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Description  |
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FIELD OF THE INVENTION
The invention relates generally to methods for identifying, sorting, and/or
tracking molecules, especially polynucleotides, with oligonucleotide
labels, and more particularly, to a method of sorting polynucleotides by
specific hybridization to oligonucleotide tags.
BACKGROUND
Specific hybridization of oligonucleotides and their analogs is a
fundamental process that is employed in a wide variety of research,
medical, and industrial applications, including the identification of
disease-related polynucleotides in diagnostic assays, screening for clones
of novel target polynucleotides, identification of specific
polynucleotides in blots of mixtures of polynucleotides, amplification of
specific target polynucleotides, therapeutic blocking of inappropriately
expressed genes, DNA sequencing, and the like, e.g. Sambrook et al,
Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor
Laboratory, New York, 1989); Keller and Manak, DNA Probes, 2nd Edition
(Stockton Press, New York, 1993); Milligan et al, J. Med. Chem., 36:
1923-1937 (1993); Drmanac et al, Science, 260: 1649-1652 (1993); Bains, J.
DNA Sequencing and Mapping, 4: 143-150 (1993).
Specific hybridization has also been proposed as a method of tracking,
retrieving, and identifying compounds labeled with oligonucleotide tags.
For example, in multiplex DNA sequencing oligonucleotide tags are used to
identify electrophoretically separated bands on a gel that consist of DNA
fragments generated in the same sequencing reaction. In this way, DNA
fragments from many sequencing reactions are separated on the same lane of
a gel which is then blotted with separate solid phase materials on which
the fragment bands from the separate sequencing reactions are visualized
with oligonucleotide probes that specifically hybridize to complementary
tags, Church et al, Science, 240: 185-188 (1988). Similar uses of
oligonucleotide tags have also been proposed for identifying explosives,
potential pollutants, such as crude oil, and currency for prevention and
detection of counterfeiting, e.g. reviewed by Dollinger, pages 265-274 in
Mullis et al, editors, The Polymerase Chain Reaction (Birkhauser, Boston,
1994). More recently, systems employing oligonucleotide tags have also
been proposed as a means of manipulating and identifying individual
molecules in complex combinatorial chemical libraries, for example, as an
aid to screening such libraries for drug candidates, Brenner and Lerner,
Proc. Natl. Acad. Sci., 89: 5381-5383 (1992); Alper, Science, 264:
1399-1401 (1994); and Needels et al, Proc. Natl. Acad. Sci., 90:
10700-10704 (1993).
The successful implementation of such tagging schemes depends in large part
on the success in achieving specific hybridization between a tag and its
complementary probe. That is, for an oligonucleotide tag to successfully
identify a substance, the number of false positive and false negative
signals must be minimized. Unfortunately, such spurious signals are not
uncommon because base pairing and base stacking free energies vary widely
among nucleotides in a duplex or triplex structure. For example, a duplex
consisting of a repeated sequence of deoxyadenine (A) and thymidine (T)
bound to its complement may have less stability than an equal-length
duplex consisting of a repeated sequence of deoxyguanidine (G) and
deoxycytidine (C) bound to a partially complementary target containing a
mismatch. Thus, if a desired compound from a large combinatorial chemical
library were tagged with the former oligonucleotide, a significant
possibility would exist that, under hybridization conditions designed to
detect perfectly matched AT-rich duplexes, undesired compounds labeled
with the GC-rich oligonucleotide--even in a mismatched duplex--would be
detected along with the perfectly matched duplexes consisting of the
AT-rich tag. In the molecular tagging system proposed by Brenner et al
(cited above), the related problem of mis-hybridizations of closely
related tags was addressed by employing a so-called "commaless" code,
which ensures that a probe out of register (or frame shifted) with respect
to its complementary tag would result in a duplex with one or more
mismatches for each of its five or more three-base words, or "codons."
Even though reagents, such as tetramethylammonium chloride, are available
to negate base-specific stability differences of oligonucleotide duplexes,
the effect of such reagents is often limited and their presence can be
incompatible with, or render more difficult, further manipulations of the
selected compounds, e.g. amplification by polymerase chain reaction (PCR),
or the like.
Such problems have made the simultaneous use of multiple hybridization
probes in the analysis of multiple or complex genetic loci, e.g. via
multiplex PCR, reverse dot blotting, or the like, very difficult. As a
result, direct sequencing of certain loci, e.g. HLA genes, has been
promoted as a reliable alternative to indirected methods employing
specific hybridization for the identification of genotypes, e.g.
Gyllensten et al, Proc. Natl. Acad. Sci., 85: 7652-7656 (1988).
The ability to sort cloned and identically tagged DNA fragments onto
distinct solid phase supports would facilitate such sequencing,
particularly when coupled with a non gel-based sequencing methodology
simultaneously applicable to many samples in parallel.
In view of the above, it would be useful if there were available an
oligonucleotide-based tagging system which provided a large repertoire of
tags, but which also minimized the occurance of false positive and false
negative signals without the need to employ special reagents for altering
natural base pairing and base stacking free energy differences. Such a
tagging system would find applications in many areas, including
construction and use of combinatorial chemical libraries, large-scale
mapping and sequencing of DNA, genetic identification, medical
diagnostics, and the like.
SUMMARY OF THE INVENTION
An object of my invention is to provide a molecular tagging system for
tracking, retrieving, and identifying compounds.
Another object of my invention is to provide a method for sorting identical
molecules, or subclasses of molecules, especially polynucleotides, onto
surfaces of solid phase materials by the specific hybridization of
oligonucleotide tags and their complements.
A further object of my invention is to provide a combinatorial chemical
library whose member compounds are identified by the specific
hybridization of oligonucleotide tags and their complements.
A still further object of my invention is to provide a system for tagging
and sorting many thousands of fragments, especially randomly overlapping
fragments, of a target polynucleotide for simultaneous analysis and/or
sequencing.
Another object of my invention is to provide a rapid and reliable method
for sequencing target polynucleotides having a length in the range of a
few hundred basepairs to several tens of thousands of basepairs.
My invention achieves these and other objects by providing a method and
materials for tracking, identifying, and/or sorting classes or
subpopulations of molecules by the use of oligonucleotide tags. An
oligonucleotide tag of the invention consists of a plurality of subunits,
each subunit consisting of an oligonucleotide of 3 to 6 nucleotides in
length. Subunits of an oligonucleotide tag are selected from a minimally
cross-hybridizing set. In such a set, a duplex or triplex consisting of a
subunit of the set and the complement of any other subunit of the set
contains at least two mismatches. In other words, a subunit of a minimally
cross-hybridizing set at best forms a duplex or triplex having two
mismatches with the complement of any other subunit of the same set. The
number of oligonucleotide tags available in a particular embodiment
depends on the number of subunits per tag and on the length of the
subunit. The number is generally much less than the number of all possible
sequences the length of the tag, which for a tag n nucleotides long would
be 4.sup.n. More preferably, subunits are oligonucleotides from 4 to 5
nucleotides in length.
In one aspect of my invention, complements of oligonucleotide tags attached
to a solid phase support are used to sort polynucleotides from a mixture
of polynucleotides each containing a tag. In this embodiment, complements
of the oligonucleotide tags are synthesized on the surface of a solid
phase support, such as a microscopic bead or a specific location on an
array of synthesis locations on a single support, such that populations of
identical sequences are produced in specific regions. That is, the surface
of each support, in the case of a bead, or of each region, in the case of
an array, is derivatized by only one type of complement which has a
particular sequence. The population of such beads or regions contains a
repertoire of complements with distinct sequences, the size of the
repertoire depending on the number of subunits per oligonucleotide tag and
the length of the subunits employed. Similarly, the polynucleotides to be
sorted each comprises an oligonucleotide tag in the repertoire, such that
identical polynucleotides have the same tag and different polynucleotides
have different tags. Thus, when the populations of supports and
polynucleotides are mixed under conditions which permit specific
hybridization of the oligonucleotide tags with their respective
complements, subpopulations of identical polynucleotides are sorted onto
particular beads or regions. The subpopulations of polynucleotides can
then be manipulated on the solid phase support by micro-biochemical
techniques.
Generally, the method of my invention comprises the following steps: (a)
attaching an oligonucleotide tag from a repertoire of tags to each
molecule in a population of molecules (i) such that substantially all the
same molecules or same subpopulation of molecules in the population have
the same oligonucleotide tag attached and substantially all different
molecules or different subpopulations of molecules in the population have
different oligonucleotide tags attached and (ii) such that each
oligonucleotide tag from the repertoire comprises a plurality of subunits
and each subunit of the plurality consists of an oligonucleotide having a
length from three to six nucleotides or from three to six basepairs, the
subunits being selected from a minimally cross-hybridizing set; and (b)
sorting the molecules or subpopulations of molecules of the population by
specifically hybridizing the oligonucleotide tags with their respective
complements.
An important aspect of my invention is the use of the oligonucleotide tags
to sort polynucleotides for parallel sequence determination. Preferably,
such sequencing is carried out by the following steps: (a) generating from
the target polynucleotide a plurality of fragments that cover the target
polynucleotide; (b) attaching an oligonucleotide tag from a repertoire of
tags to each fragment of the plurality (i) such that substantially all the
same fragments have the same oligonucleotide tag attached and
substantially all different fragments have different oligonucleotide tags
attached and (ii) such that each oligonucleotide tag from the repertoire
comprises a plurality of subunits and each subunit of the plurality
consists of an oligonucleotide having a length from three to six
nucleotides or from three to six basepairs, the subunits being selected
from a minimally cross-hybridizing set;
sorting the fragments by specifically hybridizing the oligonucleotide tags
with their respective complements; (c) determining the nucleotide sequence
of a portion of each of the fragments of the plurality, preferably by a
single-base sequencing methodology as described below; and (d) determining
the nucleotide sequence of the target polynucleotide by collating the
sequences of the fragments.
My invention overcomes a key deficiency of current methods of tagging or
labeling molecules with oligonucleotides: By coding the sequences of the
tags in accordance with the invention, the stability of any mismatched
duplex or triplex between a tag and a complement to another tag is far
lower than that of any perfectly matched duplex between the tag and its
own complement. Thus, the problem of incorrect sorting because of mismatch
duplexes of GC-rich tags being more stable than perfectly matched AT-rich
tags is eliminated.
When used in combination with solid phase supports, such as microscopic
beads, my invention provides a readily automated system for manipulating
and sorting polynucleotides, particularly useful in large-scale parallel
operations, such as large-scale DNA sequencing, wherein many target
polynucleotides or many segments of a single target polynucleotide are
sequenced and/or analyzed simultaneously.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a-1c illustrates structures of labeled probes employed in a
preferred method of "single base" sequencing which may be used with the
invention.
FIG. 2 illustrates the relative positions of the nuclease recognition site,
ligation site, and cleavage site in a ligated complex (SEQ ID NO:16)
formed between a target polynucleotide and a probe used in a preferred
"single base" sequencing method.
FIG. 3 is a flow chart illustrating a general algorithm for generating
minimally cross-hybridizing sets.
FIG. 4 illustrates a scheme for synthesizing and using a combinatorial
chemical library in which member compounds are labeled with
oligonucleotide tags in accordance with the invention.
FIG. 5 diagrammatically illustrates an apparatus for carrying out parallel
operations, such as polynucleotide sequencing, in accordance with the
invention.
DEFINITIONS
"Complement" or "tag complement" as used herein in reference to
oligonucleotide tags refers to an oligonucleotide to which a
oligonucleotide tag specifically hybridizes to form a perfectly matched
duplex or triplex. In embodiments where specific hybridization results in
a triplex, the oligonucleotide tag may be selected to be either double
stranded or single stranded. Thus, where triplexes are formed, the term
"complement" is meant to encompass either a double stranded complement of
a single stranded oligonucleotide tag or a single stranded complement of a
double stranded oligonucleotide tag.
The term "oligonucleotide" as used herein includes linear oligomers of
natural or modified monomers or linkages, including deoxyribonucleosides,
ribonucleosides, .alpha.-anomeric forms thereof, peptide nucleic acids
(PNAs), and the like, capable of specifically binding to a target
polynucleotide by way of a regular pattern of monomer-to-monomer
interactions, such as Watson-Crick type of base pairing, base stacking,
Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually
monomers are linked by phosphodiester bonds or analogs thereof to form
oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to
several tens of monomeric units. Whenever an oligonucleotide is
represented by a sequence of letters, such as "ATGCCTG," it will be
understood that the nucleotides are in 5'.fwdarw.3' order from left to
right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G"
denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoranilidate, phosphoramidate, and the like.
Usually oligonucleotides of the invention comprise the four natural
nucleotides; however, they may also comprise non-natural nucleotide
analogs. It is clear to those skilled in the art when oligonucleotides
having natural or non-natural nucleotides may be employed, e.g. where
processing by enzymes is called for, usually oligonucleotides consisting
of natural nucleotides are required.
"Perfectly matched" in reference to a duplex means that the poly- or
oligonucleotide strands making up the duplex form a double stranded
structure with one other such that every nucleotide in each strand
undergoes Watson-Crick basepairing with a nucleotide in the other strand.
The term also comprehends the pairing of nucleoside analogs, such as
deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may
be employed. In reference to a triplex, the term means that the triplex
consists of a perfectly matched duplex and a third strand in which every
nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a
basepair of the perfectly matched duplex. Conversely, a "mismatch" in a
duplex between a tag and an oligonucleotide means that a pair or triplet
of nucleotides in the duplex or triplex fails to undergo Watson-Crick
and/or Hoogsteen and/or reverse Hoogsteen bonding.
As used herein, "nucleoside" includes the natural nucleosides, including
2'-deoxy and 2'-hydroxyl forms, e.g. as described in Kornberg and Baker,
DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" in
reference to nucleosides includes synthetic nucleosides having modified
base moieties and/or modified sugar moieties, e.g. described by Scheit,
Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,
Chemical Reviews, 90: 543-584 (1990), or the like, with the only proviso
that they are capable of specific hybridization. Such analogs include
synthetic nucleosides designed to enhance binding properties, reduce
degeneracy, increase specificity, and the like.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method of labeling and sorting molecules,
particularly polynucleotides, by the use of oligonucleotide tags. The
oligonucleotide tags of the invention comprise a plurality of "words" or
subunits selected from minimally cross-hybridizing sets of subunits.
Subunits of such sets cannot form a duplex or triplex with the complement
of another subunit of the same set with less than two mismatched
nucleotides. Thus, the sequences of any two oligonucleotide tags of a
repertoire that form duplexes will never be "closer" than differing by two
nucleotides. In particular embodiments, sequences of any two
oligonucleotide tags of a repertoire can be even "further" apart, e.g. by
designing a minimally cross-hybridizing set such that subunits cannot form
a duplex with the complement of another subunit of the same set with less
than three mismatched nucleotides, and so on. The invention is
particularly useful in labeling and sorting polynucleotides for parallel
operations, such as sequencing, fingerprinting or other types of analysis.
Constructing Oligonucleotide Tags from Minimally Cross-Hybridizing Sets of
Subunits
The nucleotide sequences of the subunits for any minimally
cross-hybridizing set are conveniently enumerated by simple computer
programs following the general algorithm illustrated in FIG. 3, and as
exemplified by program minhx whose source code is listed in Appendix I.
Minhx computes all minimally cross-hybridizing sets having subunits
composed of three kinds of nucleotides and having length of four.
The algorithm of FIG. 3 is implemented by first defining the characteristic
of the subunits of the minimally cross-hybridizing set, i.e. length,
number of base differences between members, and composition, e.g. do they
consist of two, three, or four kinds of bases. A table M.sub.n, n=1, is
generated (100) that consists of all possible sequences of a given length
and composition. An initial subunit S.sub.1 is selected and compared (120)
with successive subunits S.sub.i for i=n+1 to the end of the table.
Whenever a successive subunit has the required number of mismatches to be
a member of the minimally cross-hybridizing set, it is saved in a new
table M.sub.n+1 (125), that also contains subunits previously selected in
prior passes through step 120. For example, in the first set of
comparisons, M.sub.2 will contain S.sub.1 ; in the second set of
comparisons, M.sub.3 will contain S.sub.1 and S.sub.2 ; in the third set
of comparisons, M.sub.4 will contain S.sub.1, S.sub.2, and S.sub.3 ; and
so on. Similarly, comparisons in table M.sub.j will be between S.sub.j and
all successive subunits in M.sub.j. Note that each successive table
M.sub.n+1 is smaller than its predecessors as subunits are eliminated in
successive passes through step 130. After every subunit of table M.sub.n
has been compared (140) the old table is replaced by the new table
M.sub.n+1, and the next round of comparisons are begun. The process stops
(160) when a table M.sub.n is reached that contains no successive subunits
to compare to the selected subunit S.sub.i, i.e. M.sub.n =M.sub.n+1.
Preferably, minimally cross-hybridizing sets comprise subunits that make
approximately equivalent contributions to duplex stability as every other
subunit in the set. In this way, the stability of perfectly matched
duplexes between every subunit and its complement is appoximately equal.
Guidance for selecting such sets is provided by published techniques for
selecting optimal PCR primers and calculating duplex stabilities, e.g.
Rychlik et al, Nucleic Acids Research, 17: 8543-8551 (1989) and 18:
6409-6412 (1990); Breslauer et al, Proc. Natl. Acad. Sci., 83: 3746-3750
(1986); Wetmur, Crit. Rev. Biochem. Mol. Biol., 26: 227-259 (1991); and
the like. For shorter tags, e.g. about 30 nucleotides or less, the
algorithm described by Rychlik and Wetmur is preferred, and for longer
tags, e.g. about 30-35 nucleotides or greater, an algorithm disclosed by
Suggs et al, pages 683-693 in Brown, editor, ICN-UCLA Symp. Dev. Biol.,
Vol. 23 (Academic Press, New York, 1981) may be conveniently employed.
A preferred embodiment of minimally cross-hybridizing sets are those whose
subunits are made up of three of the four natural nucleotides. As will be
discussed more fully below, the absence of one type of nucleotide in the
oligonucleotide tags permits target polynucleotides to be loaded onto
solid phase supports by use of the 5'.fwdarw.3' exonuclease activity of a
DNA polymerase. The following is an exemplary minimally cross-hybridizing
set of subunits each comprising four nucleotides selected from the group
consisting of A, G, and T:
TABLE I
______________________________________
Word: w.sub.1 w.sub.2 w.sub.3
w.sub.4
Sequence: GATT TGAT TAGA TTTG
Word: w.sub.5 w.sub.6 w.sub.7
w.sub.8
Sequence: GTAA AGTA ATGT AAAG
______________________________________
In this set, each member would form a duplex having three mismatched bases
with the complement of every other member.
Further exemplary minimally cross-hybridizing sets are listed below in
Table I. Clearly, additional sets can be generated by substituting
different groups of nucleotides, or by using subsets of known minimally
cross-hybridizing sets.
TABLE II
______________________________________
Exemplary Minimally Cross-Hybridizing Sets of 4-mer Subunits
______________________________________
CATT ACCC AAAC AAAG AACA AACG
CTAA AGGG ACCA ACCA ACAC ACAA
TCAT CACG AGGG AGGC AGGG AGGC
ACTA CCGA CACG CACC CAAG CAAC
TACA CGAC CCGC CCGG CCGC CCGG
TTTC GAGC CGAA CGAA CGCA CGCA
ATCT GCAG GAGA GAGA GAGA GAGA
AAAC GGCA GCAG GCAC GCCG GCCC
AAAA GGCC GGCG GGAC GGAG
AAGA AAGC AAGG ACAG ACCG ACGA
ACAC ACAA ACAA AACA AAAA AAAC
AGCG AGCG AGCC AGGC AGGC AGCG
CAAG CAAG CAAC CAAC CACC CACA
CCCA CCCC CCCG CCGA CCGA CCAG
CGGC CGGA CGGA CGCG CGAG CGGC
GACC GACA GACA GAGG GAGG GAGG
GCGG GCGG GCGC GCCC GCAC GCCC
GGAA GGAC GGAG GGAA GGCA GGAA
______________________________________
The oligonucleotide tags of the invention and their complements are
conveniently synthesized on an automated DNA synthesizer, e.g. an Applied
Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA
Synthesizer, using standard chemistries, such as phosphoramidite
chemistry, e.g. disclosed in the following references: Beaucage and Iyer,
Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. Pat. No. 4,980,460;
Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos.
4,415,732; 4,458,066; and 4,973,679; and the like. Alternative
chemistries, e.g. resulting in non-natural backbone groups, such as
phosphorothioate, phosphoramidate, and the like, may also be employed
provided that the resulting oligonucleotides are capable of specific
hybridization. In some embodiments, tags may comprise naturally occuring
nucleotides that permit processing or manipulation by enzymes, while the
corresponding tag complements may comprise non-natural nucleotide analogs,
such as peptide nucleic acids, or like compounds, that promote the
formation of more stable duplexes during sorting.
When microparticles are used as supports, repertoires of oligonucleotide
tags and tag complements are preferably generated by subunit-wise
synthesis via "split and mix" techniques, e.g. as disclosed in Shortle et
al, International patent application PCT/US93/03418. Briefly, the basic
unit of the synthesis is a subunit of the oligonucleotide tag. Preferably,
phosphoramidite chemistry is used and 3' phosphoramidite oligonucleotides
are prepared for each subunit in a minimally cross-hybridizing set, e.g.
for the set first listed above, there would be eight 4-mer
3'-phosphoramidites. Synthesis proceeds as disclosed by Shortle et al or
in direct analogy with the techniques employed to generate diverse
oligonucleotide libraries using nucleosidic monomers, e.g. as disclosed in
Telenius et al, Genomics, 13: 718-725 (1992); Welsh et al, Nucleic Acids
Research, 19: 5275-5279 (1991); Grothues et al, Nucleic Acids Research,
21: 1321-1322 (1993); Hartley, European patent application 90304496.4; Lam
et al, Nature, 354: 82-84 (1991); Zuckerman et al, Int. J. Pept. Protein
Research, 40: 498-507 (1992); and the like. Generally, these techniques
simply call for the application of mixtures of the activated monomers to
the growing oligonucleotide during the coupling steps.
Double stranded forms of tags are made by separately synthesizing the
complementary strands followed by mixing under conditions that permit
duplex formation. Such duplex tags may then be inserted into cloning
vectors along with target polynucleotides for sorting and manipulation of
the target polynucleotide in accordance with the invention.
In embodiments where specific hybridization occurs via triplex formation,
coding of tag sequences follows the same principles as for duplex-forming
tags; however, there are further constraints on the selection of subunit
sequences. Generally, third strand association via Hoogsteen type of
binding is most stable along homopyrimidine-homopurine tracks in a double
stranded target. Usually, base triplets form in T-A*T or C-G*C motifs
(where "-" indicates Watson-Crick pairing and "*" indicates Hoogsteen type
of binding); however, other motifs are also possible. For example,
Hoogsteen base pairing permits parallel and antiparallel orientations
between the third strand (the Hoogsteen strand) and the purine-rich strand
of the duplex to which the third strand binds, depending on conditions and
the composition of the strands. There is extensive guidance in the
literature for selecting appropriate sequences, orientation, conditions,
nucleoside type (e.g. whether ribose or deoxyribose nucleosides are
employed), base modifications (e.g. methylated cytosine, and the like) in
order to maximize, or otherwise regulate, triplex stability as desired in
particular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci., 88:
9397-9401 (1991); Roberts et al, Science, 258: 1463-1466 (1992); Distefano
et al, Proc. Natl. Acad. Sci., 90: 1179-1183 (1993); Mergny et al,
Biochemistry, 30: 9791-9798 (1991); Cheng et al, J. Am. Chem. Soc., 114:
4465-4474 (1992); Beal and Dervan, Nucleic Acids Research, 20: 2773-2776
(1992); Beal and Dervan, J. Am. Chem. Soc., 114: 4976-4982 (1992);
Giovannangeli et al, Proc. Natl. Acad. Sci., 89: 8631-8635 (1992); Moser
and Dervan, Science, 238: 645-650 (1987); McShan et al, J. Biol. Chem.,
267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci., 89: 3840-3844
(1992); Blume et al, Nucleic Acids Research, 20: 1777-1784 (1992); Thuong
and Helene, Angew. Chem. Int. Ed. Engl. 32: 666-690 (1993); and the like.
Conditions for annealing single-stranded or duplex tags to their
single-stranded or duplex complements are well known, e.g. Ji et al, Anal.
Chem. 65: 1323-1328 (1993).
Oligonucleotide tags of the invention may range in length from 12 to 60
nucleotides or basepairs. Preferably, oligonucleotide tags range in length
from 18 to 40 nucleotides or basepairs. More preferably, oligonucleotide
tags range in length from 25 to 40 nucleotides or basepairs. Most
preferably, oligonucleotide tags are single stranded and specific
hybridization occurs via Watson-Crick pairing with a tag complement.
Attaching Tags to Molecules
Oligonucleotide tags may be attached to many different classes of molecules
by a variety of reactive functionalities well known in the art, e.g.
Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular
Probes, Inc., Eugene, 1992); Khanna et al, U.S. Pat. No. 4,318,846; or the
like. Table III provides exemplary functionalities and counterpart
reactive groups that may reside on oligonucleotide tags or the molecules
of interest. When the functionalities and counterpart reactants are
reacted together, after activation in some cases, a linking group is
formed. Moreover, as described more fully below, tags may be synthesized
simultaneously with the molecules undergoing selection to form
combinatorial chemical libraries.
TABLE III
__________________________________________________________________________
Reactive Functionalities and Their Counterpart Reactants
and Resulting Linking Groups
Reactive Counterpart Linking
Functionality
Functionality
Group
__________________________________________________________________________
NH.sub.2 COOH CONH
NH.sub.2 NCO NHCONH
NH.sub.2 NCS NHCSNH
NH.sub.2
##STR1##
##STR2##
SH CCCO SCCCO
NH.sub.2 CHO CH.sub.2 NH
NH.sub.2 SO.sub.2 Cl SO.sub.2 NH
OH OP(NCH(CH.sub.3).sub.2).sub.2
OP(O)(O)O
OP(O)(O)S NHC(O)CH.sub.2 Br
NHC(O)CH.sub.2 SP(O)(O)O
__________________________________________________________________________
A class of molecules particularly convenient for the generation of
combinatorial chemical libraries includes linear polymeric molecules of
the form:
--(M--L).sub.n --
wherein L is a linker moiety and M is a monomer that may selected from a
wide range of chemical structures to provide a range of functions from
serving as an inert non-sterically hindering spacer moiety to providing a
reactive functionality which can serve as a branching point to attach
other components, a site for attaching labels; a site for attaching
oligonucleotides or other binding polymers for hybridizing or binding to a
therapeutic target; or as a site for attaching other groups for affecting
solubility, promotion of duplex and/or triplex formation, such as
intercalators, alkylating agents, and the like. The sequence, and
therefore composition, of such linear polymeric molecules may be encoded
within a polynucleotide attached to the tag, as taught by Brenner and
Lerner (cited above). However, after a selection event, instead of
amplifying then sequencing the tag of the selected molecule, the tag
itself or an additional coding segment can be sequenced directly--using a
so-called "single base" approach described below--after releasing the
molecule of interest, e.g. by restriction digestion of a site engineered
into the tag. Clearly, any molecule produced by a sequence of chemical
reaction steps compatible with the simultaneous synthesis of the tag
moieties can be used in the generation of combinatorial libraries.
Conveniently there is a wide diversity of phosphate-linked monomers
available for generating combinatorial libraries. The following references
disclose several phosphoramidite and/or hydrogen phosphonate monomers
suitable for use in the present invention and provide guidance for their
synthesis and inclusion into oligonucleotides: Newton et al, Nucleic Acids
Research, 21: 1155-1162 (1993); Griffin et al, J. Am. Chem. Soc., 114:
7976-7982 (1992); Jaschke et al, Tetrahedron Letters, 34: 301-304 (1992);
Ma et al, International application PCT/CA92/00423; Zon et al,
International application PCT/US90/06630; Durand et al, Nucleic Acids
Research, 18: 6353-6359 (1990); Salunkhe et al, J. Am. Chem. Soc., 114:
8768-8772 (1992); Urdea et al, U.S. Pat. No. 5,093,232; Ruth, U.S. Pat.
No. 4,948,882; Cruickshank, U.S. Pat. No. 5,091,519; Haralambidis et al,
Nucleic Acids Research, 15: 4857-4876 (1987); and the like. More
particularly, M may be a straight chain, cyclic, or branched organic
molecular structure containing from 1 to 20 carbon atoms and from 0 to 10
heteroatoms selected from the group consisting of oxygen, nitrogen, and
sulfur. Preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1
to 16 carbon atoms; a heterocycle having from 3 to 8 carbon atoms and from
1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen,
and sulfur; glycosyl; or nucleosidyl. More preferably, M is alkyl, alkoxy,
alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or
nucleosidyl.
Preferably, L is a phosphorus(V) linking group which may be phosphodiester,
phosphotriester, methyl or ethyl phosphonate, phosphorothioate,
phosphorodithioate, phosphoramidate, or the like. Generally, linkages
derived from phosphoramidite or hydrogen phosphonate precursors are
preferred so that the linear polymeric units of the invention can be
conveniently synthesized with commercial automated DNA synthesizers, e.g.
Applied Biosystems, Inc. (Foster City, Calif.) model 394, or the like.
n may vary significantly depending on the nature of M and L. Usually, n
varies from about 3 to about 100. When M is a nucleoside or analog thereof
or a nucleoside-sized monomer and L is a phosphorus(V) linkage, then n
varies from about 12 to about 100. Preferably, when M is a nucleoside or
analog thereof or a nucleoside-sized monomer and L is a phosphorus(V)
linkage, then n varies from about 12 to about 40.
Peptides are another preferred class of molecules to which tags of the
invention are attached. Synthesis of peptide-oligonucleotide conjugates
which may be used in the invention is taught in Nielsen et al, J. Am.
Chem. Soc., 115: 9812-9813 (1993); Haralambidis et al (cited above) and
International patent application PCT/AU88/004417; Truffert et al,
Tetrahedron Letters, 35: 2353-2356 (1994); de la Torre et al, Tetrahedron
Letters, 35: 2733-2736 (1994); and like references. Preferably,
peptide-oligonucleotide conjugates are synthesized as described below.
Peptides synthesized in accordance with the invention may consist of the
natural amino acid monomers or non-natural monomers, including the D
isomers of the natural amino acids and the like.
Combinatorial Chemical Libraries
Combinatorial chemical libraries employing tags of the invention are
preferably prepared by the method disclosed in Nielsen et al (cited above)
and illustrated in FIG. 4 for a particular embodiment. Briefly, a solid
phase support, such as CPG, is derivatized with a cleavable linker that is
compatible with both the chemistry employed to synthesize the tags and the
chemistry employed to synthesize the molecule that will undergo some
selection process. Preferably, tags are synthesized using phosphoramidite
chemistry as described above and with the modifications recommended by
Nielsen et al (cited above); that is, DMT-5'-O-protected
3'-phosphoramidite-derivatized subunits having methyl-protected phosphite
and phosphate moities are added in each synthesis cycle. Library compounds
are preferably monomers having Fmoc--or equivalent--protecting groups
masking the functionality to which successive monomer will be coupled. A
suitable linker for chemistries employing both DMT and Fmoc protecting
groups (referred to herein as a sarcosine linker) is disclosed by Brown et
al, J. Chem. Soc. Chem. Commun., 1989: 891-893, which reference is
incorporated by reference.
FIG. 4 illustrates a scheme for generating a combinatorial chemical library
of peptides conjugated to oligonucleotide tags. Solid phase support 200 is
derivatized by sarcosine linker 205 (exemplified in the formula below) as
taught by Nielsen et al (cited above), which has an extended linking
moiety to facilitate reagent access.
(CPG)-NHC(O)CN(CH.sub.3)C(O)CH.sub.2 CH.sub.2 C(O)O(CH.sub.2).sub.6
NHC(O)CH.sub. | | |
