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This application is based on U. The present invention relates to compositions and methods for synthesizing a DNA-templated combinatorial chemistry library of compounds, the selection of compounds with a desired activity and genetic recombination and in vitro evolution of selected compounds.
The invention further includes the nucleic acid tags which direct the synthesis of the compound library, and the library of compounds produced by the methods of the invention.
There is widespread interest in efficient synthesis and screening of large numbers of compounds to identify candidate compounds with a given desired activity. Combinatorial libraries of random-sequence oligonucleotides, polypeptides, synthetic oligomers and small organic molecules have been described and their utility in identifying active compounds or as a starting point for developing related molecules with more desirable properties has been proposed Ellman, J A and Gallop, M A, One method for formation of combinatorial libraries involves preparation of high density position-addressable oligomer arrays on a planar substrate.
In this method, a substrate having photoprotective groups is irradiated, using photolithographic mask techniques, in selected regions only, to deprotect surface active groups in those selected regions. The entire surface is then treated with a solution of a selected subunit, which itself has a photoprotected group, to react this subunit with the surface groups in the photodeprotected regions. This process is repeated to i add a selected subunit at each region of the surface, and ii build up different-sequence oligomers at known, addressable regions of the surface.
This method has the advantage that reaction sites do not have to be physically separated during subunit addition, and therefore massive parallel subunit addition is possible by applying subunit-addition reagents over the entire surface of the array.
Greater site density is therefore feasible than in systems where physical separation of reagents is required from one reaction site to another, and where individual reagents are spotted or deposited in defined array regions. A related approach wherein the library is produced in capillary tubes has also been described wherein a method for producing, high-density, position-addressable combinatorial library of different-sequence oligomer or different-substituent small molecule compounds.
The disclosed invention includes massive parallel synthesis of subunits and known, addressable library positions in a dense array of capillary tubes, and the screening of individual library compounds in either solution phase or solid phase. In a related approach, a traditional split-and-recombine strategy for synthesis of combinatorial libraries has been described. In one application of this approach, beads containing successive precursors to the target compounds that form the library may be alternately mixed and separated, with one of a selected number of reagents being added to each group of separated beads at each step [Furka, A.
An advantage of this method is that each bead contains only one chemical species, allowing the beads themselves to be used for screening. However, the identity of the species on each bead must be independently determined. Although several methods have been reported for tagging the support beads with molecules more readily analyzable than the library members themselves [e.
Replacements for the conventional bead support for combinatorial synthesis have also been described, e. The various polymer supports useful in combinatorial library formation of same molecules have been recently reviewed. Another general approach involves the synthesis of a combinatorial library as a physically segregated array of compounds [Geysen, H.
Libraries of compounds have been synthesized on functionalized resins either coated on Geysen, et al, , ; Bunin, et al. Southern used arrays of spots laid down on a substrate such as glass by a pen plotter. A key advantage of this approach is that the chemical identity of each library element on the array is associated with an addressable position on the array.
In cases where the compounds may be screened for biological activity while still attached to the substrate, this method also allows for massive and rapid screening, by binding a reporter-labeled target to the surface and determining the positions of bound target. Surface arrays of this type may be used both for combinatorial library screening Fodor, S. In a further approach, two alternating parallel combinatorial syntheses are performed such that a genetic tag is chemically linked to the chemical structure being synthesized.
A library is built up by the repeating the process after pooling and division of the reaction products obtained at each step. One limitation in the early methods of combinatorial library formation is that large-library planar arrays are necessarily limited in the amount number of molecules of each library species, since the planar region available to each species is quite small, e. As a consequence, the ability to detect binding species on the array may be limited. Further, it is not feasible to carry out solution-phase screening on a planar array, because of the difficulty of physically separating different array regions carrying different library members.
It would thus be desirable to provide a method for preparing a large combinatorial library of compounds which has the advantages of i massive parallel synthesis of subunits and known, addressable library positions, ii adaptable to virtually any oligomer or small-molecule chemistry, iii a relatively large area for synthesis of each library member, iv capable of being screened either as a mixture or as individual library compounds in either solution phase or solid phase, and v capable of amplifying and modifying selected library compounds.
The present invention provides methods and compositions for iterative synthesis and screening of a plurality of compounds wherein a nucleic acid tag directs and encodes the synthesis of the compound to which it is covalently attached, and the tag is a DNA molecule which can be amplified biochemically.
The methods of the present invention provide for synthesis of a plurality of compounds in a combinatorial library by way of a split and combine synthesis strategy, wherein synthesis is directed by the nucleic acid tag. The library may be provided in solution or attached to a solid support.
The nucleic acid tags useful in the methods of the present invention comprise nucleic acid sequences having a plurality of different first hybridization sequences, a mixture of different second hybridization sequences, and a chemical reaction site. The present invention further provides a library of nucleic acid tags, also termed nucleic acid supports for use in directing the synthesis of a plurality of compounds wherein each tag has a first segment having a selected one of a plurality of different first hybridization sequences, a mixture of different second hybridization sequences, and a chemical reaction site; and a second segment having a selected one of a plurality of different second hybridization sequences and a mixture of different first hybridization sequences.
The methods of the present invention provide subsets of nucleic acid tags generated by base-specific duplex formation between each different first hybridization sequence and a complementary oligonucleotides or oligonucleotide analogs. The chemical reaction sites in each of the subsets are reacted with a selected reagent to form a reagent-specific compound intermediate. The methods of the present invention further provide that the steps of formation of subsets of nucleic acid sequences by base-specific duplex formation be repeated and a chemical subunit added to the chemical reaction site or last added chemical subunit within each subset until synthesis of the plurality of compounds is complete.
In an exemplary aspect of the present invention, the nucleic acid tags include alternating spacer and hybridization sequences, wherein the spacer sequences are the same for all subsets of nucleic acid sequences and the hybridization sequences are different for each subset of nucleic acid sequences.
In a related aspect, the spacer sequence portion of each nucleic acid sequence has a restriction enzyme site which is unique to a given spacer sequence. The methods of the present invention provide for the synthesis of small molecules with different chemical sequences, catalysts useful for the synthesis of complex molecules from simple substrates, inorganic compounds with useful properties as materials, non-ribosomally produced polypeptides, peptoids, polyketide-based natural products or subunit oligomers, e.
In one aspect, the invention provides compound libraries wherein the compounds of such libraries can be subjected to enrichment for one or more desired activities on a continuously amplifying population. In the methods of the present invention compounds having one or more desired activities are enriched to yield a subpopulation of nucleic acid sequences.
The enriched subpopulation s of nucleic acid sequences serve as the starting material for repeating the step-wise synthesis of additional compounds. Alternatively, the enriched subpopulation of nucleic acid sequences is amplified by non-specific polymerase chain reaction PCR , and a new chemical reaction site added prior to repeating the step-wise synthesis of additional compounds.
In such a process, the enriched subpopulation of nucleic acid sequences is treated with one or more restriction enzymes under conditions effective to produce a partial digest by cleavage at a sequence-specific restriction enzyme site within each spacer sequence. The partially digested nucleic acid sequences are rejoined and a new chemical reaction site added prior to repeating the step-wise synthesis of additional compounds. Compound libraries which are synthesized under the direction of compound-specific synthesis-directing nucleic acid tags are also provided by the present invention.
In this aspect, the nucleic acid sequences which direct the synthesis of the compounds can be subjected to genetic recombination or in vitro evolution by repeated cycles of enrichment and step-wise synthesis; enrichment, PCR amplification and step-wise synthesis; or enrichment, partial digestion, rejoining of fragments and stepwise synthesis to yield a highly enriched subpopulation of synthesis-directing nucleic acid sequences.
The invention also provides a method for library splitting on the basis of sequence hybridization post-synthesis. In this aspect, a complete library is synthesized, split by hybridization based on the different sequence directing nucleic acid tag attached to each library member and further step performed on the split library.
Preferred types of compounds in the compound libraries of the present invention include, but are not limited to, small molecules with different chemical sequences, catalysts useful for the synthesis of complex molecules from simple substrates, inorganic compounds with useful properties as materials, non-ribosomally produced polypeptides, peptoids, polyketide-based natural products or subunit oligomers, e.
Further, the invention provides a method to perform all genetic manipulations possible with natural biopolymers through the manipulation of DNA instructions on such DNA-templated combinatorial libraries of compounds as a means to provide a method to identify useful compounds from large combinatorial libraries of compounds, as described above.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. The degenerate family of DNA fragments consists of catenated 20 base-pair nucleotide sequences, which are either constant z 1 -z 5 or variable a 1 -j 4. The letters a 1 through j 4 in the variable regions of the DNA fragments denote distinct 20 nucleotide sequences with orthogonal hybridization properties.
To carry out the first split, the degenerate family of fragments are passed over a set of ten different affinity resins displaying the sequences a 1 c -j 1 c , which are complementary to the sequences a 1 -j 1 in the first variable region one affinity resin is represented by the shaded ball.
Ten sub-pools of the original family of fragments result. Each sub-pool is coupled to a distinct chemical monomer at the chemical reaction site. The sub-pools are recombined, and the library is split into a new set of sub-pools based on the sequences a 2 -j 2 , etc.
The process can be repeated many times, and with a variety of amino acids, to produce polypeptide libraries. Such conditions are preferably stringent enough to prevent or largely prevent hybridization of two nearly-complementary strands that have one or more internal base mismatches. Preferably the region of identity between two sequences forming a base-specific duplex is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.
The sequential annealing, extension and denaturation steps are controlled by varying the temperature of the reaction container, normally in a repeating cyclical manner. In the context of oligos which direct the synthesis of the library compounds of the present invention, the oligos may include or be composed primarily of nucleotide analog subunits, or other subunits capable of forming sequence-specific Watson-Crick base pairing, when assembled in a linear polymer, with the proviso that the free ends of the oligos are ribonucleotide or deoxyribonucleotide subunits capable of providing a suitable substrate for strand-directed polymerization in the presence of a DNA polymerase and one or more nucleotide triphosphates, e.
An oligonucleotide analog will generally contain phosphodiester bonds, although in some cases, oligonucleotide analogs are included that may have alternate backbones. Modifications of the ribose-phosphate backbone may facilitate the addition of additional moieties such as labels, or may be done to increase the stability and half-life of such molecules.
In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. The oligonucleotides may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The oligonucleotide may be DNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo-and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
Such compounds are usually non-oligomeric that is, do not consist of sequences of repeating similar subunits and may be similar in terms of basic structure and functional groups, but vary in such aspects as chain length, ring size or number, or patterns of substitution. In another aspect, the invention provides combinatorial compound libraries which can be subjected to genetic recombination or in vitro evolution by repeated cycles of enrichment and step-wise synthesis, enrichment, PCR amplification and step-wise synthesis or enrichment, partial digestion, reformation and stepwise synthesis to yield a highly enriched subpopulation of nucleic acids which are bound to compounds having one or more desired activities.
Receptors can be attached, covalently or non-covalently, to a binding member, either directly or via a specific binding substance.
Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants such as on viruses, cells, or other materials , cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors. As one of skill in the art will recognize, a molecule or macromolecular complex can be both a receptor and a ligand.
In general, the binding partner having a smaller molecular weight is referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor. Modulation may relate to an increase or a decrease in biological activity, binding characteristics, or any other biological, functional, or immunological property of the molecule.
Agonists may themselves be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of the receptor. Antagonists may themselves be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of the receptor.
Other terms used herein should be construed to take on meanings customary in the art, unless otherwise defined herein. The present invention provides encoded combinatorial chemical libraries which comprise a plurality of species of bifunctional molecules that each define a different chemical structure and that each contain a unique identifier nucleic acid sequence whose sequence defines, and directs the synthesis of the corresponding chemical structure.
The invention is based on the traditional split-and-recombine strategy for synthesis of combinatorial libraries comprising two or more synthetic steps.
For example, in a combinatorial synthesis consisting of i steps, for which j different chemical coupling reactions are performed at each step, j i compounds will be present in the final library. The traditional split and-recombine strategy is carried out using the following steps; i at the beginning of each of the i steps, the pool of solid tags is randomly split into j subsets, ii each of the j subsets of solid tags is subjected to a different chemical coupling step, and iii after the chemical coupling step, the subsets are recombined into a single pool.
This recombined pool is again randomly divided into j subsets specifically as in i above at the beginning of the next step in the library synthesis. In the synthesis of peptide libraries, for example, the coupling step is the addition of an amino-acid active ester to a free amine group on the solid tag. Each of the j subsets is coupled to a different amino acid e.
For example, a split-and-recombine synthesis of 10 synthetic steps, with 10 coupling reactions at each step, would yield a final library size of 10 The invention further provides a library of nucleic acid tags, also termed nucleic acid supports for use in directing the synthesis of a plurality of compounds wherein each tag has a first segment having a selected one of a plurality of different first hybridization sequences, a mixture of different second hybridization sequences, and a chemical reaction site; and a second segment having a selected one of a plurality of different second hybridization sequences and a mixture of different first hybridization sequences.
The methods of the present invention provide subsets of nucleic acid tags generated by base-specific duplex formation between each different first hybridization sequence and a complementary oligonucleotide or oligonucleotide analog. In one preferred embodiment, the nucleic acid tag comprises a first hybridization sequence, a second hybridization sequence and a chemical reaction site.
In a preferred aspect, a complementary oligonucleotide or oligonucleotide analog useful in the methods of the present invention is bound to one of a plurality of surface bound reagents. The methods of the present invention provide that the steps of formation of subsets of nucleic acid sequences by base-specific duplex formation be repeated and a chemical subunit added to the chemical reaction site or last added chemical subunit within each subset until synthesis of the plurality of compounds is complete.
In general, the synthesis of a plurality of compounds requires two or more synthetic steps. In a preferred aspect, each subset of nucleic acid sequences includes at least 5 separate hybridization sequences. In a more preferred aspect each subset of nucleic acid sequences includes at least 10 separate hybridization sequences.