggaagac
60559DNAArtificial Sequencesource1..59/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 5gccttgccag
cccgctcagg caagtcttac agccgatcag tcttccttgg tgcctgaag
59620DNAArtificial Sequencesource1..20/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 6ctgttcgatc
ttgggcgtat 20719DNAArtificial Sequencesource1..19/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 7gccttgccag
cccgctcag 19819DNAArtificial Sequencesource1..19/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 8gccttgccag
cccgctcag 19916DNAArtificial Sequencesource1..16/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 9tggtccctgg cagtct
161012DNAArtificial Sequencesource1..12/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 10gaacaggacc ga
121120DNAArtificial Sequencesource1..20/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 11ctgttcgatc
ttgggcgtat 201218DNAArtificial Sequencesource1..18/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 12acgcccaaga
tcgaacag 181361DNAArtificial Sequencesource1..61/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 13gccttgccag
cccgctcagg ggaaggacgt tggtgtagaa gcgttcactt ggtggaagta 60t
611420DNAArtificial Sequencesource1..20/mol_type="DNA"
/note="Primer" /organism="Artificial Sequence" 14acttccacca
agtgaacgct 201560DNAartificial sequencessource1..60/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 15ctgttcgatc
ttgggcgtat tgttttagct gccccaactc cttcaggcac caaggaagac
601620DNAartificial sequencessource1..20/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 16gcaagtctta
cagccgatca 201718DNAartificial sequencessource1..18/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 17tggtccctgg
cagtctcc 181850DNAartificial sequencessource1..50/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 18cctatcccct
gtgtgccttg gcagtctcag gtcttccttg gtgcctgaag 501952DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 19ccatctcatc cctgcgtgtc tccgactcag
aggttggtcc ctggcagtct cc 522052DNAartificial
sequencessource1..52/mol_type="DNA" /note="PRIMER"
/organism="artificial sequences" 20ccatctcatc cctgcgtgtc tccgactcag
atcgtggtcc ctggcagtct cc 522152DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 21ccatctcatc cctgcgtgtc tccgactcag
atgctggtcc ctggcagtct cc 522252DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 22ccatctcatc cctgcgtgtc tccgactcag
cacttggtcc ctggcagtct cc 522352DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 23ccatctcatc cctgcgtgtc tccgactcag
cagatggtcc ctggcagtct cc 522452DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 24ccatctcatc cctgcgtgtc tccgactcag
ccattggtcc ctggcagtct cc 522518DNAartificial
sequencessource1..18/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 25tggtccctgg cagtctcc
182636DNAartificial sequencessource1..36/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 26caccacgatg
gcaatgcatt cttcgctgcc attctg 362720DNAartificial
sequencessource1..20/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 27cgatgtcctg aggtggaagt
202840DNAartificial sequencessource1..40/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 28ggcaagtgat
tgtccatgtg catgagaaga ggcccacatt 402920DNAartificial
sequencessource1..20/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 29cacatggaca atcacttgcc
203020DNAartificial sequencessource1..20/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 30aatgtgggcc
tcttctcatg 203118DNAartificial sequencessource1..18/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 31tccacatcct
ccagttca 183240DNAartificial sequencessource1..40/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 32acttccacct
caggacatcg agctggagct tgctgttagc 403340DNAartificial
sequencessource1..40/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 33aggttcgctc cctccttaag tcaggaggat
gtgacaccaa 403440DNAartificial sequencessource1..40/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 34cgatgtcctg
aggtggaagt tgaactggag gatgtggaca 403540DNAartificial
sequencessource1..40/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 35cttaaggagg gagcgaacct gctaacagca
agctccagct 403620DNAartificial sequencessource1..20/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 36ttggtgtcac
atcctcctga 203720DNAartificial sequencessource1..20/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 37atgaaagacg
tggccattgc 203850DNAartificial sequencessource1..50/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 38ctgacatggt
ccctggcagt ctcctgtcag gaccgactcc gctcgaagac 503940DNAartificial
sequencessource1..40/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 39ctatcggttt taccgatagg tcttcgagct
gtacctgcgc 404040DNAartificial sequencessource1..40/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 40agctaggttt
tacctagctg cgcaggtact gtgcatcgac 404140DNAartificial
sequencessource1..40/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 41ctatcggttt taccgatagg tcgatgcact
ggagtcggtc 404218DNAartificial sequencessource1..18/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 42cttatgctgg
cagtttca 184340DNAartificial sequencessource1..40/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 43acttccacct
caggacatcg agctggagct tgctgttagc 404440DNAartificial
sequencessource1..40/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 44aggttcgctc cctccttaag ccagcagtgg
taattcgaca 404540DNAartificial sequencessource1..40/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 45cgatgtcctg
aggtggaagt tgaaactgcc agcataagga 404640DNAartificial
sequencessource1..40/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 46cttaaggagg gagcgaacct gctaacagca
agctccagct 404720DNAartificial sequencessource1..20/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 47tgtcgaatta
ccactgctgg 204850DNAartificial sequencessource1..50/mol_type="DNA"
/note="Primer" /organism="artificial sequences" 48cctatcccct
gtgtgccttg gcagtctcag cgatgtcctg aggtggaagt 504952DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 49ccatctcatc cctgcgtgtc tccgactcag
aacctggtcc ctggcagtct cc 525052DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 50ccatctcatc cctgcgtgtc tccgactcag
aaggtggtcc ctggcagtct cc 525152DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 51ccatctcatc cctgcgtgtc tccgactcag
acactggtcc ctggcagtct cc 525252DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 52ccatctcatc cctgcgtgtc tccgactcag
actgtggtcc ctggcagtct cc 525352DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 53ccatctcatc cctgcgtgtc tccgactcag
agagtggtcc ctggcagtct cc 525452DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 54ccatctcatc cctgcgtgtc tccgactcag
agcatggtcc ctggcagtct cc 525552DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 55ccatctcatc cctgcgtgtc tccgactcag
agtctggtcc ctggcagtct cc 525652DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 56ccatctcatc cctgcgtgtc tccgactcag
cctatggtcc ctggcagtct cc 525752DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 57ccatctcatc cctgcgtgtc tccgactcag
cgaatggtcc ctggcagtct cc 525852DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 58ccatctcatc cctgcgtgtc tccgactcag
ctactggtcc ctggcagtct cc 525952DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 59ccatctcatc cctgcgtgtc tccgactcag
ctcatggtcc ctggcagtct cc 526052DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 60ccatctcatc cctgcgtgtc tccgactcag
ctgttggtcc ctggcagtct cc 526152DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 61ccatctcatc cctgcgtgtc tccgactcag
cttgtggtcc ctggcagtct cc 526252DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 62ccatctcatc cctgcgtgtc tccgactcag
gaactggtcc ctggcagtct cc 526352DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 63ccatctcatc cctgcgtgtc tccgactcag
gacatggtcc ctggcagtct cc 526452DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 64ccatctcatc cctgcgtgtc tccgactcag
gagttggtcc ctggcagtct cc 526552DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 65ccatctcatc cctgcgtgtc tccgactcag
gatgtggtcc ctggcagtct cc 526652DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 66ccatctcatc cctgcgtgtc tccgactcag
gcaatggtcc ctggcagtct cc 526752DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 67ccatctcatc cctgcgtgtc tccgactcag
gtcttggtcc ctggcagtct cc 526852DNAartificial
sequencessource1..52/mol_type="DNA" /note="Pprimer"
/organism="artificial sequences" 68ccatctcatc cctgcgtgtc tccgactcag
gtgatggtcc ctggcagtct cc 526952DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 69ccatctcatc cctgcgtgtc tccgactcag
tacgtggtcc ctggcagtct cc 527052DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 70ccatctcatc cctgcgtgtc tccgactcag
tagctggtcc ctggcagtct cc 527152DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 71ccatctcatc cctgcgtgtc tccgactcag
tcagtggtcc ctggcagtct cc 527252DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 72ccatctcatc cctgcgtgtc tccgactcag
tccatggtcc ctggcagtct cc 527352DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 73ccatctcatc cctgcgtgtc tccgactcag
tcgttggtcc ctggcagtct cc 527452DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 74ccatctcatc cctgcgtgtc tccgactcag
tctctggtcc ctggcagtct cc 527552DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 75ccatctcatc cctgcgtgtc tccgactcag
tgactggtcc ctggcagtct cc 527652DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 76ccatctcatc cctgcgtgtc tccgactcag
tgtgtggtcc ctggcagtct cc 527752DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 77ccatctcatc cctgcgtgtc tccgactcag
ttcctggtcc ctggcagtct cc 527852DNAartificial
sequencessource1..52/mol_type="DNA" /note="Primer"
/organism="artificial sequences" 78ccatctcatc cctgcgtgtc tccgactcag
ttggtggtcc ctggcagtct cc 52
* * * * *