U.S. patent application number 10/103944 was filed with the patent office on 2002-08-01 for transcription-free selex.
This patent application is currently assigned to Gilead Sciences. Invention is credited to Gold, Larry, Smith, Jonathan Drew.
Application Number | 20020102599 10/103944 |
Document ID | / |
Family ID | 23426653 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102599 |
Kind Code |
A1 |
Smith, Jonathan Drew ; et
al. |
August 1, 2002 |
Transcription-free SELEX
Abstract
Methods are provided for the production of nucleic acid ligands
against target molecules using a procedure known as
Transcription-free Systematic Evolution of Ligands by EXponential
enrichment (Transcription-free SELEX). The Transcription-free SELEX
method assembles nucleic acid ligands from fragments of synthetic
nucleic acids by annealing those fragments to a complementary
template, and then ligating the fragments together.
Inventors: |
Smith, Jonathan Drew;
(Boulder, CO) ; Gold, Larry; (Boulder,
CO) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Assignee: |
Gilead Sciences
Foster City
CA
|
Family ID: |
23426653 |
Appl. No.: |
10/103944 |
Filed: |
March 22, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10103944 |
Mar 22, 2002 |
|
|
|
09362578 |
Jul 28, 1999 |
|
|
|
6387620 |
|
|
|
|
Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 1/6811 20130101;
C12Q 2521/501 20130101; C12Q 2531/113 20130101; C12Q 1/6811
20130101; C12Q 2537/155 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of identifying nucleic acid ligands that photocrosslink
to a target from a candidate mixture of ribonucleic acids, wherein
each member of said candidate mixture comprises one or more
photoreactive groups, the method comprising: a) contacting said
candidate mixture of nucleic acids with target, wherein nucleic
acids having an increased affinity to target relative to the
candidate mixture form nucleic acid-protein complexes with the
target; b) irradiating said complexes, wherein said nucleic acid
and target photocrosslink; c) partitioning the photocrosslinked
nucleic acid-target complexes from the candidate mixture; and d)
identifying a nucleic acid ligand that photocrosslinked to the
target wherein each member of said candidate mixture is assembled
from fragments of RNA comprising randomized sequence.
2. The method of claim 1 wherein said candidate mixture of nucleic
acids is comprised of single stranded nucleic acids.
3. The method of claim 1 further comprising after step c): i)
repeating steps a), b) and c); and ii) amplifying the nucleic acid
ligands that photocrosslinked to the target.
4. The method of claim 3 wherein said photoreactive group is
5-bromouracil.
5. The method of claim 1 wherein said RNA fragments comprise
synthetic RNA molecules.
6. The method of claim 5 wherein said synthetic RNA molecules
comprise at least one non-naturally occurring ribonucleotide.
7. The method of claim 6 wherein said non-naturally-occurring
ribonucleotide is a 2'-OMe ribonucleotide.
8. The method of claim 1 wherein said candidate mixture of nucleic
acids is assembled from fragments of RNA by annealing said RNA
fragments to a complementary DNA template, and then ligating said
annealed RNA fragments.
9. A method for the preparation of nucleic acid ligands that
photocrosslink to in a target, the method comprising: a) providing
a DNA template library comprising fixed 3' and 5' sequence regions,
and random internal sequences; b) contacting said DNA template
library with one or more RNA libraries, each said library
comprising synthetic randomized RNA fragments, each RNA fragment
comprising one or more photoreactive groups, wherein said RNA
fragments anneal to said DNA template, and wherein each said RNA
fragment is shorter than said DNA template; c) ligating said RNA
fragments together to form a candidate mixture of RNA nucleic acid
ligands; d) purifying said candidate mixture of RNA nucleic acid
ligands from said DNA template library and contacting said
candidate mixture of RNA nucleic acid ligands with a target,
wherein RNA nucleic acids ligands having an increased affinity to
target relative to the candidate mixture form nucleic acid-protein
complexes with the target; e) irradiating said complexes, wherein
said RNA nucleic acid ligand and said target photocrosslink; f)
partitioning the photocrosslinked nucleic acid-target complexes
from the candidate mixture; g) reverse transcribing those RNA
nucleic acid ligands that photocrosslinked to the target to form
DNA templates; h) amplifying those DNA templates using the
Polymerase Chain Reaction with primers that hybridize to said fixed
5' and 3' sequence regions to form a new DNA template library; i)
optionally repeating steps (b)-(h) for a desired number of
repetitions.
10. The method of claim 9 wherein a first, second and a third
library comprising synthetic randomized RNA fragments are used in
step (b), wherein said first library further comprises a fixed RNA
sequence complementary to the 5' fixed regions of said DNA
template, and wherein said second library further comprises a fixed
RNA sequence complementary to the 3' fixed sequence region of said
DNA template.
11. The method of claim 10 wherein said first library comprises X
ribonucleotides of fixed sequence and Y ribonucleotides of
randomized sequence; wherein said second library comprises A
ribonucleotides of fixed sequence and B ribonucleotides of
randomized sequence; wherein said third library comprises Z
ribonucleotides of randomized sequence; and wherein X+Y>Z,
A+B>Z, Y>X, B>A, Z>Y, and Z>B.
12. A method for the preparation of RNA nucleic acid ligands that
photocrosslink to a target, the method comprising: a) providing a
DNA template library comprising fixed 3' and 5' sequence regions,
and random internal sequences; b) contacting said DNA template
library with one or more RNA libraries, each said RNA library
comprising synthetic randomized RNA fragments, each RNA fragment
comprising one or more photoreactive groups, wherein said RNA
fragments anneal to said DNA template, and wherein each said RNA
fragment is shorter than said DNA template c) ligating said RNA
fragments together to form a candidate mixture of RNA nucleic acid
ligands; d) purifying said candidate mixture of RNA nucleic acid
ligands from said DNA template library and contacting said
candidate mixture of RNA nucleic acid ligands with a target,
wherein RNA nucleic acids ligands having an increased affinity to
target relative to the candidate mixture form nucleic acid-protein
complexes with the target; e) irradiating said complexes, wherein
said RNA nucleic acid ligand and said target photocrosslink; f)
partitioning the photocrosslinked nucleic acid-target complexes
from the candidate mixture; g) contacting those RNA nucleic acid
ligands that photocrosslinked to the target with one or more DNA
libraries, each said DNA library comprising synthetic randomized
DNA fragments, wherein said DNA fragments anneal to said RNA
nucleic acid ligands, and wherein each said DNA fragment is shorter
than said RNA nucleic acid ligands; h) ligating said DNA fragments
together to form new DNA templates; i) amplifying those new DNA
templates using the Polymerase Chain Reaction with primers that
hybridize to said fixed 5' and 3' sequence regions to form a new
DNA template library; j) optionally repeating steps (b)-(i) for the
desired number of repetitions.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/362,578, filed Jul. 28, 1999, entitled
"Transcription-Free SELEX."
FIELD OF THE INVENTION
[0002] This invention is directed to a method for the generation of
nucleic acid ligands having specific functions against target
molecules using the SELEX process. The invention provides a method
of producing candidate mixtures of RNA nucleic acid ligands without
using transcription. The instant methods allow SELEX to be
performed using modified ribonucleotides that cannot serve as
efficient substrates for RNA polymerases.
BACKGROUND OF THE INVENTION
[0003] The dogma for many years was that nucleic acids had
primarily an informational role. Through a method known as
Systematic Evolution of Ligands by EXponential enrichment, termed
the SELEX process, it has become clear that nucleic acids have
three dimensional structural diversity not unlike proteins. The
SELEX process is a method for the in vitro evolution of nucleic
acid molecules with highly specific binding to target molecules and
is described in U.S. patent application Ser. No. 07/536,428, filed
Jun. 11, 1990, entitled "Systematic Evolution of Ligands by
EXponential Enrichment," now abandoned, U.S. Pat. No. 5,475,096
entitled "Nucleic Acid Ligands", U.S. Pat. No. 5,270,163 (see also
WO 91/19813) entitled "Nucleic Acid Ligands" each of which is
specifically incorporated by reference herein. Each of these
applications, collectively referred to herein as the SELEX Patent
Applications, describes a fundamentally novel method for making a
nucleic acid ligand to any desired target molecule. The SELEX
process provides a class of products which are referred to as
nucleic acid ligands or aptamers, each having a unique sequence,
and which has the property of binding specifically to a desired
target compound or molecule. Each SELEX-identified nucleic acid
ligand is a specific ligand of a given target compound or molecule.
The SELEX process is based on the unique insight that nucleic acids
have sufficient capacity for forming a variety of two- and
three-dimensional structures and sufficient chemical versatility
available within their monomers to act as ligands (form specific
binding pairs) with virtually any chemical compound, whether
monomeric or polymeric. Molecules of any size or composition can
serve as targets in the SELEX method. The SELEX method applied to
the application of high affinity binding involves selection from a
mixture of candidate oligonucleotides and stepwise iterations of
binding, partitioning and amplification, using the same general
selection scheme, to achieve virtually any desired criterion of
binding affinity and selectivity. Starting from a mixture of
nucleic acids, preferably comprising a segment of randomized
sequence, the SELEX method includes steps of contacting the mixture
with the target under conditions favorable for binding,
partitioning unbound nucleic acids from those nucleic acids which
have bound specifically to target molecules, dissociating the
nucleic acid-target complexes, amplifying the nucleic acids
dissociated from the nucleic acid-target complexes to yield a
ligand-enriched mixture of nucleic acids, then reiterating the
steps of binding, partitioning, dissociating and amplifying through
as many cycles as desired to yield highly specific high affinity
nucleic acid ligands to the target molecule.
[0004] It has been recognized by the present inventors that the
SELEX method demonstrates that nucleic acids as chemical compounds
can form a wide array of shapes, sizes and configurations, and are
capable of a far broader repertoire of binding and other functions
than those displayed by nucleic acids in biological systems.
[0005] The basic SELEX method has been modified to achieve a number
of specific objectives. For example, U.S. patent application Ser.
No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat.
No. 5,707,796, both entitled "Method for Selecting Nucleic Acids on
the Basis of Structure," describe the use of the SELEX process in
conjunction with gel electrophoresis to select nucleic acid
molecules with specific structural characteristics, such as bent
DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17,
1993, entitled "Photoselection of Nucleic Acid Ligands,", now
abandoned, U.S. Pat. No. 5,763,177 entitled "Systematic Evolution
of Nucleic Acid Ligands by Exponential Enrichment: Photoselection
of Nucleic Acid Ligands and Solution SELEX" and U.S. patent
application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled
"Systematic Evolution of Nucleic Acid Ligands by Exponential
Enrichment: Photoselection of Nucleic Acid Ligands and Solution
SELEX," now U.S. Pat. No. 6,001,577, describe a SELEX based method
for selecting nucleic acid ligands containing photoreactive groups
capable of binding and/or photocrosslinking to and/or
photoinactivating a target molecule. U.S. Pat. No. 5,580,737
entitled "High-Affinity Nucleic Acid Ligands That Discriminate
Between Theophylline and Caffeine," describes a method for
identifying highly specific nucleic acid ligands able to
discriminate between closely related molecules, which can be
non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588
entitled "Systematic Evolution of Ligands by EXponential
Enrichment: Solution SELEX," describes a SELEX-based method which
achieves highly efficient partitioning between oligonucleotides
having high and low affinity for a target molecule.
[0006] The SELEX method encompasses the identification of
high-affinity nucleic acid ligands containing modified nucleotides
conferring improved characteristics on the ligand, such as improved
in vivo stability or improved delivery characteristics. Examples of
such modifications include chemical substitutions at the ribose
and/or phosphate and/or base positions. SELEX process-identified
nucleic acid ligands containing modified nucleotides are described
in U.S. Pat. No. 5,660,985 entitled "High Affinity Nucleic Acid
Ligands Containing Modified Nucleotides," that describes
oligonucleotides containing nucleotide derivatives chemically
modified at the 5- and 2'-positions of pyrimidines. U.S. Pat. No.
5,580,737, supra, describes highly specific nucleic acid ligands
containing one or more nucleotides modified with 2'-amino
(2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S.
patent application Ser. No. 08/264,029, filed Jun. 22, 1994,
entitled "Novel Method of Preparation of 2' Modified Pyrimidine
Intramolecular Nucleophilic Displacement," describes
oligonucleotides containing various 2'-modified pyrimidines.
[0007] The SELEX method encompasses combining selected
oligonucleotides with other selected oligonucleotides and
non-oligonucleotide functional units as described in U.S. Pat. No.
5,637,459 entitled "Systematic Evolution of Ligands by EXponential
Enrichment: Chimeric SELEX," and U.S. Pat. No. 5,683,867 entitled
"Systematic Evolution of Ligands by EXponential Enrichment: Blended
SELEX," respectively. These applications allow the combination of
the broad array of shapes and other properties, and the efficient
amplification and replication properties, of oligonucleotides with
the desirable properties of other molecules.
[0008] The SELEX method further encompasses combining selected
nucleic acid ligands with lipophilic compounds or non-immunogenic,
high molecular weight compounds in a diagnostic or therapeutic
complex as described in U.S. patent application Ser. No.
08/434,465, filed May 4, 1995, entitled "Nucleic Acid Complexes".
Each of the above described patent applications which describe
modifications of the basic SELEX procedure are specifically
incorporated by reference herein in their entirety.
[0009] The central method for identifying nucleic acid ligands to a
target is called the SELEX process, an acronym for Systematic
Evolution of Ligands by Exponential enrichment and involves (a)
contacting the candidate mixture of nucleic acids the target, (b)
partitioning between members of said candidate mixture on the basis
of affinity to the target, and (c) amplifying the selected
molecules to yield a mixture of nucleic acids enriched for nucleic
acid sequences with a relatively higher affinity for binding to the
target.
[0010] In typical embodiments of the SELEX process, the candidate
mixture of nucleic acid ligands comprises RNA molecules. Following
partitioning step (b) above, the RNA molecules that have higher
affinity for the target are reverse transcribed to form a DNA
template. This DNA template is then amplified by the Polymerase
Chain Reaction (PCR), and the amplified DNA molecules are
transcribed in order to provide a new RNA candidate mixture for the
next round of the SELEX process.
[0011] Although the transcription of DNA templates during the SELEX
process to form RNA nucleic acid ligand candidate mixtures is
generally efficient, problems can arise when attempting to
incorporate modified ribonucleotides into the RNA molecules during
transcription. Such modified ribonucleotides increase the
functionality and stability of candidate nucleic acid ligands, but
are often poor substrates for RNA polymerase. As a result,
transcription in the presence of such modified ribonucleotides is
often inefficient, leading to poor yields, or does not take place
at all. For example, it is often desirable to incorporate 2'-O
alkyl ribonucleotides (ribonucleotides that have an alkyl grouping
at the 2' oxygen), such as 2'-OMe (a methyl group at the 2'
oxygen), into the candidate RNA nucleic acid ligands because such
ribonucleotides confer great stability to the RNA. However, no
transcription takes place in the presence of 2'-OMe ribonucleotides
because these modified ribonucleotides are not substrates for RNA
polymerase.
[0012] Typical SELEX procedures permit the incorporation, at most,
of 5 different modified nucleotides into a candidate mixture (one
for each of the 4 NTPs incorporated during the elongation phase of
transcription, and one nucleoside, NMP or NDP incorporated at the
5' end at initiation of transcription). With the exception of the
5' modification, these modifications are distributed randomly, with
respect to both number and position, throughout the randomized
portion of the transcript. This random distribution can be a
disadvantage, particularly when chemically reactive modifications,
or modifications which reduce the solubility or stability of the
transcript are introduced.
[0013] It is an object of the present invention to provide a method
for performing the SELEX process in which modified ribonucleotides,
such as 2'-OMe ribonucleotides, are efficiently incorporated into
RNA candidate mixtures. It is a further object of the invention to
provide a method for performing the SELEX process in which modified
ribonucleotides can be incorporated at specific positions.
SUMMARY OF THE INVENTION
[0014] The instant invention provides novel methods for performing
the SELEX process to obtain nucleic acid ligands to target
compounds. In particular, methods are provided for obtaining
candidate mixtures of nucleic acid ligands comprised of RNA without
requiring transcription. Instead of transcription, the candidate
RNA nucleic acid ligands are prepared by annealing at least
partially randomized RNA fragments to at least partially randomized
DNA templates. The annealed RNA fragments are then ligated together
to form the candidate nucleic acid ligands. The RNA fragments can
be fully synthetic, and so can be comprised of modified
ribonucleotide subunits that cannot be incorporated into RNA by RNA
polymerase during transcription. Thus, the instant invention allows
the SELEX process to be performed with more diverse nucleic acid
chemistries than was previously possible.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a schematic representation of one embodiment of
the Transcription-free SELEX process. A DNA template library
comprising randomized (R) and fixed (F) sequence regions is
contacted with a 3 libraries of RNA fragments comprising randomized
regions and fixed regions complementary to the fixed regions of the
DNA template. After annealing in the correct register, the RNA
fragments are ligated together, and the RNA is partitioned from the
DNA template to provide a candidate mixture of nucleic acid
ligands. The candidate mixture of nucleic acid ligands is contacted
with a target molecule of interest; nucleic acid ligands that
interact with the target in the desired manner are partitioned from
those that do not. The nucleic acid ligands that interact with the
target in the desired manner are then reverse transcribed to yield
complementary DNA templates. Alternatively, if the nucleic acid
ligands are comprised of ribonucleotides that are not compatible
with reverse transcriptase, then the DNA templates are assembled by
annealing DNA fragments to the nucleic acid ligands, and then
ligating those DNA fragments together. In either case, these DNA
templates can then serve as templates for RNA fragment annealing in
a further optional cycle of the Transcription-free SELEX
method.
[0016] FIG. 2 shows a schematic representation of 3 random and
partly random RNA libraries annealed to a typical SELEX DNA
template. The relative sizes of the fixed and random sequence
regions of the RNA libraries insures that at equilibrium, the most
stable configuration of the individual library fragments is the one
illustrated.
[0017] FIG. 3 shows a schematic representation of 3 possible RNA
libraries that could be used in a Transcription-free SELEX
procedure using a DNA template with a 29 nt randomized region. The
individual RNA fragments are shown annealed to the DNA template in
the most thermodynamically favorable configuration.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Definitions
[0019] Various terms are used herein to refer to aspects of the
present invention. To aid in the clarification of the description
of the components of this invention, the following definitions are
provided:
[0020] As used herein, "nucleic acid ligand" is a non-naturally
occurring nucleic acid having a desirable action on a target.
Nucleic acid ligands are often referred to as "aptamers". A
desirable action includes, but is not limited to, binding of the
target, catalytically changing the target, reacting with the target
in a way which modifies/alters the target or the functional
activity of the target, covalently attaching to the target as in a
suicide inhibitor, facilitating the reaction between the target and
another molecule. In the preferred embodiment, the action is
specific binding affinity for a target molecule, such target
molecule being a three dimensional chemical structure other than a
polynucleotide that binds to the nucleic acid ligand through a
mechanism which predominantly depends on Watson/Crick base pairing
or triple helix binding, wherein the nucleic acid ligand is not a
nucleic acid having the known physiological function of being bound
by the target molecule. Nucleic acid ligands include nucleic acids
that are identified from a candidate mixture of nucleic acids, said
nucleic acid ligand being a ligand of a given target, by the method
comprising: a) contacting the candidate mixture with the target,
wherein nucleic acids having an increased affinity to the target
relative to the candidate mixture may be partitioned from the
remainder of the candidate mixture; b) partitioning the increased
affinity nucleic acids from the remainder of the candidate mixture;
and c) amplifying the increased affinity nucleic acids to yield a
ligand-enriched mixture of nucleic acids.
[0021] As used herein, "candidate mixture" is a mixture of nucleic
acids of differing sequence from which to select a desired ligand.
The source of a candidate mixture can be from naturally-occurring
nucleic acids or fragments thereof, chemically synthesized nucleic
acids, enzymatically synthesized nucleic acids or nucleic acids
made by a combination of the foregoing techniques. In a preferred
embodiment, each nucleic acid has fixed sequences surrounding a
randomized region to facilitate the amplification process. In
preferred embodiments of the instant invention, the candidate
mixture is comprised of synthetic RNA molecules that are assembled
from smaller RNA fragments.
[0022] "SELEX target" or "target" means any compound or molecule of
interest for which a ligand is desired. A target can be a protein,
peptide, carbohydrate, polysaccharide, glycoprotein, hormone,
receptor, antigen, antibody, virus, substrate, metabolite,
transition state analog, cofactor, inhibitor, drug, dye, nutrient,
growth factor, etc. without limitation.
[0023] As used herein, "nucleic acid" means either DNA, RNA,
single-stranded or double-stranded, and any chemical modifications
thereof. Modifications include, but are not limited to, those which
provide other chemical groups that incorporate additional charge,
polarizability, hydrogen bonding, electrostatic interaction, and
fluxionality to the nucleic acid ligand bases or to the nucleic
acid ligand as a whole. Such modifications include, but are not
limited to, 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
exocyclic amines, substitution of 4-thiouridine, substitution of
5-bromo or 5-iodo-uracil; backbone modifications, methylations,
unusual base-pairing combinations such as the isobases isocytidine
and isoguanidine and the like. Modifications can also include 3'
and 5' modifications such as capping. In the instant invention, one
preferred modification is the positioning of a methyl group at the
2'-oxygen of ribonucleotides.
[0024] "SELEX" methodology involves the combination of selection of
nucleic acid ligands which interact with a target in a desirable
manner, for example binding to a protein, with amplification of
those selected nucleic acids. Optional iterative cycling of the
selection/amplification steps allows selection of one or a small
number of nucleic acids which interact most strongly with the
target from a pool which contains a very large number of nucleic
acids. Cycling of the selection/amplification procedure is
continued until a selected goal is achieved. The SELEX process is
described in U.S. patent application Ser. No. 07/536,428, entitled
Systematic Evolution of Ligands by Exponential Enrichment, now
abandoned, U.S. Pat. No. 5,475,096 entitled nucleic acid ligands,
U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled nucleic
acid ligands. These patents and applications, each specifically
incorporated herein by reference, are collectively called the SELEX
Patent Applications.
[0025] In its most basic form, the SELEX process may be defined by
the following series of steps:
[0026] 1) A candidate mixture of nucleic acids of differing
sequence is prepared. The candidate mixture generally includes
regions of fixed sequences (i.e., each of the members of the
candidate mixture contains the same sequences in the same location)
and regions of randomized sequences. The fixed sequence regions are
selected either: (a) to assist in the amplification steps described
below, (b) to mimic a sequence known to bind to the target, or (c)
to enhance the concentration of a given structural arrangement of
the nucleic acids in the candidate mixture. The randomized
sequences can be totally randomized (i.e., the probability of
finding a base at any position being one in four) or only partially
randomized (e.g., the probability of finding a base at any location
can be selected at any level between 0 and 100 percent).
[0027] 2) The candidate mixture is contacted with the selected
target under conditions favorable for binding between the target
and members of the candidate mixture. Under these circumstances,
the interaction between the target and the nucleic acids of the
candidate mixture can be considered as forming nucleic acid-target
pairs between the target and those nucleic acids having the
strongest affinity for the target.
[0028] 3) The nucleic acids with the highest affinity for the
target are partitioned from those nucleic acids with lesser
affinity to the target. Because only an extremely small number of
sequences (and possibly only one molecule of nucleic acid)
corresponding to the highest affinity nucleic acids exist in the
candidate mixture, it is generally desirable to set the
partitioning criteria so that a significant amount of the nucleic
acids in the candidate mixture (approximately 5-50%) are retained
during partitioning.
[0029] 4) Those nucleic acids selected during partitioning as
having the relatively higher affinity for the target are then
amplified to create a new candidate mixture that is enriched in
nucleic acids having a relatively higher affinity for the
target.
[0030] 5) By repeating the partitioning and amplifying steps above,
the newly formed candidate mixture contains fewer and fewer unique
sequences, and the average degree of affinity of the nucleic acids
to the target will generally increase. Taken to its extreme, the
SELEX process will yield a candidate mixture containing one or a
small number of unique nucleic acids representing those nucleic
acids from the original candidate mixture having the highest
affinity to the target molecule.
[0031] The basic SELEX method has been modified to achieve a number
of specific objectives. For example, U.S. patent application Ser.
No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat.
No. 5,707,796 both entitled "Method for Selecting Nucleic Acids on
the Basis of Structure," describe the use of the SELEX process in
conjunction with gel electrophoresis to select nucleic acid
molecules with specific structural characteristics, such as bent
DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17,
1993, entitled "Photoselection of Nucleic Acid Ligands," now
abandoned, U.S. Pat. No. 5,763,177 entitled "Systematic Evolution
of Nucleic Acid Ligands by Exponential Enrichment: Photoselection
of Nucleic Acid Ligands and Solution SELEX" and U.S. patent
application Ser. No. 09/093,293, filed Jun. 8 1998, entitled
"Systematic Evolution of Nucleic Acid Ligands by Exponential
Enrichment: Photoselection of Nucleic Acid Ligands and Solution
SELEX," now U.S. Pat. No. 6,001,577, all describe a SELEX based
method for selecting nucleic acid ligands containing photoreactive
groups capable of binding and/or photocrosslinking to and/or
photoinactivating a target molecule. U.S. Pat. No. 5,580,737
entitled "High-Affinity Nucleic Acid Ligands That Discriminate
Between Theophylline and Caffeine," describes a method for
identifying highly specific nucleic acid ligands able to
discriminate between closely related molecules, termed
Counter-SELEX. U.S. Pat. No. 5,567,588 entitled "Systematic
Evolution of Ligands by Exponential Enrichment: Solution SELEX,"
describes a SELEX-based method which achieves highly efficient
partitioning between oligonucleotides having high and low affinity
for a target molecule. U.S. Pat. No. 5,496,938 entitled "Nucleic
Acid Ligands to HIV-RT and HIV-1 Rev," describes methods for
obtaining improved nucleic acid ligands after SELEX has been
performed. U.S. Pat. No. 5,705,337 entitled "Systematic Evolution
of Ligands by Exponential Enrichment: Chemi-SELEX," describes
methods for covalently linking a ligand to its target.
[0032] The SELEX method encompasses the identification of
high-affinity nucleic acid ligands containing modified nucleotides
conferring improved characteristics on the ligand, such as improved
in vivo stability or improved delivery characteristics. Examples of
such modifications include chemical substitutions at the ribose
and/or phosphate and/or base positions. SELEX-identified nucleic
acid ligands containing modified nucleotides are described in U.S.
Pat. No. 5,660,985 entitled "High Affinity Nucleic Acid Ligands
Containing Modified Nucleotides," that describes oligonucleotides
containing nucleotide derivatives chemically modified at the 5- and
2'-positions of pyrimidines. U.S. Pat. No. 5,637,459, supra,
describes highly specific nucleic acid ligands containing one or
more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro
(2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser.
No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of
Preparation of Known and Novel 2' Modified Nucleosides by
Intramolecular Nucleophilic Displacement," describes
oligonucleotides containing various 2'-modified pyrimidines.
[0033] The SELEX method encompasses combining selected
oligonucleotides with other selected oligonucleotides and
non-oligonucleotide functional units as described in U.S. Pat. No.
5,637,459 entitled "Systematic Evolution of Ligands by Exponential
Enrichment: Chimeric SELEX," and U.S. Pat. No. 5,683,867 entitled
"Systematic Evolution of Ligands by Exponential Enrichment: Blended
SELEX," respectively. These applications allow the combination of
the broad array of shapes and other properties, and the efficient
amplification and replication properties, of oligonucleotides with
the desirable properties of other molecules.
[0034] In U.S. Pat. No. 5,496,938, methods are described for
obtaining improved nucleic acid ligands after the SELEX process has
been performed. This patent, entitled, "Methods of Producing
nucleic acid ligands," is specifically incorporated herein by
reference.
[0035] The SELEX process provides a class of products which are
nucleic acid molecules, each having a unique sequence, and each of
which has the property of binding specifically to a desired target
compound or molecule. Target molecules are preferably proteins, but
can also include among others carbohydrates, peptidoglycans and a
variety of small molecules. SELEX methodology can also be used to
target biological structures, such as cell surfaces or viruses,
through specific interaction with a molecule that is an integral
part of that biological structure.
[0036] One potential problem encountered in the diagnostic use of
nucleic acids is that oligonucleotides in their phosphodiester form
may be quickly degraded in body fluids by intracellular and
extracellular enzymes such as endonucleases and exonucleases before
the desired effect is manifest. Certain chemical modifications of
the nucleic acid ligand can be made to increase the in vivo
stability of the nucleic acid ligand or to enhance or to mediate
the delivery of the nucleic acid ligand. See, e.g., U.S. patent
application Ser. No. 08/117,991, filed Sep. 9, 1993, now abandoned,
and U.S. Pat. No. 5,660,985, both entitled "High Affinity Nucleic
Acid Ligands Containing Modified Nucleotides", which is
specifically incorporated herein by reference. Modifications of the
nucleic acid ligands contemplated in this invention include, but
are not limited to, those which provide other chemical groups that
incorporate additional charge, polarizability, hydrophobicity,
hydrogen bonding, electrostatic interaction, and fluxionality to
the nucleic acid ligand bases or to the nucleic acid ligand as a
whole. Such modifications include, but are not limited to,
2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
exocyclic amines, substitution of 4-thiouridine, substitution of
5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate
or alkyl phosphate modifications, methylations, methyl
phosphonates, H-phosphonates, peptide modifications, unusual
base-pairing combinations such as the isobases isocytidine and
isoguanidine and the like. Modifications can also include 3' and 5'
modifications such as capping, 3' or 5' sulfurs, and 3' or 5'
amines. Any nucleic acid chemistry in which the modified nucleic
acid is still able to form a double-helix with a complementary
sequence is contemplated in the instant invention.
[0037] The modifications can be pre- or post-SELEX process
modifications. PreSELEX process modifications yield nucleic acid
ligands with both specificity for their SELEX target and improved
in vivo stability. Post-SELEX process modifications made to 2'-OH
nucleic acid ligands can result in improved in vivo stability
without adversely affecting the binding capacity of the nucleic
acid ligand. The instant invention provides methods for performing
SELEX using modified nucleic acids in the candidate mixture.
[0038] Other modifications are known to one of ordinary skill in
the art. Such modifications may be made post-SELEX process
(modification of previously identified unmodified ligands) or by
incorporation into the SELEX process.
[0039] In some embodiments, the nucleic acid ligands become
covalently attached to their targets upon irradiation of the
nucleic acid ligand with light having a selected wavelength.
Methods for obtaining such nucleic acid ligands are detailed in
U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993,
entitled "Photoselection of Nucleic Acid Ligands,", now abandoned,
U.S. Pat. No. 5,763,177 entitled "Systematic Evolution of Nucleic
Acid Ligands by Exponential Enrichment: Photoselection of Nucleic
Acid Ligands and Solution SELEX" and U.S. patent application Ser.
No. 09/093,293, filed Jun. 8, 1998, entitled "Systematic Evolution
of Nucleic Acid Ligands by Exponential Enrichment: Photoselection
of Nucleic Acid Ligands and Solution SELEX," now U.S. Pat. No.
6,001,577, each of which is specifically incorporated herein by
reference in its entirety.
[0040] As used herein "synthetic RNA" means a ribonucleotide
polymer that is assembled in vitro without the use of an enzyme.
Any chemical system known in the art for ribonucleotide polymer
synthesis is contemplated, including both solid-phase and
solution-phase chemistries.
[0041] As used herein "library" means a population of nucleic acid
molecules of constant length in which the individual members of the
population differ in sequence from one another at predetermined
positions. An individual library can have one or more fixed
sequence regions--where every member of the library has the same
bases at particular positions--and randomized regions. The
randomized regions can be partly or completely randomized.
Preferred embodiments of the instant invention uses three
libraries: a first library in which the individual nucleic acid
molecules have a 5' randomized region adjacent to a 3' fixed
region; a second library in which the individual nucleic acid
molecules have a 5' fixed region adjacent to a 3' randomized
region; and a third library in which the individual nucleic acid
molecules are randomized throughout their length.
[0042] As used herein "fragment" means an individual nucleic acid
molecule obtained from a library. A fragment is typically shorter
than an individual nucleic acid ligand.
[0043] Transcription-free SELEX
[0044] In some embodiments of the SELEX process, the candidate
mixture of nucleic acid ligands comprises RNA molecules. In such
embodiments, the SELEX process may comprise the following
steps:
[0045] (a) providing a DNA template library comprising fixed 3' and
5' sequences, and random internal sequences;
[0046] (b) transcribing said DNA library from one of said fixed
sequences to form a candidate mixture of RNA nucleic acid
ligands;
[0047] (c) purifying said candidate mixture of RNA nucleic acid
ligands from the DNA template library, and contacting said
candidate mixture of RNA nucleic acid ligands with a target;
[0048] (d) partitioning RNA nucleic acid ligands that interact with
the target in the desired manner from those that do not;
[0049] (e) reverse transcribing those RNA nucleic acid ligands that
interact with the target in the desired manner to form DNA
templates;
[0050] (f) amplifying said DNA templates using the Polymerase Chain
Reaction with primers that hybridize to said fixed 5' and 3'
sequences; and optionally;
[0051] (g) repeating steps (b)-(f) for the desired number of
cycles.
[0052] The present invention accomplishes the SELEX process
outlined above without requiring that transcription occurs at step
(b) and, optionally, without requiring that reverse transcription
occurs at step (e). Instead, the instant invention uses one or more
randomized libraries of synthetic RNA molecules to directly
assemble complementary RNA molecules on the DNA template of step
(b). When contacted with the DNA template of step (b), the
randomized synthetic RNA molecules anneal to the template. The
individual RNA fragments can be ligated together, and the resulting
RNA molecules can then be purified for use as the candidate mixture
of nucleic acid ligands as described above. In turn, the candidate
mixture of nucleic acid ligands can serve as templates for assembly
of DNA using DNA fragments that anneal to the RNA molecules and are
then ligated together. The resulting DNA can be PCR amplified, and
then serve as the DNA template for the next round of the SELEX
method. In this way, it is possible to produce candidate mixtures
of RNA nucleic acid ligands from DNA templates without requiring
transcription, and then optionally to produce DNA templates from
RNA nucleic acid ligands without requiring reverse transcription.
The method is termed Transcription-free SELEX.
[0053] In one embodiment of the Transcription-free SELEX method,
the following steps take place (FIG. 1):
[0054] (a) providing a DNA library comprising fixed 3' and 5'
sequences, and random internal sequences;
[0055] (b) contacting said DNA library with one or more synthetic
libraries comprising randomized RNA fragments, wherein said
fragments anneal to said DNA library to form substantially
contiguous RNA molecules complementary to individual members of
said DNA library;
[0056] (c) ligating said RNA fragments together to form a candidate
mixture of RNA nucleic acid ligands;
[0057] (d) purifying said candidate mixture of RNA nucleic acid
ligands from said DNA library, and contacting said candidate
mixture of RNA nucleic acid ligands with a target;
[0058] (e) partitioning RNA nucleic acid ligands that interact with
the target in the desired manner from those that do not;
[0059] (f) reverse transcribing those RNA nucleic acid ligands that
interact with the target in the desired manner to form DNA
templates;
[0060] (g) amplifying those DNA templates using the Polymerase
Chain Reaction with primers that hybridize to said fixed 5' and 3'
sequences; and optionally
[0061] (h) repeating steps (b)-(g) for the desired number of
cycles.
[0062] The primary advantage of using synthetic RNA fragments to
assemble candidate mixtures of RNA nucleic acid ligands, rather
than transcription, is that modified ribonucleotides can be more
readily incorporated into the nucleic acid ligands. Such modified
ribonucleotides are often poor substrates for RNA polymerase, and
so yields of transcription are poor or non-existent. For example,
in some embodiments it is desirable to use RNA nucleic acid ligands
that are 2'-OMe. 2'-OMe ribonucleotides confer stability from
ribonucleases upon RNA nucleic acid ligands. However, RNA
polymerase does not incorporate 2'-OMe ribonucleotides into RNA, so
no RNA is produced. In the Transcription-free SELEX method, by
contrast, the RNA fragments can be chemically synthesized with
2'-OMe ribonucleotides by any technique known in the art. By
ligating 2'-OMe RNA fragments together, a 2'-OMe candidate mixture
of RNA nucleic acid ligands is efficiently produced without
transcription. The Transcription-free SELEX method will allow
functional activities to be included at defined sites, including
but not limited to nucleophiles, RGD peptides, cages, and PEG
groups (see below).
[0063] Preferred embodiments of the invention use libraries of
randomized and partly randomized synthetic RNA fragments. The
partly randomized RNA fragments comprise random sequence regions
and fixed sequence regions, wherein the fixed sequence regions are
complementary in sequence to the fixed sequence regions of the DNA
template. Thus, partly randomized fragments anneal to the fixed
regions of the DNA template and to the adjacent random sequence
region of the DNA template. The fixed sequence regions anneal
rapidly (due to their higher concentration relative to each random
sequence) and thus set the "register" for annealing the fully
randomized RNA molecules. This favors products of the correct size,
and is necessary for position specific modification (see below).
The individual randomized and partly randomized RNA molecules that
have annealed along the length of the DNA template can then be
ligated together to form a continuous RNA strand, complementary in
sequence to the DNA template.
[0064] In preferred embodiments of the invention, at least 3
separate RNA libraries are used. A schematic representation of
these libraries is given in FIG. 2. A first library has a
randomized sequence 3' region X nucleotides long and a fixed
sequence 5' region Y nucleotides long that is complementary to the
3' fixed sequence region of the DNA template; the second library
has a randomized sequence 5' region A nucleotides long and a fixed
sequence 3' region B nucleotides long that is complementary to the
fixed 5' region of the DNA template; the third library is Z
nucleotides of random sequence. The total length of the randomized
portions of the three libraries is equal to the length of the
randomized portion of the DNA template (X+A+Z-DNA template random
region). The total length of each RNA molecule in the first and
second library is greater than that of each molecule in the third
library (X+Y>Z; A+B>Z); the randomized portions of the first
and second libraries are preferably shorter than the totally
randomized molecules of the third population (X<Z; A<Z).
These sequences anneal to DNA templates as shown in FIG. 2.
[0065] By using a plurality of randomized RNA fragments, rather
than using randomized RNA molecules of the same size as the DNA
template, the time taken for hybridization along the entire length
of the DNA template is dramatically reduced. For example, the time
taken to allow a random library of 30 mer RNA molecules to anneal
to a 30 mer DNA template is approximately 10.sup.4 years, whereas
using three random libraries of shorter RNA fragments takes only
1-2 hours to go to completion. Furthermore, by endowing the first
and second populations with regions complementary to the fixed
sequences in the DNA template, these RNA fragments are kept in
proper register. Because the randomized portions of the first and
second libraries are shorter than in the third library (X<Z;
A<Z), the annealing of the third library to the DNA template
will displace any first and second library RNA fragments that have
annealed to the randomized region of the DNA template without
annealing to the fixed regions also. Similarly, because the first
and second library RNA fragments are longer than the third
population RNA fragments, correctly annealed first and second
library RNA fragments will not be displaced by third library
molecules that fortuitously hybridize to the same sequence. Thus,
the relative lengths of the random and fixed sequence regions of
the three libraries insures that the thermodynamically most stable
configuration occurs when the three RNA fragments anneal to the DNA
template as shown in FIG. 2.
[0066] Although the embodiment described above uses 3 populations,
any number of RNA fragments can be used. Indeed, in some
applications it may be desirable to use a greater number. For
example, it is known that in some SELEX reactions, a single
dominant sequence may comprise .about.10% of the library. If the
third population RNA fragments (fully randomized) are, for example,
11 nucleotides long, and if there is a 5-fold excess of these RNA
fragments over DNA template, then only 1/4.sup.11=1/(4.times.10-
.sup.6) of the RNA fragments will possess the correct sequence to
anneal to the dominant sequence. This may mean that the dominant
sequence will be poorly amplified in later SELEX rounds. Using RNA
fragments shorter than 11 nucleotides that are present at a much
larger excess than 5-fold will allow more efficient amplification
of the dominant sequence.
[0067] It will be appreciated from the foregoing that there are a
number of variables that may be readily adjusted in order to obtain
maximum efficiency in a particular Transcription-free SELEX
application. The ability to manipulate such variables gives the
Transcription-free SELEX method a high degree of flexibility.
Manipulable variables include without limitation: the number of
populations of randomized and partly randomized RNA fragments; the
length of the individual RNA molecules in each population; the
concentrations of the RNA molecules in each population; the length
of the fixed sequence regions in partly randomized RNA fragment
populations; and the time and temperature at which the
hybridization occurs. The determination of these variables requires
only routine experimentation for those skilled in the art.
[0068] The Transcription-free SELEX method provides a means to
control the number and position of the modified nucleotides
introduced into a candidate mixture of nucleic acid ligands.
Consider a candidate mixture of RNA nucleic acid ligands assembled
according to the example shown in FIG. 2. Each of the library RNA
fragments is the product of a separate synthesis, and each occupies
a unique site on the DNA template. It is possible to specify, for
instance, that all of the uridines in the RNA fragment that anneals
to the 5' end of the DNA template are modified in one way (e.g.,
2'OMe), all of the uridines in the central RNA fragment are
modified in a second way (e.g., BrdU), and all of the uridines in
the RNA fragment that anneals to the 3' end of the DNA template are
modified in a third way (e.g., 5-amino-benzoyl). This method can be
used for all four nucleotides in each library of RNA fragments.
[0069] This concept can be extended to the level of the individual
nucleotide, limited in practice only by the number of ports
available on the synthesizer. That is, one could specify that
uridines at position 1 of an RNA fragment are modification 1,
uridines at position 2 are modification 2 etc. The number of
different modifications incorporated into the library is thus
limited only by the ability to synthesize the oligo libraries. The
present invention contemplates the use of any nucleic acid
chemistry in which the modified nucleic acid is able to form a
double helix with a complementary nucleic acid sequence.
[0070] In preferred embodiments of the invention, the individual
RNA fragments are ligated together following annealing by adding T4
DNA ligase to the reaction mixture. It is preferable to add ligase
at the end of the annealing process, otherwise kinetic
intermediates (incompletely and inappropriately annealed RNA
fragments) will be ligated together.
[0071] Some of the modified ribonucleotides contemplated may serve
as poor substrates for ligase; RNA fragments containing such
modified ribonucleotides may therefore be ligated together with low
efficiency. In such cases, the synthetic RNA molecules can be
designed such that the particular ribonucleotide modification is
not present at the critical positions of the RNA fragment needed
for efficient RNA ligase function. In other embodiments, ligation
may be achieved chemically without the use of ligase. A variety of
chemical ligation procedures have been described in the scientific
literature, including: carbodiimide condensation, as described in
Dolinnaya, N. G., N. I. Sokolova, et al. (1988). "Site-directed
modification of DNA duplexes by chemical ligation." Nucleic Acids
Res. 16(9): 3721-38; cyanogen bromide condensation as described in
Dolinnaya, N. G., N. I. Sokolova, et al. (1991). "The use of BrCN
for assembling modified DNA duplexes and DNA-RNA hybrids;
comparison with water-soluble carbodiimide." Nucleic Acids Res.
19(11): 3067-72; and sulfur-halide nucleophilic displacement as
described in Xu, Y. and E. T. Kool (1999). "High sequence fidelity
in a non-enzymatic DNA autoligation reaction." Nucleic Acids Res.
27(3): 875-81. Each of the foregoing references is incorporated
herein by reference in its entirety. The fidelity of the reactions,
and the activity of the resultant products has also been
demonstrated as described in Housby, J. N. and E. M. Southern
(1998). "Fidelity of DNA ligation: a novel experimental approach
based on the polymerisation of libraries of oligonucleotides." NAR
26: 4259-4266; James, K. D., A. R. Boles, et al. (1998). "The
fidelity of template-directed oligonucleotide ligation and its
relevance to DNA computation." Nucleic Acids Research 26(22):
5203-5211; James, K. D. and A. D. Ellington (1997). "Surprising
fidelity of template-directed chemical ligation of
oligonucleotides." Chem. Biol. 4(8): 595-605; and Shabarova, Z. A.,
I. N. Merenkova, et al. (1991). "Chemical ligation of DNA: the
first non-enzymatic assembly of a biologically active gene."
Nucleic Acids Res. 19(15): 4247-51, each of which is incorporated
herein by reference in its entirety.
[0072] In the embodiments described above, the synthetic RNA
candidate mixture that is assembled through ligation must still be
capable of serving as a template for reverse transcription.
Although some modified ribonucleotides, such as 2'-OMe, can serve
as templates for reverse transcriptase, other useful modified
ribonucleotides cannot. This may limit somewhat the identity of the
modified ribonucleotides that can be incorporated into the
candidate RNA mixture. However, the present invention also
contemplates embodiments where reverse transcription is not used to
provide the DNA template required for PCR. In these embodiments, a
DNA template for PCR is assembled on partitioned RNA candidate
mixture molecules (those RNA molecules that interact with the
target in the desired manner) in the same way that the RNA
candidate mixture molecules themselves were assembled i.e., by
contacting the partitioned RNA candidate mixture with libraries
comprising randomized and partly randomized DNA fragments, allowing
these DNA fragments to anneal to the RNA templates, followed by
ligation of the DNA fragments, and PCR amplification of the ligated
fragments. As described above, ligation of the DNA fragments can be
performed chemically without the use of a DNA ligase. By performing
DNA template assembly without reverse transcriptase, it is possible
to expand the repertoire of modified bases employed in SELEX even
further. Modified bases that are incompatible with RNA polymerase
and also with reverse transcriptase can still be incorporated into
candidate mixtures using this method. This method will greatly
enhance the utility of the SELEX technique by increasing even
further the diversity of nucleic acid structure, chemistry and
functionality that can comprise a candidate mixture.
[0073] The embodiments described above contemplate the use of
candidate mixtures of nucleic acid ligands comprising single
stranded RNA molecules. However, it will be apparent to those
skilled in the art that the methods described herein are readily
applicable to candidate mixtures comprising double stranded RNA,
single stranded DNA and double stranded DNA.
[0074] The Transcription-free SELEX method has a number of
additional advantages over the typical SELEX methods. For example,
Transcription-free SELEX can produce smaller nucleic acid ligands
than typical SELEX methods. This is due to two factors: (1)
Increased chemical activity of modified nucleotides, as compared to
standard nucleotides; (2) Increased stability of smaller structural
motifs, though reduction of backbone charge repulsion. These will
be discussed in turn.
[0075] The modem, standard set of nucleotides does not provide
strong nucleophilic or electrophilic centers, nor does it provide
acid-base transitions in neutral pH environments. This lack of
reactivity can be compensated somewhat by size: many weak
interactions can sum to replace a single strong interaction. This
is a disadvantage for nucleic acid ligands, as sequences which
specify these many weak interactions will be correspondingly rare
and hard to select, and will result in large, hard-to-synthesize
sequences if they are selected. Modified fragment libraries which
contain highly reactive nucleotides may achieve a smaller nucleic
acid ligand size by replacing many weak interactions with one
strong one. For instance, a positive charge could be provided by a
single modified nucleotide; this could replace the several standard
nucleotides in a standard nucleic acid ligand which must fold to
form a metal-binding pocket which provides the equivalent
charge.
[0076] The second path by which Transcription-free SELEX using
chemically synthesized fragment libraries can reduce nucleic acid
ligand size is by reduction of electrostatic repulsion. Standard
phosphodiester nucleic acids contain a single net negative charge
per residue. The repulsion between phosphate groups is substantial,
even in a standard double helix, and requires many hydrogen-bonding
and stacking interactions to compensate, and allow a stable
structure to form. Backbone modifications which eliminate this
negative charge form much more stable helices. For instance,
DNA-PNA (peptide nucleic acid) helices are about 30% more stable
than the corresponding DNA-DNA helix. A modified fragment library
which incorporates uncharged residues can therefore be expected to
have helices and other structural motifs which are more stable than
that of a standard nucleic acid library. This increased stability
will result in smaller structural motifs, and therefore smaller
nucleic acid ligands than the corresponding agents derived from
standard SELEX methods.
[0077] One of the limiting factors in commercializing nucleic acid
ligands, and indeed all oligonucleotide agents, is cost.
Considerable effort goes into minimizing the size of candidate
oligonucleotide agents in order to minimize this cost. Because the
Transcription-free SELEX method can produce smaller nucleic acid
ligands than the typical SELEX methods, the method of the instant
invention should greatly facilitate the development of more
cost-effective nucleic acid ligands.
EXAMPLES
[0078] The following examples are described solely for the purpose
of illustrating various embodiments of the invention. These
examples are not to be construed as limiting the scope of the
invention in any sense.
Example 1
Annealing of Random 11-mer RNA to DNA Template
[0079] FIG. 3 shows a typical SELEX DNA template library comprising
5' and a 3' fixed sequence regions, and an internal 29 nucleotide
random sequence region. Three libraries of RNA are then
synthesized:
[0080] 1. (5+9) nt: each RNA molecule in this library comprises a
5' 5 nucleotide fixed sequence complementary to the 3' fixed
sequence region of the DNA template; immediately 3' to the 5
nucleotide fixed region, each molecule has a 9 nucleotide random
sequence. The 5' end of the individual molecules in the (5+9) nt
RNA library bear a hydroxyl (OH) group.
[0081] 2. (9+5) nt: each RNA molecule in this library comprises a
3' 5 nucleotide fixed sequence complementary to the 5' fixed
sequence region of the DNA template; immediately 5' to the 5
nucleotide fixed region, each molecule has a 9 nucleotide random
sequence. The 5' end of the individual molecules in the (9+5) nt
RNA library bear a phosphate (P) group.
[0082] 3. 11 nt: this comprises a randomized 11 nucleotide RNA
sequence; The 5' end of the individual molecules in 11 nt RNA
library bear a phosphate (P) group.
[0083] Thus, at equilibrium, an 11 nt RNA molecule from the 11 nt
library, and 9 nt sequences from the (5+9) and (9+5) nt libraries
will completely cover the 29 nt region of each DNA template
molecule.
Example 2
Using RNA Libraries to Assemble an RNA Candidate Mixture
[0084] The annealing rates for the libraries of Example 1 are
calculated as follows:
[0085] Number of sequences in a random 11-mer:
4.sup.11=4.times.10.sup.6
[0086] Annealing rate: 1.times.10.sup.7 M.sup.-1 s.sup.-1 (in 0.1
mM CTAB, 65.degree.)
[0087] Concentration of DNA template: 1 nmol/50
.mu.l=2.times.10.sup.-5 M
[0088] Concentration of 11 mer library: 5 nmol/50
.mu.l=1.times.10.sup.-4 M
[0089] The rate at which a random 11 mer hybridizes is:
1.times.10.sup.7 M.sup.1 s.sup.-1.times.1.times.10.sup.-4
M/4.times.10.sup.6=3.times.10.su- p.-4 s.sup.-1.
[0090] At this rate, the annealing reaction is over in 1-2
hours.
[0091] Because the RNA libraries are in excess, their concentration
drives the reaction. The relevant number is the concentration of
each sequence, which is the total concentration divided by the
complexity. The 9-mers are present at 16-fold higher concentration,
and so anneal that much faster. This forces the 11-mer into the
proper register. Although misannealing will occur (there are more
ways to misanneal than to anneal properly), annealing in the proper
register maximizes the number and fraction of bases paired, and so
is the most favorable configuration.
* * * * *