NZ722289B2 - Methods for tagging dna-encoded libraries - Google Patents

Abstract

Methods for tagging DNA-encoded libraries are provided. One embodiment provides a method of tagging a library comprising an oligonucleotide-encoded small molecule or peptide, the method comprises (i) providing a headpiece having a first functional group and a second functional group; (ii) binding the first functional group of the headpiece to a first component of the small molecule or peptide, wherein the headpiece is directly connected to the first component or the headpiece is indirectly connected to the first component by a bifunctional linker; and (iii) ligating the second functional group of the headpiece to a first building block tag to form a complex, wherein the ligating comprises chemical ligation which results in the formation of a linkage that does not comprise a phosphodiester or a phosphorothioate. Steps (ii) and (iii) can be performed in any order and said oligonucleotide headpiece does not comprise a 2'-substituted nucleotide at the 5'-terminus and/or the 3'-terminus. e first functional group of the headpiece to a first component of the small molecule or peptide, wherein the headpiece is directly connected to the first component or the headpiece is indirectly connected to the first component by a bifunctional linker; and (iii) ligating the second functional group of the headpiece to a first building block tag to form a complex, wherein the ligating comprises chemical ligation which results in the formation of a linkage that does not comprise a phosphodiester or a phosphorothioate. Steps (ii) and (iii) can be performed in any order and said oligonucleotide headpiece does not comprise a 2'-substituted nucleotide at the 5'-terminus and/or the 3'-terminus.

Description

METHODS FOR TAGGING DNA-ENCODED LIBRARIES Cross-reference to Related Applications This application is a divisional of New Zealand patent application 621592, which is the national phase entry in New Zealand of PCT international ation (published as ), claims benefit of U.S. Provisional Application Nos. 61/531,820, filed ber 7, 2011, and 61/536,929, filed September 20, 2011, all of which is hereby incorporated by reference.

Background of the Invention In general, this invention s lly to DNA-encoded libraries of compounds and methods of using and creating such libraries. Also described herein are compositions for use in such libraries.

DNA-encoded combinatorial libraries afford many benefits for drug discovery. These libraries can provide a large number of diverse compounds that can be rapidly screened and interrogated. To r increase complexity, various steps of the discovery s can be programmed and automated.

These steps include the use of multi-step, split-and-pool synthesis to add ng blocks to atomic or polyatomic scaffolds and the use of tic and/or chemical ligation to add DNA tags that encode both the synthetic steps and the building blocks.

Despite these benefits, numerous issues can arise when very large or x libraries must be sized and deconvoluted. As the size of the library increases, improved methods may be needed to provide high yields of tag ligation. To create libraries under diverse reaction conditions, stable d nucleotide constructs would be beneficial, such as constructs that are stable under conditions of high pH and elevated ature. To simplify deconvolution of tags, the sequence of the tags could be recognized by DNA- or RNA-dependent polymerases, such that tag population demographics can be determined by template-dependent polymerization and sequence determination. Difficulties may arise when creating a y having all of these beneficial attributes. Accordingly, there exists a need for improved, more robust methods of screening and identifying small compounds in DNA-encoded libraries.

Summary of the Invention Disclosed herein are s of creating libraries, where the method includes one or more conditions that improve single-stranded ligation of tags, and compositions for use in ng libraries.

Exemplary conditions include the use of one or more 2’-substituted bases within the tags, such as 2’-O- methyl or 2’-fluoro; the use of tags of particular length; the use of one or more enzymes; optionally, the inclusion of error-recognition capabilities in the tag design; and/or the use of one or more agents during ligation.

Accordingly, in one aspect the invention relates to a method of tagging a library comprising an oligonucleotide-encoded small molecule or peptide, said method comprising: (i) providing an oligonucleotide headpiece having a first functional group and a second functional group; (ii) binding said first functional group of said ucleotide headpiece to a first component of said small molecule or peptide, wherein said headpiece is directly connected to said first component or said headpiece is indirectly connected to said first component by a bifunctional linker; and (iii) ligating said second functional group of said oligonucleotide headpiece to a first building block tag to form a complex, wherein said ligating comprises chemical ligation, which results in the ion of a linkage that does not comprise a phosphodiester or a phosphorothioate, wherein steps (ii) and (iii) can be med in any order, n said oligonucleotide headpiece does not comprise a 2'-substituted nucleotide at the 5'-terminus and/or the 3'-terminus.

Certain statements that appear below are broader than what appears in the statements of the invention above. These statements are provided in the interests of providing the reader with a better understanding of the invention and its practice. The reader is directed to the accompanying claim set which defines the scope of the invention.

Also described herein is a method of tagging a first library including an ucleotide-encoded chemical , the method including: (i) providing a headpiece having a first functional group and a second onal group, where the headpiece includes at least one 2’-substituted nucleotide; (ii) binding the first functional group of the headpiece to a first component of the al entity, where the headpiece is directly connected to the first ent or the headpiece is indirectly connected to the first component by a bifunctional linker (e.g., a poly ethylene glycol linker or –(CH2CH2O)nCH2CH2-, where n is an integer from 1 to 50); and (iii) binding the second functional group of the headpiece to a first ng block tag to form a complex, where steps (ii) and (iii) can be performed in any order and where the first building block tag encodes for the binding reaction of step (ii), thereby providing a tagged library.

In some embodiments, the headpiece includes a 2’-substituted nucleotide at one or more of the ’-terminus, the 3’-terminus, or the internal on of the headpiece. In particular embodiments, the headpiece includes the 2’-substituted nucleotide and the second functional group at the 5’-terminus or at the 3’-terminus.

In other embodiments, the first building block tag includes at least one (e.g., at least two, three, four, five, or more) 2’-substituted nucleotides. In particular ments, the first building block tag includes a 2’-substituted nucleotide at one or more of the 5’-terminus, the 3’-terminus, or the internal position of the first building block tag (e.g., a 2’-O-methyl nucleotide or a oro tide at both of the 5’- and 3’-termini). In some embodiments, the first building block tag includes a protecting group at the 3’-terminus or at the 5’-terminus.

In any of the embodiments described herein, the 2’-substituted nucleotide is a ethyl nucleotide (e.g., 2’-O-methyl guanine or 2’-O-methyl ) or a 2’-fluoro nucleotide (e.g., 2’-fluoro guanine, or 2’-fluoro uracil).

In any of the above embodiments, step (ii) may include joining, binding, or operatively associating the headpiece directly to the first component (e.g., a scaffold or a first building block). In yet other embodiments, step (ii) includes binding the headpiece indirectly to the first component (e.g., a scaffold or a first building block) via a bifunctional linker (e.g., the method includes g the headpiece with the first onal group of the linker and binding the first component with the second functional group of the linker).

In any of the above embodiments, the method may further include (iv) binding a second building block tag to the 5’-terminus or minus of the complex; and (v) binding a second component (e.g., a first building block or a second building block) of the al library to the first component, where steps (iv) and (v) can be performed in any order. In some embodiments, the second building block tag encodes for the binding reaction of step (v). In other embodiments, step (iv) may include binding the second building block tag to the 5’-terminus of the complex; the complex includes a phosphate group at the 5’- terminus; and the second building block tag includes a hydroxyl group at both of the 3’- and 5’-termini.

In other embodiments, step (iv) may further include purifying the complex and reacting the complex with a polynucleotide kinase to form a ate group on the 5’-terminus prior to g the second building block tag. In other embodiments, step (iv) may include binding the second building block tag to the minus of the complex; the complex es a protecting group at the 3’-terminus; and the second ng block tag includes a phosphate group at the 5’-terminus and a protecting group at the 3’- terminus. In yet other embodiments, step (iv) may further include reacting the complex with a hydrolyzing agent to release the protecting group from the complex prior to binding the second building block tag to the complex.

In further embodiments, the second building block tag includes a 2’-substituted tide (e.g., a 2’-O-methyl nucleotide or a 2’-fluoro nucleotide) at one or more of the minus, the minus, or the internal position of the second building block tag (e.g., a 2’-O-methyl nucleotide and/or a 2’-fluoro nucleotide at both of the 5’- and 3’-termini).

In some ments, step (iv) may include the use of an RNA ligase (e.g., T4 RNA ) and/or a DNA ligase (e.g., a ssDNA ligase) to bind the second building block tag to the complex (e.g., may include the use of both RNA ligase and the DNA ligase).

In other embodiments, step (iii) may include the use of an RNA ligase (e.g., T4 RNA ligase) and/or a DNA ligase (e.g., ssDNA ligase) to bind the headpiece to the first building block tag (e.g., may include the use of both RNA ligase and the DNA ligase).

In further embodiments, step (iii) and/or step (iv), if present, may include the use of poly ethylene glycol and/or one or more soluble multivalent cations (e.g., magnesium chloride, manganese (II) chloride, or hexamine cobalt (III) chloride). In some embodiments, the poly ethylene glycol is in an amount from about 25% (w/v) to about 35% (w/v) (e.g., from about 25% (w/v) to about 30 % (w/v), from about 30 % (w/v) to about 35% (w/v), or about 30% (w/v)). In other embodiments, the poly ethylene glycol has an average molecular weight from about 3,000 to about 5,500 Daltons (e.g., about 4,600 s). In other embodiments, the one or more soluble multivalent cations are in an amount of from about 0.05 mM to about 10.5 mM (e.g., from 0.05 mM to 0.5 mM, from 0.05 mM to 0.75 mM, from 0.05 mM to 1.0 mM, from 0.05 mM to 1.5 mM, from 0.05 mM to 2.0 mM, from 0.05 mM to 3.0 mM, from 0.05 mM to 4.0 mM, from 0.05 mM to 5.0 mM, from 0.05 mM to 6.0 mM, from 0.05 mM to 7.0 mM, from 0.05 mM to 8.0 mM, from 0.05 mM to 9.0 mM, from 0.05 mM to 10.0 mM, from 0.1 mM to 0.5 mM, from 0.1 mM to 0.75 mM, from 0.1 mM to 1.0 mM, from 0.1 mM to 1.5 mM, from 0.1 mM to 2.0 mM, from 0.1 mM to 3.0 mM, from 0.1 mM to 4.0 mM, from 0.1 mM to 5.0 mM, from 0.1 mM to 6.0 mM, from 0.1 mM to 7.0 mM, from 0.1 mM to 8.0 mM, from 0.1 mM to 9.0 mM, from 0.1 mM to 10.0 mM, from 0.1 mM to .5 mM, from 0.5 mM to 0.75 mM, from 0.5 mM to 1.0 mM, from 0.5 mM to 1.5 mM, from 0.5 mM to 2.0 mM, from 0.5 mM to 3.0 mM, from 0.5 mM to 4.0 mM, from 0.5 mM to 5.0 mM, from 0.5 mM to 6.0 mM, from 0.5 mM to 7.0 mM, from 0.5 mM to 8.0 mM, from 0.5 mM to 9.0 mM, from 0.5 mM to .0 mM, from 0.5 mM to 10.5 mM, from 0.75 mM to 1.0 mM, from 0.75 mM to 1.5 mM, from 0.75 mM to 2.0 mM, from 0.75 mM to 3.0 mM, from 0.75 mM to 4.0 mM, from 0.75 mM to 5.0 mM, from 0.75 mM to 6.0 mM, from 0.75 mM to 7.0 mM, from 0.75 mM to 8.0 mM, from 0.75 mM to 9.0 mM, from 0.75 mM to 10.0 mM, from 0.75 mM to 10.5 mM, from 1.0 mM to 1.5 mM, from 1.0 mM to 2.0 mM, from 1.0 mM to 3.0 mM, from 1.0 mM to 4.0 mM, from 1.0 mM to 5.0 mM, from 1.0 mM to 6.0 mM, from 1.0 mM to 7.0 mM, from 1.0 mM to 8.0 mM, from 1.0 mM to 9.0 mM, from 1.0 mM to 10.0 mM, from 1.0 mM to 10.5 mM, from 1.5 mM to 2.0 mM, from 1.5 mM to 3.0 mM, from 1.5 mM to 4.0 mM, from 1.5 mM to 5.0 mM, from 1.5 mM to 6.0 mM, from 1.5 mM to 7.0 mM, from 1.5 mM to 8.0 mM, from 1.5 mM to 9.0 mM, from 1.5 mM to 10.0 mM, from 1.5 mM to 10.5 mM, from 2.0 mM to 3.0 mM, from 2.0 mM to 4.0 mM, from 2.0 mM to 5.0 mM, from 2.0 mM to 6.0 mM, from 2.0 mM to 7.0 mM, from 2.0 mM to 8.0 mM, from 2.0 mM to 9.0 mM, from 2.0 mM to 10.0 mM, and from 2.0 mM to 10.5 mM). In some embodiments, one or more multivalent cations are in an amount of about 1 mM (e.g., from 0.5 mM to 1.5 mM). In a particular embodiment, the multivalent cation is in the form of hexamine cobalt (III) chloride.

In other embodiments, the method further includes separating the complex from any unreacted tag or unreacted headpiece before any one of binding steps v). In other embodiments, the method further includes purifying the complex before any one of binding steps (ii)-(v). In other embodiments, the method further includes binding one or more additional components (e.g., a scaffold or a first building block) and one or more additional building block tags, in any order and after any one of binding step (ii)- (v).

Also described herein is a method of tagging a first library including an oligonucleotide-encoded chemical entity, the method including: (i) providing a headpiece having a first onal group and a second functional group, where the headpiece includes a 2’-substituted nucleotide at the 5’-terminus, ally one or more nucleotides at the internal position of the headpiece, and a protecting group at the 2’-position and/or the 3’-position at the 3’-terminus; (ii) binding the first functional group of the ece to a first ent of the chemical entity, where the headpiece is directly connected to the first component or the headpiece is indirectly connected to the first component by a tional ; and (iii) binding the second functional group of the headpiece to a first building block tag, where the first building block tag includes a 2’-substituted tide and a hydroxyl group at the minus, optionally one or more nucleotides at the internal on of the tag, and a 2’-substituted nucleotide and a hydroxyl group at the 3’-terminus; where steps (ii) and (iii) can be performed in any order and where the first building block tag encodes for the binding reaction of step (ii), y providing a tagged library.

In some embodiments, the 2’-substituted nucleotide is a 2’-O-methyl nucleotide (e.g., 2’-O- methyl guanine) or a 2’-fluoro nucleotide (e.g., 2’-fluoro e). In other embodiments, one or more nucleotides at the internal position of the headpiece are 2’-deoxynucleotides. In yet other embodiments, the bifunctional linker is a poly ethylene glycol linker (e.g., -(CH2CH2O)n CH2CH2-, where n is an integer from 1 to 50).

In other embodiments, one or more tides (e.g., one or more 2’-deoxynucleotides) are present at the internal position of the headpiece or the tag.

In some embodiments, step (iii) may include the use of one or more soluble multivalent cations (e.g., magnesium chloride, manganese (II) chloride, or hexamine cobalt (III) chloride), poly ethylene glycol (e.g., having an average molecular weight of about 4,600 Daltons), and RNA ligase (e.g., T4 RNA ligase).

Also described herein are methods to identify and/or discover a chemical entity, the method including tagging a first library including an oligonucleotide-encoded al entity (e.g., including steps (i) to (iii) and optionally including steps (iv) to (v)) and selecting for a particular characteristic or function (e.g., selecting for binding to a protein target including exposing the oligonucleotide-encoded chemical entity or chemical entity to the protein target and ing the one or more oligonucleotideencoded chemical entities or chemical entities that bind to the protein target (e.g., by using size exclusion tography)). Also described herein is a complex including a headpiece and a building block tag, where the tag includes from 5 to 20 nucleotides, a 2’-substituted tide at the 5’-terminus, and a 2’- substituted tide at the 3’-terminus. In some embodiments, the 2’-substituted nucleotide at the 5’- terminus and/or 3’-terminus is a 2’-O-methyl nucleotide (e.g., 2’-O-methyl e or 2’-O-methyl uracil) or a 2’-fluoro nucleotide (e.g., 2’-fluoro guanine or 2’-fluoro ). In ular embodiments, the headpiece includes a hairpin structure. In some embodiments, the headpiece includes a 2’-substituted tide at one or more of the 5’-terminus, the 3’-terminus, or the internal position of the ece. In other embodiments, the headpiece further includes a preadenylated 5’-terminus. In yet other embodiments, the headpiece includes from 5 to 20 nucleotides.

In any of the above embodiments, the headpiece, the first building block tag, the second building block tag, or the one or more onal building block tags, if present, includes a preadenylated 5’- terminus.

In any of the above embodiments, the method further includes binding one or more (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) additional building block tags to the complex and binding one or more (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) additional components (e.g., scaffolds or building blocks) to the complex, where the one or more additional building block tag encodes for the one or more additional components or encodes for the g reaction of one or more additional components, thereby providing a tagged library.

In any of the above embodiments, the 2’-substituted nucleotide is a 2’-O-methyl nucleotide, such as 2’-O-methyl guanine, 2’-O-methyl uracil, 2’-O-methyl adenosine, 2’-O-methyl thymidine, 2’-O- methyl inosine, 2’-O-methyl cytidine, or 2’-O-methyl diamino purine. Alternatively, in any of the above embodiments, the 2’-substituted nucleotide is a 2’-fluoro nucleotide, such as 2’-fluoro e, 2’-fluoro uracil, 2’-fluoro adenosine, 2’-fluoro thymidine, 2’-fluoro inosine, 2’-fluoro cytidine, or 2’-fluoro diamino purine.

In any of the above embodiments, the RNA ligase is T4 RNA ligase and/or the DNA ligase is a ssDNA ligase.

In any of the above embodiments, the method includes a plurality of headpieces. In some embodiments of this method, each ece of the plurality of headpieces includes an identical sequence region and a different encoding region. In ular embodiments, the identical sequence region is a primer binding region. In other embodiments, the different encoding region is an initial building block tag that encodes for the headpiece or for an addition of an initial component.

In any of the above ments, binding in at least one of steps (ii)-(iv), if present, es enzyme on and/or chemical ligation. In some ments, enzymatic ligation es use of an RNA ligase (e.g., T4 RNA ligase) or a DNA ligase (e.g., ssDNA ligase). In other embodiments, tic ligation includes use of an RNA ligase (e.g., T4 RNA ligase) and a DNA ligase (e.g., ssDNA ligase). In some embodiments, chemical ligation includes use of one or more chemically co-reactive pairs (e.g., a pair including an optionally substituted alkynyl group with an optionally substituted azido group; a pair ing an optionally substituted diene having a 4π electron system (e.g., an optionally substituted 1,3-unsaturated compound, such as optionally substituted 1,3-butadiene, 1-methoxy hylsilyloxy-1,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) with an ally tuted dienophile or an optionally substituted heterodienophile having a 2π on system (e.g., an optionally substituted alkenyl group or an optionally substituted alkynyl group); a pair ing a nucleophile (e.g., an optionally substituted amine or an optionally substituted thiol) with a strained heterocyclyl electrophile (e.g., optionally substituted epoxide, aziridine, aziridinium ion, or episulfonium ion); a pair including a phosphorothioate group with an iodo group (e.g., a phosphorothioate group at the 3’-terminus and an iodo group at the 5’-terminus); or a pair including an aldehyde group with an amino group (e.g., a primary amino or a secondary amino group, including a hydrazido group)). In particular ments, the chemically co-reactive pair produces a resultant spacer having a length from about 4 to about 24 atoms (e.g., from about 4 to about 10 atoms). In other embodiments, chemical ligation includes use of a phosphorothioate group (e.g., at the 3’-terminus) and an iodo group (e.g., at the 5’-terminus). In further embodiments, chemical ligation includes a splint oligonucleotide in the g reaction. In some embodiments, the chemical ligation es use of a phosphorothioate group (e.g., at the 3’-terminus of the headpiece, the first building block tag, the second ng block tag, the one or more additional building block tags, the library-identifying tag, the use tag, and/or the origin tag, if present), an iodo group (e.g., at the minus of the headpiece, the first building block tag, the second ng block tag, the one or more onal building block tags, the library-identifying tag, the use tag, and/or the origin tag, if present), and a splint oligonucleotide in the binding reaction, where the use avoids use of one or more protecting groups. In other embodiments, chemical ligation of multiple tags comprises alternating use of orthogonal chemically co-reactive pairs (e.g., any two or more chemically co-reactive pairs described ) for ligating successive tags.

In any of the above embodiments, the headpiece may include a single-stranded (e.g., hairpin) structure.

In any of the above embodiments, the headpiece, the first building block tag, the second building block tag, the one or more additional building block tags, the library-identifying tag, the use tag, and/or the origin tag, if present, includes a sequence that is substantially identical (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any ce described herein (e.g., the sequence in any one of SEQ ID NOs: 6-21, 26, 27, or 29-31), or a sequence that is complementary to a sequence that is substantially cal (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any sequence described herein (e.g., the sequence in any one of SEQ ID NOs: 6-21, 26, 27, or 29-31). In particular embodiments, the first building block tag, the second ng block tag, the one or more onal building block tags, the library-identifying tag, the use tag, and/or the origin tag, if present, further includes a sequence that is substantially identical (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In any of the above embodiments, the methods or complexes include only single-stranded molecules, where the headpiece, the first building block tag, the second building block tag, and/or the one or more additional building block tags are single-stranded. In some embodiments, one or more of the -stranded molecules have a hairpin structure. In particular embodiments, the headpiece includes a hairpin structure and the one or more building block tags do not include a hairpin structure.

In any of the above embodiments, the method further comprises one or more optional steps to diversify the library or to interrogate the members of the y, as described herein. In some embodiments, the method further comprises identifying a small ike library member that binds or inactivates a protein of therapeutic interest. In other embodiments, the method further comprises contacting a member of the library with a biological target under conditions suitable for at least one member of the library to bind to the target, removing one or more y members that do not bind to the target, and analyzing the one or more oligonucleotide tags associated with them.

As described herein, the use of single-stranded molecules (e.g., including hairpin molecules) could have numerous benefits. Accordingly, in any of the embodiments described herein, the s and complexes include a headpiece, one or more building block tags, a x, a chemical entity, a molecule, or any member of a tagged library having decreased mass, sed solubility (e.g., in an organic solvent), decreased cost, increased reactivity, increased target accessibility, decreased hydrodynamic radius, and/or increased accuracy of analytical assessments, as compared to a method including one or more double-stranded molecules (e.g., a double-stranded headpiece or a double-stranded building block tag). In some embodiments, each of the building block tags (e.g., the first building block tag, the second building block tag, and/or one or more onal building block tags, if present) has about the same mass (e.g., each ng block tag has a mass that is about +/- 10% from the average mass between two or more building block tags). In particular embodiments, the building block tag has a decreased mass (e.g., less than about 15,000 Daltons, about 14,000 s, about 13,000 Daltons, about 12,000 Daltons, about 11,000 Daltons, about 10,000 Daltons, about 9,000 Daltons, about 8,000 Daltons, about 7,500 Daltons, about 7,000 Daltons, about 6,000 Daltons, about 6,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 4,000 Daltons, about 4,500 Daltons, or about 3,000 Daltons) compared to a double-stranded tag (e.g., a double-stranded tag having a mass of about 15,000 Daltons, about 14,000 Daltons, about 13,000 Daltons, or about 12,000 Daltons). In other embodiments, the building block tag has a reduced length ed to a double-stranded tag (e.g., a double-stranded tag having a length of less than about 20 nucleotides, less than about 19 nucleotides, less than about 18 nucleotides, less than about 17 nucleotides, less than about 16 nucleotides, less than about 15 nucleotides, less than about 14 nucleotides, less than about 13 nucleotides, less than about 12 nucleotides, less than about 11 nucleotides, less than about 10 nucleotides, less than about 9 nucleotides, less than about 8 nucleotides, or less than about 7 nucleotides). In some ments, one or more building block tags or s of the library lack a primer binding region and/or a constant region (e.g., during a selection step, such as selection using size exclusion chromatography). In some embodiments, one or more building block tags or members of the library have a reduced constant region (e.g., a length less than about 30 nucleotides, less than about 25 nucleotides, less than about 20 nucleotides, less than about 19 nucleotides, less than about 18 nucleotides, less than about 17 nucleotides, less than about 16 nucleotides, less than about 15 nucleotides, less than about 14 tides, less than about 13 nucleotides, less than about 12 nucleotides, less than about 11 nucleotides, less than about 10 nucleotides, less than about 9 nucleotides, less than about 8 nucleotides, or less than about 7 nucleotides). In other embodiments, the methods include a headpiece that encodes for a molecule, a portion of a chemical entity, a binding reaction (e.g., chemical or enzymatic ligation) of a step, or the identity of a library, where the encoding headpiece eliminates the need of an additional building block tag to encode such ation.

In any of the above embodiments, an oligonucleotide (e.g., the ece, the first building block tag, the second building block tag, and/or one or more additional building block tags, if present) encodes for the ty of the y. In some embodiments, the ucleotide (e.g., the ece, the first building block tag, the second building block tag, and/or one or more additional building block tags, if present) includes a first library-identifying sequence, where the sequence encodes for the identity of the first library. In ular embodiments, the ucleotide is a first y-identifying tag. In some embodiments, the method includes providing a first library-identifying tag, where the tag includes a sequence that encodes for a first library, and/or binding the first library-identifying tag to the complex. In some embodiments, the method includes providing a second library and combining the first library with a second library. In further embodiments, the method includes providing a second library-identifying tag, where the tag includes a sequence that encodes for a second library.

In any of the above embodiments, an oligonucleotide (e.g., a headpiece and/or one or more building blocks) encodes for the use of the member of the library (e.g., use in a selection step or a binding step, as described herein). In some embodiments, the oligonucleotide (e.g., the headpiece, the first building block tag, the second building block tag, and/or one or more onal ng block tags, if present) includes a use sequence, where the sequence encodes for use of a subset of members in the library in one or more steps (e.g., a ion step and/or a binding step). In particular embodiments, the oligonucleotide is a use tag including a use sequence. In some embodiments, an oligonucleotide (e.g., a headpiece and/or one or more building ) s for the origin of the member of the library (e.g., in a particular part of the library). In some embodiments, the oligonucleotide (e.g., the headpiece, the first building block tag, the second building block tag, and/or one or more additional building block tags, if present) includes an origin sequence (e.g., a random degenerate ce having a length of about 10, 9, 8, 7, or 6 nucleotides), where the sequence encodes for the origin of the member in the library. In particular embodiments, the oligonucleotide is an origin tag including an origin sequence. In some embodiments, the method r includes joining, binding, or operatively associating a use tag and/or an origin tag to the complex.

In any of the above embodiments, the methods, itions, and complexes optionally include a tailpiece, where the tailpiece includes one or more of a library-identifying sequence, a use sequence, or an origin sequence, as described herein. In particular embodiments, the methods further include joining, binding, or operatively associating the tailpiece (e.g., including one or more of a library-identifying sequence, a use sequence, or an origin sequence) to the complex.

In any of the above ments, the methods, itions, and complexes, or portions thereof (e.g., the headpiece, the first building block tag, the second building block tag, and/or the one or more additional building block tags, if present), es a modified phosphate group (e.g., a phosphorothioate or a hosphoramidite linkage) between the terminal nucleotide at the minus and the nucleotide adjacent to the al nucleotide. In particular embodiments, the modified ate group minimizes shuffling during enzymatic ligation between two ucleotides (e.g., minimizes inclusion of an additional nucleotide or excision of a nucleotide in the final product or complex, as compared to the sequences of two oligonucleotides to be ligated, such as between a headpiece to a building block tag or between a first building block tag and a second building block tag), as compared to ligation between two oligonucleotides (e.g., a headpiece and a building block tag or a first ng block tag and a second building block tag) lacking the modified phosphate group. In some embodiments, the complex may include a phosphorothioate or a triazole group.

In any of the above embodiments, the methods, compositions, and complexes, or portions thereof (e.g., the ece, the first building block tag, the second building block tag, and/or the one or more additional building block tags, if present), includes a modification that supports solubility in semi-, reduced-, or non-aqueous (e.g., organic) ions. In some embodiments, the bifunctional linker, headpiece, or one or more building block tags is modified to increase solubility of a member of said DNA-encoded chemical library in organic conditions In some embodiments, the modification is one or more of an alkyl chain, a polyethylene glycol unit, a branched species with positive charges, or a hydrophobic ring structure. In some embodiments, the modification includes one or more modified nucleotides having a hydrophobic moiety (e.g., modified at the C5 positions of T or C bases with aliphatic chains, such as in 5’-dimethoxytrityl-N4-diisobutylaminomethylidene(1-propynyl)-2’- ytidine,3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5’-dimethoxytrityl(1- propynyl)-2’-deoxyuridine,3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5’-dimethoxytrityl fluoro-2’-deoxyuridine,3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; and 5’-dimethoxytrityl- enyl-ethynyl)-2’-deoxyuridine, or 3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) or an ion having a hydrophobic moiety (e.g., an azobenzene). In some embodiments, the member of the library has an octanol:water coefficient from about 1.0 to about 2.5 (e.g., about 1.0 to about 1.5, about 1.0 to about 2.0, about 1.3 to about 1.5, about 1.3 to about 2.0, about 1.3 to about 2.5, about 1.5 to about 2.0, about 1.5 to about 2.5, or about 2.0 to about 2.5).

In any of the above embodiments, the headpiece, the tailpiece, the first building block tag, the second building block tag, the one or more additional building block tags, the library-identifying tag, the use tag, and/or the origin tag, if present, may include from 5 to 20 nucleotides (e.g., from 5 to 7 nucleotides, from 5 to 8 tides, from 5 to 9 tides, from 5 to 10 nucleotides, from 5 to 11 nucleotides, from 5 to 12 nucleotides, from 5 to 13 nucleotides, from 5 to 14 nucleotides, from 5 to 15 nucleotides, from 5 to 16 nucleotides, from 5 to 17 nucleotides, from 5 to 18 nucleotides, from 5 to 19 nucleotides, from 6 to 7 nucleotides, from 6 to 8 nucleotides, from 6 to 9 nucleotides, from 6 to 10 nucleotides, from 6 to 11 nucleotides, from 6 to 12 nucleotides, from 6 to 13 tides, from 6 to 14 nucleotides, from 6 to 15 nucleotides, from 6 to 16 nucleotides, from 6 to 17 nucleotides, from 6 to 18 nucleotides, from 6 to 19 nucleotides, from 6 to 20 nucleotides, from 7 to 8 nucleotides, from 7 to 9 nucleotides, from 7 to 10 nucleotides, from 7 to 11 nucleotides, from 7 to 12 tides, from 7 to 13 nucleotides, from 7 to 14 nucleotides, from 7 to 15 tides, from 7 to 16 nucleotides, from 7 to 17 nucleotides, from 7 to 18 nucleotides, from 7 to 19 tides, from 7 to 20 tides, from 8 to 9 nucleotides, from 8 to 10 nucleotides, from 8 to 11 nucleotides, from 8 to 12 nucleotides, from 8 to 13 nucleotides, from 8 to 14 nucleotides, from 8 to 15 nucleotides, from 8 to 16 nucleotides, from 8 to 17 nucleotides, from 8 to 18 nucleotides, from 8 to 19 tides, from 8 to 20 nucleotides, from 9 to 10 nucleotides, from 9 to 11 nucleotides, from 9 to 12 nucleotides, from 9 to 13 nucleotides, from 9 to 14 nucleotides, from 9 to 15 nucleotides, from 9 to 16 nucleotides, from 9 to 17 nucleotides, from 9 to 18 nucleotides, from 9 to 19 nucleotides, from 9 to 20 nucleotides, from 10 to 11 nucleotides, from 10 to 12 nucleotides, from 10 to 13 nucleotides, from 10 to 14 nucleotides, from 10 to 15 nucleotides, from 10 to 16 nucleotides, from 10 to 17 nucleotides, from 10 to 18 nucleotides, from 10 to 19 nucleotides, from 10 to 20 tides, from 11 to 12 nucleotides, from 11 to 13 nucleotides, from 11 to 14 nucleotides, from 11 to 15 nucleotides, from 11 to 16 nucleotides, from 11 to 17 nucleotides, from 11 to 18 tides, from 11 to 19 nucleotides, from 11 to 20 nucleotides, from 12 to 13 nucleotides, from 12 to 14 nucleotides, from 12 to 15 nucleotides, from 12 to 16 nucleotides, from 12 to 17 nucleotides, from 12 to 18 nucleotides, from 12 to 19 nucleotides, from 12 to 20 nucleotides, from 13 to 14 nucleotides, from 13 to 15 nucleotides, from 13 to 16 nucleotides, from 13 to 17 nucleotides, from 13 to 18 nucleotides, from 13 to 19 nucleotides, from 13 to 20 nucleotides, from 14 to 15 nucleotides, from 14 to 16 nucleotides, from 14 to 17 nucleotides, from 14 to 18 nucleotides, from 14 to 19 nucleotides, from 14 to 20 nucleotides, from 15 to 16 tides, from 15 to 17 nucleotides, from 15 to 18 nucleotides, from 15 to 19 nucleotides, from 15 to 20 nucleotides, from 16 to 17 nucleotides, from 16 to 18 nucleotides, from 16 to 19 nucleotides, from 16 to 20 nucleotides, from 17 to 18 nucleotides, from 17 to 19 nucleotides, from 17 to 20 nucleotides, from 18 to 19 nucleotides, from 18 to 20 nucleotides, and from 19 to 20 nucleotides). In particular embodiments, the ece, the first building block tag, the second building block tag, the one or more additional building block tags, the library-identifying tag, the use tag, and/or the origin tag, if present, have a length of less than 20 nucleotides (e.g., less than 19 tides, less than 18 nucleotides, less than 17 nucleotides, less than 16 tides, less than 15 nucleotides, less than 14 nucleotides, less than 13 nucleotides, less than 12 nucleotides, less than 11 nucleotides, less than 10 nucleotides, less than 9 nucleotides, less than 8 nucleotides, or less than 7 nucleotides).

In particular embodiments, the first building block tag and the second building block tag include the same number of nucleotides. In other embodiments, either the first building block tag or the second building block tag includes more than 8 nucleotides (e.g., more than 9 nucleotides, more than 10 nucleotides, more than 11 nucleotides, more than 12 nucleotides, more than 13 nucleotides, more than 14 nucleotides, and more than 15 tides). In some embodiments, the first building block tag is a donor tag (e.g., as defined herein) having from 8 to 20 nucleotides (e.g., from 8 to 9 nucleotides, from 8 to 10 nucleotides, from 8 to 11 nucleotides, from 8 to 12 nucleotides, from 8 to 13 nucleotides, from 8 to 14 nucleotides, from 8 to 15 nucleotides, from 8 to 16 nucleotides, from 8 to 17 nucleotides, from 8 to 18 nucleotides, from 8 to 19 nucleotides, from 8 to 20 nucleotides, from 9 to 10 nucleotides, from 9 to 11 nucleotides, from 9 to 12 nucleotides, from 9 to 13 nucleotides, from 9 to 14 nucleotides, from 9 to 15 nucleotides, from 9 to 16 nucleotides, from 9 to 17 nucleotides, from 9 to 18 tides, from 9 to 19 nucleotides, from 9 to 20 nucleotides, from 10 to 11 nucleotides, from 10 to 12 nucleotides, from 10 to 13 nucleotides, from 10 to 14 nucleotides, from 10 to 15 nucleotides, from 10 to 16 nucleotides, from 10 to 17 nucleotides, from 10 to 18 nucleotides, from 10 to 19 nucleotides, from 10 to 20 nucleotides, from 11 to 12 nucleotides, from 11 to 13 tides, from 11 to 14 nucleotides, from 11 to 15 nucleotides, from 11 to 16 nucleotides, from 11 to 17 nucleotides, from 11 to 18 nucleotides, from 11 to 19 nucleotides, from 11 to 20 nucleotides, from 12 to 13 tides, from 12 to 14 nucleotides, from 12 to 15 nucleotides, from 12 to 16 tides, from 12 to 17 nucleotides, from 12 to 18 nucleotides, from 12 to 19 nucleotides, from 12 to 20 tides, from 13 to 14 nucleotides, from 13 to 15 nucleotides, from 13 to 16 nucleotides, from 13 to 17 nucleotides, from 13 to 18 nucleotides, from 13 to 19 nucleotides, from 13 to 20 tides, from 14 to 15 nucleotides, from 14 to 16 nucleotides, from 14 to 17 nucleotides, from 14 to 18 nucleotides, from 14 to 19 tides, from 14 to 20 nucleotides, from 15 to 16 nucleotides, from 15 to 17 tides, from 15 to 18 nucleotides, from 15 to 19 nucleotides, from 15 to nucleotides, from 16 to 17 nucleotides, from 16 to 18 nucleotides, from 16 to 19 nucleotides, from 16 to 20 nucleotides, from 17 to 18 nucleotides, from 17 to 19 nucleotides, from 17 to 20 nucleotides, from 18 to 19 nucleotides, from 18 to 20 nucleotides, and from 19 to 20 nucleotides).

Definitions By “2’-substituted nucleotide” is meant a nucleotide base having a tution at the 2’-position of ribose in the base.

By “about” is meant +/- 10% of the recited value.

By “bifunctional” is meant having two ve groups that allow for binding of two chemical moieties. For example, a bifunctional linker is a linker, as described herein, having two reactive groups that allow for g of a ece and a chemical entity By “binding” is meant attaching by a covalent bond or a non-covalent bond. Non-covalent bonds include those formed by van der Waals forces, hydrogen bonds, ionic bonds, entrapment or physical encapsulation, tion, adsorption, and/or other intermolecular forces. Binding can be effectuated by any useful means, such as by enzymatic binding (e.g., tic ligation) or by chemical binding (e.g., chemical ligation).

By “building block” is meant a structural unit of a chemical , where the unit is directly linked to other al structural units or indirectly linked through the scaffold. When the chemical entity is polymeric or oligomeric, the building blocks are the monomeric units of the polymer or oligomer. Building blocks can have one or more diversity nodes that allow for the addition of one or more other building blocks or scaffolds. In most cases, each diversity node is a functional group capable of reacting with one or more building blocks or scaffolds to form a chemical entity. Generally, the building blocks have at least two diversity nodes (or reactive functional groups), but some building blocks may have one diversity node (or ve functional group). Alternatively, the encoded chemical or binding steps may include several chemical components (e.g., multi-component condensation reactions or multi-step processes). Reactive groups on two different ng blocks should be complementary, i.e., capable of reacting together to form a covalent or a non-covalent bond.

By “building block tag” is meant an oligonucleotide portion of the library that s the addition (e.g., by a binding reaction) of a component (i.e., a scaffold or a building block), the headpiece in the library, the identity of the library, the use of the library, and/or the origin of a library member. By “acceptor tag” is meant a building block tag having a reactive entity (e.g., a yl group at the 3’- terminus in the case of enzymatic on). By “donor tag” is meant a building block tag having an entity capable of reacting with the ve entity on the acceptor tag (e.g., a phosphoryl group at the 5’- us in the case of enzymatic ligation).

By “chemical entity” is meant a nd comprising one or more building blocks and ally a scaffold. The chemical entity can be any small molecule or peptide drug or drug candidate designed or built to have one or more desired characteristics, e.g., capacity to bind a biological target, lity, availability of hydrogen bond donors and acceptors, rotational degrees of freedom of the bonds, positive charge, negative charge, and the like. In certain embodiments, the chemical entity can be reacted r as a bifunctional or trifunctional (or greater) .

By “chemically co-reactive pair” is meant a pair of reactive groups that participates in a modular reaction with high yield and a high thermodynamic gain, thus producing a spacer. Exemplary reactions and chemically ctive pairs include a Huisgen polar cycloaddition reaction with a pair of an optionally substituted alkynyl group and an optionally substituted azido group; a Diels-Alder reaction with a pair of an optionally substituted diene having a 4π electron system and an optionally substituted dienophile or an optionally substituted heterodienophile having a 2π electron system; a ring opening reaction with a nucleophile and a strained heterocyclyl electrophile; a splint ligation reaction with a phosphorothioate group and an iodo group; and a reductive amination reaction with an aldehyde group and an amino group, as described .

By “complex” or “ligated complex” is meant a headpiece that is operatively associated with a chemical entity and/or one or more oligonucleotide tags by a covalent bond or a non-covalent bond. The complex can optionally include a bifunctional linker between the chemical entity and the ece.

By “component” of a chemical entity is meant either a ld or a building block.

By “diversity node” is meant a functional group at a position in the scaffold or the building block that allows for adding another building block.

By “headpiece” is meant a starting oligonucleotide for library synthesis that is operatively linked to a ent of a chemical entity and to a ng block tag. Optionally, a bifunctional linker connects the headpiece to the component.

By “library” is meant a collection of molecules or chemical entities. Optionally, the molecules or chemical entities are bound to one or more oligonucleotides that encodes for the molecules or portions of the chemical entity.

By r” is meant a chemical connecting entity that links the headpiece to a chemical entity.

By “multivalent cation” is meant a cation e of forming more than one bond with more than one ligand or anion. The multivalent cation can form either an ionic complex or a coordination complex.

Exemplary multivalent cations include those from the alkali earth metals (e.g., magnesium) and transition metals (e.g., manganese (II) or cobalt (III)), and those that are optionally bound to one or more anions and/or one or more univalent or polydentate ligands, such as chloride, amine, and/or ethylenediamine.

By “oligonucleotide” is meant a polymer of nucleotides having a 5’-terminus, a 3’-terminus, and one or more tides at the internal position between the 5’- and mini. The oligonucleotide may include DNA, RNA, or any tive thereof known in the art that can be synthesized and used for basepair recognition. The oligonucleotide does not have to have contiguous bases but can be persed with linker moieties. The oligonucleotide polymer may include natural bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, deoxycytidine, inosine, or diamino purine), base analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, orouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, aguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), modified nucleotides (e.g., 2’-substituted nucleotides, such as 2’-O-methylated bases and 2’-fluoro bases), alated bases, modified sugars (e.g., ororibose, ribose, 2’-deoxyribose, arabinose, and hexose), and/or modified phosphate groups (e.g., phosphorothioates and 5’-N-phosphoramidite linkages). Other ed bases are described herein. By “acceptor oligonucleotide” is meant an oligonucleotide having a reactive entity (e.g., a hydroxyl group at the 3’-terminus in the case of enzymatic ligation or an optionally substituted azido group in the case of al ligation). By “donor oligonucleotide” is meant an oligonucleotide having an entity capable of reacting with the reactive entity on the acceptor oligonucleotide (e.g., a phosphoryl group at the 5’- terminus in the case of tic ligation or an optionally substituted alkynyl group in the case of chemical ligation).

By “operatively linked” or “operatively associated” is meant that two or more chemical structures are directly or indirectly linked er in such a way as to remain linked through the various manipulations they are expected to o. lly, the chemical entity and the headpiece are operatively linked in an indirect manner (e.g., covalently via an appropriate linker). For example, the linker may be a bifunctional moiety with a site of attachment for chemical entity and a site of attachment for the headpiece. In on, the chemical entity and the oligonucleotide tag can be operatively linked directly or indirectly (e.g., ntly via an appropriate linker).

By “protecting group” is a meant a group intended to protect the 3’-terminus or 5’-terminus of an ucleotide against undesirable reactions during one or more binding steps of tagging a DNA- encoded library. Commonly used protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 4th Edition (John Wiley & Sons, New York, 2007), which is incorporated herein by reference. ary protecting groups include irreversible protecting groups, such as dideoxynucleotides and dideoxynucleosides (ddNTP or ddN), and, more preferably, reversible protecting groups for hydroxyl , such as ester groups (e.g., O-(α-methoxyethyl)ester, O-isovaleryl ester, and O-levulinyl ester), trityl groups (e.g., dimethoxytrityl and monomethoxytrityl), xanthenyl groups (e.g., 9- phenylxanthenyl and 9-(p-methoxyphenyl)xanthenyl), acyl groups (e.g., yacetyl and ), and silyl groups (e.g., t-butyldimethylsilyl).

By “purifying” is meant removing any unreacted product or any agent present in a reaction mixture that may reduce the activity of a chemical or biological agent to be used in a successive step.

Purifying can include one or more of chromatographic separation, ophoretic separation, and precipitation of the unreacted product or reagent to be removed.

By “scaffold” is meant a chemical moiety that displays one or more diversity nodes in a ular special geometry. Diversity nodes are lly attached to the scaffold during library synthesis, but in some cases one diversity node can be attached to the scaffold prior to library synthesis (e.g., addition of one or more building blocks and/or one or more tags). In some embodiments, the scaffold is derivatized such that it can be orthogonally deprotected during library synthesis and subsequently reacted with ent diversity nodes.

By “small molecule” drug or “small le” drug candidate is meant a molecule that has a molecular weight below about 1,000 Daltons. Small molecules may be organic or inorganic, isolated (e.g., from compound libraries or natural sources), or obtained by derivatization of known compounds.

By antial ty” or “substantially identical” is meant a polypeptide or polynucleotide sequence that has the same polypeptide or polynucleotide sequence, respectively, as a reference sequence, or has a specified percentage of amino acid residues or nucleotides, respectively, that are the same at the corresponding location within a reference ce when the two sequences are optimally aligned. For example, an amino acid sequence that is “substantially identical” to a reference ce has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference amino acid sequence. For polypeptides, the length of comparison sequences will generally be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids, more preferably at least 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids, and most preferably the full-length amino acid sequence. For nucleic acids, the length of comparison sequences will generally be at least 5 contiguous tides, preferably at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous tides, and most preferably the full length nucleotide sequence. Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the cs Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

By “tailpiece” is meant an ucleotide portion of the library that is attached to the complex after the addition of all of the building block tags and encodes for the identity of the library, the use of the library, and/or the origin of a library member.

Other es and advantages of the invention will be apparent from the following Detailed Description and the claims.

Brief Description of the Drawings Figure 1 shows an exemplary method for the general synthesis of al libraries using single- ed DNA tags that are joined sequentially by means of enzymatic and/or chemical ligation. “BB” refers to building block.

Figures 2A-2B show exemplary methods for single-stranded DNA tagging of libraries using enzymatic ligation. Figure 2A shows an ary method for tagging libraries using single-stranded enzymatic ligation with a protected (re-installed) 5’-monophosphate (5’-P) oligonucleotide, where gray boxes refer to 2’-OMe nucleotides, “X” refers to a ting group or a component of a chemical entity, and “PNK” refers to polynucleotide kinase. Figure 2B shows an ary method for tagging libraries using single-stranded ligation with a protected 3’-OH oligonucleotide, where black boxes attached to -O- refer to a protecting group of the 3’-OH terminus and “LC” refers to liquid chromatographic separation of the protecting group.

Figure 3 shows an exemplary method for tagging libraries using single-stranded on with a ’-preadenylated (labeled “5’-App”) oligonucleotide (headpiece) with a 3’-terminus that is blocked, e.g., by a chemical entity (labeled “X-3’”). This method can be used to ligate a 5’-phosphorylated oligonucleotide tag (labeled “Tag A”) to the headpiece and additional tags having a 3’-OH terminus (labeled “Tag B” and “Tag C”) to the complex in the ce of ATP.

Figures 4A-4E show ary complexes, each having a headpiece, a linker, and a small molecule including a scaffold (“S”) and diversity nodes A, B, and C. The dark gray boxes refer to 2’- OMe nucleotides, and the dotted lines refer to the presence of one or more complementary bases. Figures 4A-4B are schematics for complexes having a single-stranded linear oligonucleotide headpiece, where the linker and small molecule are connected to the 3’-terminus (Figure 4A) or the 5’-terminus (Figure 4B) of the headpiece. Figures 4C-4D are schematics for complexes having a single-stranded hairpin oligonucleotide headpiece, where the linker and small le are connected to the internal position (Figure 4C) or the 3’-terminus (Figure 4D) of the headpiece. Figure 4E shows an ary method for tagging libraries having a hairpin oligonucleotide headpiece, where the star refers to a chemical moiety and “Y” at the 3’-terminus refers to a protecting group. Oligonucleotide tags are labeled 1-4, and the r ce is the black line at the 5’-terminus.

Figures 5A-5C show oligonucleotide ligation by T4 RNA ligase or CircLigase™ ssDNA ligase.

Figure 5A is a schematic of the enzymatic ligation reaction. The donor oligonucleotide is 5’- phosphorylated and carries a 3’-fluorescein label, imitating a headpiece with a chemical y at 3’ end.

The acceptor oligonucleotide is not phosphorylated. Figure 5B shows gel electrophoresis analysis of a ligation reaction on an 8M urea/ 15% polyacrylamide gel (PAAG). “SM” refers to fluorescently labeled donor, “Product” refers to ligation product, and “Adenylated donor” refers to 5’App-Donor, as described above. Figure 5C shows high yield ligation achieved for T4 RNA ligase at high enzyme and oligonucleotide concentrations.

Figures 6A-6B represent optimization of PEG lar weight (Figure 6A) and concentration (Figure 6B) to achieve maximal ligation yield by T4 RNA . Reaction conditions are as described above for Figures 5A-5C. Figure 6A is graph quantifying the electrophoretic analysis of a ligation reaction with MNA/DNA 15mer donor and acceptor tags after incubation for 5 hours or 20 hours with % (w/v) PEG having a molecular weight from 300 to 20,000 (20K). Figure 6B shows the effect of concentration on ligation after tion for 18-20 hours in the presence of 5% to 45% (w/v) of PEG4600.

Figures 7A-7B show a correlation between ligation efficiency by CircLigase™ (Figure 7A) and T4 RNA ligase (Figure 7B) and length of the donor or acceptor oligonucleotides. Figure 7A depicts a graph quantifying the effect of the or length on ligation yield in the CircLigase™ ligation reaction.

Figure 7B s a graph and a table quantifying the effect of tide length of the acceptor and donor A tags on single-stranded ligation with T4 RNA ligase. These data represent an average of two independent experiments obtained by densitometry of scent gels at 450 nm excitation. s 8A-8B are LC-MS spectra for a MNA/DNA tag before and after orylation. Data are shown for 15mer tag 5’-HO-mUAC GTA TAC GAC H-3’ (SEQ ID NO: 13) (at 250 μM) before (Figure 8A) and after (Figure 8B) reaction with T4 polynucleotide kinase (50 units per 5 nmole of tag).

Figure 9 shows an electrophoretic gel for tial -stranded ligation of tags A-C. The 3’-terminus included fluorescein to represent a library compound (or chemical entity), and the asterisk (*) indicates purification of the ligated product (or complex) prior to phosphorylation.

Figures B show schematics of a “chemically co-reactive pair” reaction between donor and acceptor oligonucleotides ing in a 5-atom “short” spacer (Figure 10A) and a 24-atom “long” spacer (Figure 10B).

Figures 11A-11E show results of reverse transcription (RT) and PCR analysis of 75mer DNA templates containing a short or a long single spacer, as depicted in Figures 10A-10B. Figure 11A is a schematic of the RT reaction. LC-MS spectra of the RT were recorded at both 260 nm and 650 nm for the l 75mer DNA template (Figure 11B), the 75mer DNA template containing a single 5-atom (“short”) spacer (Figure 11C), and the 75mer DNA template ning a single 24-atom (“long”) spacer (Figure 11D). Figure 11E shows RT-PCR analysis of the control 75mer DNA template (“templ75”), a 75mer DNA template with a 5-atom spacer (“short ), and a 75mer DNA template with a 24-atom spacer (“long click”).

Figures 12A-12G show the results of a chemical ligation reaction between a 5’-iodo-modified DNA oligonucleotide and a 3’-phosphorothioate DNA oligonucleotide in the presence or absence of a complementary splint oligonucleotide. Figure 12A shows an exemplary schematic of the reaction. The o oligonucleotide is labeled with 6-FAM at 3’-terminus, while the 3’-phosphorothioate oligonucleotide is labeled with Cy5 at the 5’-terminus. Figure 12B shows a gel electrophoresis is of the ligation reactions in the presence (+spl) or absence (-spl) of a complementary . CCy5 and CFL indicate visible bands of Cy5 and fluorescein-labeled starting material, respectively. Figure 12C shows a time course of the splinted ligation reaction under the above conditions, which was quantified using Cy5 (635 nm) and fluorescein (450 nm) detection. Figure 12D shows LC-MS analysis of the on of CFL and CCy5 in the absence (top, at 260 nm, 495 nm, and 650 nm) and presence (bottom, at 260 nm, 495 nm, and 650 nm) of a splint, where ligation reactions were incubated for seven days. Figure 12E shows LC-MS analysis of the ligation of CFL and CCy5 in the absence a splint (at 260 nm, 495 nm, and 650 nm), where ligation reactions were incubated for eight days. Figure 12F shows MS analysis of reaction of CFL oligonucleotide with piperidine, where this reaction was ed to displace iodine.

Reaction conditions included oligonucleotides at 100 µM, piperidine at 40 mM (400 equivalents) in 100 mM borate buffer, pH 9.5, for 20 hrs at room temperature ; and oligonucleotides at 400 µM, piperidine at 2 M (4,000 equivalents) in 200 mM borate buffer, pH 9.5, for 2 hrs at 65°C (right). Figure 12G shows MS analysis of a ed ligation reaction of CFL and CCy5 oligonucleotides at 50 µM performed in the presence of 400 equivalents of piperidine in 100 mM borate buffer, pH 9.5, for 20 hrs at room temperature.

Figures 13A-13C shows the use of modified oligonucleotides to minimize shuffling. Figure 13A shows an LC-MS is of a single-stranded ligation reaction of a 5’-phosphorylated headpiece ssHP (3,636 Da) and a tag (tag 15; 2,469 Da) having 2’-O methyl nucleotides. The LC-MS analysis showed three peaks: peak 1 for the tag (2,469 Da); peak 2 for the adenylated headpiece (3,965 Da); and peak 3 having two (in some ces three) sub-peaks ning products with molecular weights of 6,089 Da (expected ligation product); 5,769 Da (expected 6,089 Da -320 Da); and 6,409 Da (expected 6,089 Da + 320 Da). This mass difference of 320 Da ponds exactly to either l or addition of an extra 2’- O-Me C nucleotide. Figures 13B-1 to 13B-3 show a non-limiting, ed mechanism of the nucleotide shuffling, where about 90% of the reaction es the ed (normal) ligation product and about 10% of the reaction provides nt ligation products (“Product -1 nt” and “Product + 1 nt”). Figure 13C shows an LC-MS analysis of ligation of ece HP-PS with tag 15. The headpiece HP-PS has the sequence the headpiece ssHP but includes a phosphorothioate linkage at the 5’-terminus. LC analysis showed three peaks: peak 1 for the tag (2,469), peak 2 for the adenylated headpiece (3,984), and peak 3 for a single ligation product (6,107) with almost no nucleotide shuffling observed. Traces of +/- 320 peaks likely correspond to the oxidative conversion of the phosphorothioate e into a native phosphodiester linkage or are due to incomplete ization.

Figure 14 is a graph showing separation of library members using size exclusion chromatography, where target-bound library members (left on graph) elute at a shorter time than unbound library members (right on graph).

Figure 15A is an exemplary schematic showing the chemical ligation of encoding DNA tags using a single chemistry that is not splint-dependent, e.g. 5’-azido/3’-alkynyl. The reactive groups are present on the 3’ and 5’ ends of each tag (Tag A, B, and C), and one of the reactive groups on either end (for example, the 3’ end) is protected to prevent the cyclization, polymerization, or wrong-cycle ligation of the tags. The cycle of tag ligation includes chemical ligation, followed by deprotection of the remaining functional group to render the growing ligated entity competent for the next cycle of ligation.

Each cycle also includes addition of one or more building blocks (BBA, BBB, and BBC, which are encoded by Tag A, B, and C, respectively). The chemical ligation s can optionally include on of a tailpiece.

Figure 15B is an exemplary schematic showing the chemical ligation of encoding DNA tags using a single chemistry that is splint-dependent. The template-dependent nature of this approach reduces the frequency of occurrence of tag polymerization, tag cyclization, as well as of ging events.

Similar to Figure 15A, this tic includes tags (Tag A, B, and C) and one or more building blocks encoded by tags (BBA, BBB, and BBC).

Figure 15C is an exemplary schematic showing the use of a succession of chemically ligated tags as a template for template-dependent rization, generating cDNA that is competent for PCR amplification and sequencing, as well as using a te-dependent polymerase capable of reading through the chemically ligated junctions.

Figure 16A is an exemplary schematic showing the chemical ligation of encoding DNA tags using TIPS-protected alkynyl tags and “click” chemistry. Each cycle of library synthesis includes Cu(I)- catalyzed chemical ligation of the rotected tag to the deprotected alkyne from the previous cycle.

After the ligation, the TIPS group is removed (deprotected), thereby activating the alkyne for the next chemical ligation step.

Figure 16B shows the structure of DMT-succinyl-3’-O-TIPS-propargyl uridine CPG that is used to initiate solid-phase synthesis of oligonucleotides g IPS-propargyl uridine at the 3’- terminus.

Figure 16C is an exemplary schematic g the use of a succession of “click” chemically ligated tags as a template for template-dependent polymerization, ting cDNA that is ent for PCR ication and sequencing, as well as using a template-dependent polymerase capable of reading through the “click” chemically d junctions.

Figures 17A-17C show the synthesis of tinylated, “single-click” templates Y55 and Y185.

Figure 17A provides an exemplary schematic. Figure 17B and Figure 17C show LC-MS analysis of Y55 and Y185, respectively.

Figures 18A-18C provide an exemplary assay for the “read-through” of a “single-click” template. Figure 18A shows a schematic, where FAM-labeled primer is annealed to the biotinylated template and is incubated with the te-dependent polymerase, according to the manufacturer’s recommended ions. The complexes are subsequently incubated with streptavidin beads, washed, eluted with NaOH, and then neutralized. After neutralization the samples are analyzed by LC-MS.

Figure 18B and Figure 18C show LC-MS data of the Klenow fragment copying of templates Y55 and Y185, respectively.

Figures 19A-19D provides the synthesis of 5’-biotinylated “double-click” template YDC and “triple-click template” YTC using a TIPS-protected alkynyl tag. Figures 19A and 19B show exemplary schematics for this synthesis. Figures 19C and 19D show LC-MS analysis of the YDC and YTC templates respectively.

Figures 20A-20C provide an exemplary click “read-through” assay using “double-click” and “triple-click” tes. Figure 20A is a schematic, where FAM-labeled primer is annealed to the biotinylated template and is incubated with Klenow fragment of E.coli DNA polymerase I according to the manufacturer’s ended reaction conditions. The complexes are incubated with streptavidin beads, washed, eluted with NaOH, and neutralized. After the neutralization, the samples are d by LC-MS. s 20B and 20C show LC-MS data of the Klenow fragment g of the templates YDC and YTC, respectively.

Figure 21 is a graph showing the efficiency of the click “read-through” using “single-click”, “double-click” and “triple-click” templates in comparison to a l “no-click” DNA template. These data were obtained using the “read-through” assay described herein, and the yields were measured by LC MS analysis by comparison to an internal standard.

Figures 22A-22C provide exemplary tics of chemical ligation with onal chemistry.

Figure 22A is a schematic of the chemical on gy for DNA encoding tags that (i) utilizes two successive orthogonal chemistries for (ii) ble read-through strategies. Each tag ns two orthogonal reactive groups, indicated by differing symbols for the 5’-terminus and the 3’-terminus of each tag. In each successive cycle of chemical ligation, an onal chemistry is used. This strategy reduces the frequency of occurrence of mistagging events and may also be used without the protection of the reactive terminal groups. Figure 22B is a schematic of the template-dependent polymerization “readthough” of a template generated by the orthogonal chemical ligation of orthogonal DNA tags to generate cDNA from which the sequence of the tags can be deduced. Figure 22C is the same as Figure 22B but includes a self-priming tailpiece, which may be rendered double-stranded by restriction ion to facilitate strand-separation during PCR amplification.

Figure 23 is an exemplary schematic showing the chemical ligation strategy for DNA encoding tags that utilizes two specific successive orthogonal chemistries. Each tag contains click-reactive and phosphorothioate/iodo-reactive groups. Tags bearing orthogonal reactive groups at their 3’ and 5’ ends cannot polymerize and have a reduced frequency of occurrence of mistagging events. Without wishing to be limited, this approach may eliminate the need for the TIPS-protection of the 3’-alkyne. In cycle A, the ’-iodo/3’-alkynyl tag is ligated using splint-dependent ligation to the 3’-phosphorothioate ece, leaving a ve 3’ alkyne for the next cycle of chemical ligation to a 5’-azido/3’-phosphorothioate tag.

The orthogonal ligation cycles may be repeated as many times as is d.

Figures 24A-24B show the protection and use of 3’-phosphorothioate/5’-iodo groups on DNA tags. Figure 24A shows an exemplary schematic for using protecting groups (PG) for these tags. Figure 24B shows an exemplary scheme for use of 3’-phosphorothioate/5’-iodo tags to chemically ligate succession of encoding DNA tags that encode a chemical library covalently installed upon the 5’- terminus.

Figures 25A-25B show the protection and use of 3’-phosphorothioate groups on DNA tags.

Figure 25A shows the scheme for protection of these groups. Figure 25B shows the scheme for use of 3’- phosphorothioate/5’-azido and pargyl/5’-iodo tags to chemically ligate a sion of orthogonal ng DNA tags that encode a chemical y covalently installed upon the 5’-terminus.

Detailed Description Described herein are methods of using single-stranded on to install oligonucleotide tags onto al entity-oligonucleotide complexes. This method can be used to create diverse ies of selectable chemical es by establishing an encoded relationship between particular tags and particular chemical reactions or building blocks. To identify one or more chemical entities, the oligonucleotide tags can be amplified, cloned, sequenced, and correlated by using the established relationship. In particular, reaction ions that promote single-stranded ligation of tags were identified. These conditions include the use of one or more 2’-substituted nucleotides (e.g., 2’-O-methyl nucleotides or 2’-fluoro nucleotides) within the tags, the use of tags of particular length (e.g., between 5 and 15 nucleotides), the use of one or more enzymes (e.g., RNA ligase and/or DNA ligase), and/or the use of one or more agents during ligation (e.g., poly ne glycol and/or a soluble multivalent cation, such as Co(NH3)6Cl3).

These methods additionally include methods of chemically joining oligonucleotides, such that the sequence of the joined oligonucleotide product may be utilized as a template for a template-dependent rase reaction. Methods of ng and tagging libraries of these complexes are described in detail below. s for tagging encoded libraries Also described herein is a method for operatively linking oligonucleotide tags with chemical es, such that encoding relationships may be established between the sequence of the tag and the structural units (or building blocks) of the al entity. In ular, the identity and/or history of a chemical entity can be inferred from the ce of bases in the oligonucleotide. Using this method, a library including diverse chemical entities or members (e.g., small les or es) can be addressed with a particular tag sequence. lly, these methods include the use of a headpiece, which has at least one functional group that may be elaborated ally and at least one functional group to which a single-stranded oligonucleotide may be bound (or ligated). Binding can be effectuated by any useful means, such as by enzymatic binding (e.g., ligation with one or more of an RNA ligase and/or a DNA ligase) or by chemical binding (e.g., by a substitution reaction between two functional groups, such as a nucleophile and a leaving .

To create numerous chemical entities within the library, a solution containing the headpiece can be divided into multiple aliquots and then placed into a multiplicity of physically separate compartments, such as the wells of a multiwell plate. Generally, this is the ” step. Within each compartment or well, successive chemical reaction and ligation steps are performed with a single-stranded tag within each aliquot. The relationship between the chemical reaction conditions and the sequence of the singlestranded tag are recorded. The reaction and ligation steps may be performed in any order. Then, the reacted and ligated aliquots are combined or “pooled,” and optionally purification may be performed at this point. These split and pool steps can be optionally repeated.

Next, the library can be tested and/or selected for a particular characteristic or function, as described herein. For example, the mixture of tagged chemical entities can be separated into at least two populations, where the first population binds to a particular biological target and the second population does not. The first population can then be selectively captured (e.g., by eluting on a column providing the target of interest or by incubating the aliquot with the target of interest) and, optionally, further ed or tested, such as with optional washing, cation, negative selection, positive selection, or separation steps.

Finally, the chemical histories of one or more members (or chemical entities) within the selected tion can be determined by the sequence of the operatively linked oligonucleotide. Upon correlating the sequence with the ular building block, this method can identify the individual members of the library with the selected characteristic (e.g., an increased tendency to bind to the target protein and thereby elicit a therapeutic effect). For r testing and optimization, candidate therapeutic compounds may then be prepared by synthesizing the identified library members with or without their associated oligonucleotide tags.

Figures 1-3 provide s exemplary methods for tagging libraries using single-stranded ligation with a headpiece, where tags can be ligated on the 5’-terminus or the 3’-terminus of the headpiece. To control the order in which the tags are ligated and to reduce side reactions, these methods ensure that only one reactive 5’-terminus and one reactive 3’-terminus are present during ligation.

Furthermore, these exemplary methods use 2’-substituted nucleotides (e.g., mixed 2’-deoxy/2’-O-methyl nucleotides) in the tags, and these tags act as templates for a DNA- or RNA-dependent polymerase capable of polymerizing nucleotides in a template-dependent fashion. Without g to be limited by theory, the use of one or more 2’-substituted nucleotides (e.g., 2’-O-methyl nucleotides and/or 2’-fluoro nucleotides) within a tag could promote ligation by RNA ligase by more closely ling RNA, while preserving both the physical and chemical robustness of the recording medium as well as the ability to extract sequence information using template-dependent polymerization.

Figure 1 provides an exemplary method for reducing side reactions, where the ligated complex and tags are designed to avoid unwanted reactions between reactive 3’-OH and 5’-monophosphate (“5’- P”) groups. In particular, this scheme s the phosphorylation-ligation cycle approach. During ligation, only one 3’-OH group (in the tag) and one 5’-P group (in the ece) are available, and, thus, only one ligation event is possible. Following the ligation and purification steps, a 5’-OH group is formed in the x, and this group can be converted into a 5’-P for adding subsequent ucleotide tags. The 3’-terminus of the x is blocked by X, which can be a ting group or a component of a chemical entity (e.g., optionally including a linker that acts as a spacer between the chemical entity and the ece).

As shown in Figure 1, the exemplary method includes ligation of ng block tag 1 (“tag 1”) to the 5’-terminus of the ece, thereby creating a complex, and performing successive ligations to the ’-terminus of the complex. The reactive 5’-terminus is a phosphate group on the complex, and the reactive 3’-terminus is a hydroxyl group on the tags. After the addition of each tag, the ligated complex is separated from the unreacted, unligated ece and tags and from other reagents (e.g., phosphate, , or other reagents present during the ligation step). Separation can be accomplished by any useful method (e.g., by chromatographic or electrophoretic tion of ligated and non-ligated products or by precipitation of a reagent). Then, the ligated complex is exposed to an agent (e.g., a polynucleotide kinase or a chemical phosphorylating agent) to form a phosphate group on the 5’-terminus of the complex. The separation and phosphorylation steps may be performed in either order. In particular, if a kinase is used in the phosphorylation step, the kinase should be vated or removed prior to the addition of the subsequent tags that may also contain a 5’-OH group, or any reagents that can inhibit the kinase should be removed from the reaction mixture prior to the phosphorylation step.

In another embodiment, the method includes g successive tags from the 3’-terminus of the preceding ligated x. In this method, the ligated complex lacks a reactive 3’-OH group immediately after the ligation step but contains a group that can be converted into a 3’-OH group (e.g., by release of a ting group). Figure 2A provides a schematic showing an exemplary method for g the 3’-terminus of a complex, and Figure 2B provides an exemplary reaction scheme for a protected 3’- terminus that ns tible 3’-OH group upon release of the 3’-linked protecting group. As shown in Figure 2A, building block tag 1 (“tag 1”) has a 3’-protected group. In the first step, the exemplary method includes ligation of the tag to the 3’-terminus of the headpiece, thereby creating a complex.

Successive ligations are performed to the 3’-terminus of the complex. The reactive 5’-terminus is a phosphate group on the tag, and the reactive 3’-terminus is a yl group on the complex. After the addition of each tag, the ligated complex is deprotected (e.g., by the addition of a hydrolyzing agent) to release the 3’-protecting group.

In yet another embodiment, the method includes binding successive tags by using a 5’- preadenylated (5’-App) oligonucleotide and a ligase (e.g., T4 RNA ligase). In the presence of ATP, T4 RNA ligase will use the ATP or to form an adenylated intermediate prior to ligation. In the absence of ATP, T4 RNA ligase will only ligate preadenylated ucleotides, and possible side reactions with ’-P oligonucleotides will not occur. Thus, single-stranded ligation with reduced side reactions can be performed with a ally synthesized 5’-App oligonucleotide in the presence of 5’- monophosphorylated tag, where the 5’-App oligonucleotide can be ligated to a headpiece prior to g or to a complex formed after multiple rounds of tagging.

Figure 3 provides a schematic showing an exemplary method for tagging the 5’-terminus of a preadenylated headpiece. Adenylation of the donor nucleotide at the 5’-phosphate group is the first step in the ligation reaction, and this reaction generally requires one molecule of ATP. In the second step, the 3’-OH group of the acceptor oligonucleotide reacts with the adenylated donor and forms a diester bond between two oligonucleotides, thus ing one AMP molecule. The chemically adenylated 5’- phosphate group of the donor oligonucleotide es a product of the first step of the ligation reaction and can be ligated to the second oligonucleotide in the absence of ATP. In the following scheme, a 5’- App headpiece is ligated to the 3’-OH group of a 5’-phosphorylated oligonucleotide tag (labeled “Tag A”). Due to the presence of the adenylated 5’-terminus of the oligonucleotide, ligation can occur in the absence of ATP. Under these conditions, the 5’-phosphate group of Tag A does not serve as a ligation donor. Building block Tag B can be ligated by providing a nucleotide having a 3’-OH terminus (labeled “Tag B”) in the presence of ATP, and additional tags (labeled “Tag C”) can be ed.

In Figure 3, the 3’-terminus of the headpiece can be blocked with any protecting group (e.g., an irreversible protecting group, such as ddN, or a reversible protecting group). In the first step, the method includes on of the tag to the 5’-terminus of the headpiece in the absence of ATP, y creating a x. Successive ligations are performed to the 5’-terminus of the complex in the presence of ATP.

This method can be modified in order to perform sive ligation to the 3’-terminus of a complex. For e, the method can include the use of a adenylated tag and a headpiece having a reactive 3’- OH terminus. This method may further require blocking the 3’-terminus of the tag to avoid eactions between tags, such as the method described above and in Figure 2.

The l method provided in Figure 3 can be ed by replacing the primer with a headpiece. In this case, the headpiece has to be adenylated chemically at the minus, and Tag A is phosphorylated at 5’-terminus. Ligation of this phosphorylated Tag A to the adenylated headpiece occurs in the same standard conditions, described herein, but omitting ATP. By using this ligation ion, the ligation of phosphorylated 5’ terminus can be prevented. In the next step, on of Tag B requires that this tag have a free hydroxyl group at 5’-terminus (i.e., non-phosphorylated). Successive ligation reactions can be performed in the presence of ATP, followed by phosphorylation of the 5’-terminus of the resulting oligonucleotide if further extension of the tags (e.g., Tag C in Figure 3) is desired.

The methods described herein can include any number of optional steps to diversify the library or to interrogate the members of the library. For any tagging method described herein (e.g., as in Figures 1- 3), successive “n” number of tags can be added with additional “n” number of ligation, separation, and/or orylation steps. Exemplary optional steps include restriction of library members using one or more restriction endonucleases; ligation of one or more adapter sequences to one or both of the library i, e.g., such as one or more adapter ces to provide a priming sequence for amplification and sequencing or to provide a label, such as biotin, for immobilization of the sequence; reverse-transcription or transcription, optionally followed by reverse-transcription, of the assembled tags in the complex using a reverse transcriptase, transcriptase, or another template-dependent polymerase; amplification of the assembled tags in the complex using, e.g., PCR; generation of clonal isolates of one or more populations of assembled tags in the x, e.g., by use of bacterial transformation, emulsion formation, dilution, surface capture techniques, etc.; amplification of clonal isolates of one or more populations of assembled tag in the complex, e.g., by using clonal isolates as templates for template-dependent polymerization of nucleotides; and sequence determination of clonal es of one or more populations of assembled tags in the complex, e.g., by using clonal isolates as templates for template-dependent polymerization with fluorescently labeled nucleotides. Additional methods for amplifying and sequencing the oligonucleotide tags are described herein.

These methods can be used to identify and discover any number of chemical entities with a particular characteristic or function, e.g., in a ion step. The desired teristic or function may be used as the basis for partitioning the library into at least two parts with the concomitant enrichment of at least one of the s or related s in the library with the desired function. In particular embodiments, the method comprises identifying a small drug-like library member that binds or inactivates a protein of therapeutic interest. In another embodiment, a sequence of chemical reactions is designed, and a set of building blocks is chosen so that the reaction of the chosen building blocks under the defined chemical conditions will te a combinatorial ity of molecules (or a library of molecules), where one or more molecules may have utility as a eutic agent for a particular n.

For example, the al reactions and building blocks are chosen to create a library having structural groups ly present in kinase inhibitors. In any of these instances, the tags encode the chemical history of the library member and, in each case, a collection of chemical possibilities may be ented by any particular tag combination.

In one embodiment, the library of chemical entities, or a portion thereof, is contacted with a biological target under conditions suitable for at least one member of the library to bind to the target, followed by removal of library members that do not bind to the target, and analyzing the one or more oligonucleotide tags associated with them. This method can optionally include amplifying the tags by methods known in the art. Exemplary biological targets include enzymes (e.g., kinases, phosphatases, methylases, demethylases, proteases, and DNA repair enzymes), proteins involved in protein:protein interactions (e.g., ligands for receptors), or targets (e.g., GPCRs and RTKs), ion channels, ia, viruses, parasites, DNA, RNA, prions, and carbohydrates.

In another embodiment, the chemical entities that bind to a target are not subjected to amplification but are analyzed directly. Exemplary methods of analysis include microarray is, including evanescent resonance photonic crystal analysis; bead-based methods for deconvoluting tags (e.g., by using his-tags); label-free photonic crystal biosensor analysis (e.g., a BIND® Reader from SRU Biosystems, Inc., Woburn, MA); or hybridization-based approaches (e.g. by using arrays of immobilized oligonucleotides complementary to ces present in the library of tags).

In addition, chemically co-reactive pairs (or functional groups) can be readily included in solidphase oligonucleotide synthesis schemes and will support the efficient chemical ligation of oligonucleotides. In addition, the resultant ligated oligonucleotides can act as templates for template- ent polymerization with one or more rases. ingly, any of the g steps described herein for tagging encoded libraries can be modified to e one or more of enzymatic on and/or chemical ligation techniques. Exemplary ligation techniques include enzyme ligation, such as use of one of more RNA ligases and/or DNA ligases; and chemical ligation, such as use of chemically co-reactive pairs (e.g., a pair including optionally substituted alkynyl and azido functional groups).

Furthermore, one or more libraries can be combined in a split-and-mix step. In order to permit mixing of two or more ies, the library member may contain one or more library-identifying sequences, such as in a library-identifying tag, in a ligated ng block tag, or as part of the headpiece sequence, as described herein.

Methods having reduced mass Much of the motivation for single-stranded encoding strategies arises from the reduced mass of a single-stranded tag when compared to a double-stranded tag. Reduced mass ially confers several benefits ing increased solubility, decreased cost, increased reactivity, increased target accessibility, decreased hydrodynamic radius, increased accuracy of analytical assessments, etc. In addition to using a single-stranded tagging methodology, further reductions in mass can be achieved by including the use of one or more of the following: one or more tags having a reduced , nt mass tag sets, an encoding ece, one or more members of a library lacking a primer binding region and/or a constant region, one or more members of a library having a reduced constant region, or any other methodologies described herein.

To minimize the mass of the members in the y, the length of one or more building block tags can be reduced, such as to a length that is as short as possible to encode each split size. In particular, the tags can be less than 20 nucleotides (e.g., less than 19 nucleotides, less than 18 nucleotides, less than 17 nucleotides, less than 16 nucleotides, less than 15 nucleotides, less than 14 nucleotides, less than 13 nucleotides, less than 12 nucleotides, less than 11 nucleotides, less than 10 nucleotides, less than 9 nucleotides, less than 8 nucleotides, or less than 7 nucleotides). As described below in the Examples, shorter tags (e.g, about 10 nucleotides or shorter) can be used for tag ligation.

Constant mass strategies can also be used, which could aid in is during library synthesis.

In addition, constant mass tag sets could permit the recognition of all single error occurences (e.g., errors arising from misreading a sequence or from chemical or enzymatic ligation of a tag) and most multiple error ences. The relationship between the length of a constant mass single-stranded tag set and encoding ability (e.g., minimum lengths to t ic building block split sizes or library identities, etc.) is outlined below in Table 1. ingly, use of constant mass tag sets could be used to provide beneficial encoding ability, while maintaining error recognition during library formation.

Table 1 To minimize mass in the library, the headpiece can be used not only to link the chemical moiety and a tag but to also encode for the identity of a particular library or for a particular step. For e, the headpiece can encode information, e.g., a plurality of headpieces that encode the first split(s) or the ty of the library, such as by using a particular sequence related to a specific library.

In addition, primer g (e.g., constant) regions from the y of DNA-encoded chemical entities can be excluded during the selection step(s). Then, these regions can be added after selection by, e.g., single-stranded ligation. One exemplary strategy would include ing a chemical entity at the ’-terminus of a encoding oligonucleotide, selecting a particular chemical entity based on any useful ular characteristic or function, and ligating a tailpiece oligonucleotide to the minus of the encoding oligonucleotide that includes a primer binding sequence and may optionally contain one or more tags, e.g. a “use” tag, an n” tag, etc., as described herein. This primer binding sequence could then be used to initiate template-dependent polymerization to generate cDNA (or cRNA) that is complementary to the selected library member. The cDNA or cRNA would then be ligated at its 3’- terminus to an oligonucleotide that contains a primer binding sequence and, now that the encoding information is d on both sides by primer binding sequences, the oligonucleotide may be sequenced and/or ied using established approaches, such as any described herein.

Mass may further be minimized by omitting or reducing the size of one or more constant sequences that separate encoding tags. Single-stranded on requires no complementary relationship between the ends to be ligated or between these ends and a splint. Therefore, no fixed sequence is required to support enzymatic ligation. Short fixed regions n tags may be useful for informatic g of tags or other in silico deconvolution ses.

Oligonucleotide tags The oligonucleotide tags described herein (e.g., a building block tag or a portion of a headpiece) can be used to encode any useful information, such as a molecule, a portion of a chemical entity, the on of a component (e.g., a scaffold or a building block), a headpiece in the library, the identity of the library, the use of one or more library members (e.g., use of the members in an aliquot of a library), and/or the origin of a library member (e.g., by use of an origin sequence).

Any sequence in an oligonucleotide can be used to encode any information. Thus, one oligonucleotide sequence can serve more than one purpose, such as to encode two or more types of information or to provide a starting oligonucleotide that also encodes for one or more types of information. For example, the first building block tag can encode for the on of a first building block, as well as for the identification of the library. In another example, a headpiece can be used to provide a starting oligonucleotide that operatively links a chemical entity to a building block tag, where the headpiece additionally includes a sequence that encodes for the identity of the library (i.e., the libraryidentifying sequence). ingly, any of the information described herein can be encoded in separate oligonucleotide tags or can be combined and encoded in the same oligonucleotide sequence (e.g., an oligonucleotide tag, such as a building block tag, or a headpiece).

A building block ce encodes for the ty of a building block and/or the type of binding reaction conducted with a building block. This building block sequence is included in a building block tag, where the tag can optionally include one or more types of sequence described below (e.g., a libraryidentifying sequence, a use sequence, and/or an origin sequence).

A library-identifying sequence encodes for the identity of a ular y. In order to permit mixing of two or more libraries, a library member may contain one or more library-identifying sequences, such as in a library-identifying tag (i.e., an oligonucleotide ing a library-identifying sequence), in a ligated ng block tag, in a part of the headpiece sequence, or in a tailpiece sequence. These libraryidentifying sequences can be used to deduce encoding relationships, where the sequence of the tag is translated and correlated with chemical (synthesis) history information. Accordingly, these libraryidentifying sequences permit the mixing of two or more libraries together for selection, amplification, cation, sequencing, etc.

A use sequence encodes the history (i.e., use) of one or more library members in an individual aliquot of a library. For example, te aliquots may be treated with different reaction conditions, building blocks, and/or selection steps. In particular, this ce may be used to identify such aliquots and deduce their history (use) and thereby permit the mixing together of aliquots of the same y with ent histories (uses) (e.g., distinct selection experiments) for the purposes of the mixing together of samples together for selection, amplification, purification, sequencing, etc. These use ces can be included in a headpiece, a tailpiece, a building block tag, a use tag (i.e., an oligonucleotide including a use sequence), or any other tag described herein (e.g., a library-identifying tag or an origin tag).

An origin sequence is a rate (random) oligonucleotide sequence of any useful length (e.g., about six oligonucleotides) that encodes for the origin of the library member. This sequence serves to stochastically subdivide library s that are otherwise identical in all ts into entities distinguishable by sequence information, such that observations of amplification products derived from unique progenitor templates (e.g., selected library members) can be distinguished from observations of multiple amplification products derived from the same progenitor template (e.g., a selected library member). For example, after library ion and prior to the selection step, each library member can include a different origin sequence, such as in an origin tag. After selection, selected library members can be amplified to produce amplification products, and the portion of the library member ed to include the origin sequence (e.g., in the origin tag) can be observed and compared with the origin sequence in each of the other library s. As the origin ces are degenerate, each amplification product of each library member should have a different origin sequence. However, an observation of the same origin sequence in the amplification product could indicate a source of error, such as an amplification error or a cyclization error in the sequence that produces repeated sequences, and the starting point or source of these errors can be traced by observing the origin sequence at each step (e.g., at each selection step or amplification step) of using the library. These origin sequences can be included in a headpiece, a tailpiece, a building block tag, an origin tag (i.e., an oligonucleotide including an origin ce), or any other tag described herein (e.g., a library-identifying tag or a use tag).

Any of the types of sequences described herein can be included in the headpiece. For example, the ece can include one or more of a building block ce, a library-identifying sequence, a use sequence, or an origin ce.

Any of these sequences described herein can be included in a tailpiece. For example, the tailpiece can include one or more of a library-identifying sequence, a use sequence, or an origin sequence.

These sequences can include any modification described herein for oligonucleotides, such as one or more modifications that promote solubility in organic solvents (e.g., any described herein, such as for the ece), that provide an analog of the natural phosphodiester linkage (e.g., a phosphorothioate analog), or that provide one or more non-natural oligonucleotides (e.g., 2’-substituted nucleotides, such as 2’-O-methylated nucleotides and 2’-fluoro nucleotides, or any described herein).

These sequences can include any characteristics described herein for oligonucleotides. For example, these sequences can be included in tag that is less than 20 nucleotides (e.g., as described ). In other examples, the tags including one or more of these sequences have about the same mass (e.g., each tag has a mass that is about +/- 10% from the e mass between two or more tags); lack a primer binding (e.g., constant) region; lack a nt region; or have a constant region of reduced length (e.g., a length less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 19 nucleotides, less than 18 nucleotides, less than 17 nucleotides, less than 16 nucleotides, less than 15 nucleotides, less than 14 nucleotides, less than 13 nucleotides, less than 12 nucleotides, less than 11 nucleotides, less than 10 nucleotides, less than 9 nucleotides, less than 8 tides, or less than 7 nucleotides).

Sequencing strategies for libraries and oligonucleotides of this length may optionally include concatenation or tion strategies to increase read fidelity or sequencing depth, tively. In particular, the selection of encoded libraries that lack primer binding regions has been described in the literature for SELEX, such as described in Jarosch et al., Nucleic Acids Res. 34: e86 (2006), which is orated herein by reference. For example, a library member can be modified (e.g., after a selection step) to include a first adapter sequence on the 5’-terminus of the complex and a second adapter sequence on the 3’-terminus of the complex, where the first sequence is substantially complementary to the second sequence and result in forming a duplex. To further improve yield, two fixed dangling nucleotides (e.g., CC) are added to the 5’-terminus. In particular embodiments, the first adapter ce is 5’- GTGCTGC-3’ (SEQ ID NO: 1), and the second r sequence is 5’-GCAGCACCC-3’ (SEQ ID NO: Headpiece In the library, the headpiece operatively links each al entity to its encoding ucleotide tag. Generally, the headpiece is a starting oligonucleotide having two functional groups that can be further derivatized, where the first functional group operatively links the chemical entity (or a component thereof) to the headpiece and the second functional group operatively links one or more tags to the headpiece. A linker can optionally be used as a spacer between the headpiece and the chemical entity.

The functional groups of the headpiece can be used to form a covalent bond with a component of the chemical entity and another covalent bond with a tag. The component can be any part of the small molecule, such as a scaffold having diversity nodes or a building block. atively, the headpiece can be derivatized to provide a linker (i.e., a spacer separating the ece from the small molecule to be formed in the library) terminating in a functional group (e.g., a hydroxyl, amine, carboxyl, sulfhydryl, alkynyl, azido, or phosphate group), which is used to form the covalent linkage with a component of the al entity. The linker can be ed to the 5’-terminus, at one of the internal positions, or to the 3’-terminus of the headpiece. When the linker is attached to one of the internal positions, the linker can be operatively linked to a derivatized base (e.g., the C5 position of uridine) or placed internally within the oligonucleotide using standard techniques known in the art. Exemplary linkers are described herein.

The headpiece can have any useful structure. The headpiece can be, e.g., 1 to 100 nucleotides in length, preferably 5 to 20 tides in , and most preferably 5 to 15 nucleotides in length. The headpiece can be single-stranded or double-stranded and can consist of natural or modified nucleotides, as bed herein. Particular exemplary ments of the headpiece are bed in Figures 4A-4D.

For example, the chemical moiety can be operatively linked to the 3’-terminus (Figure 4A) or 5’-terminus (Figure 4B) of the headpiece. In particular embodiments, the headpiece includes a hairpin structure formed by mentary bases within the sequence. For example, the chemical moiety can be operatively linked to the internal position (Figure 4C), the 3’-terminus (Figure 4D), or the 5’-terminus of the headpiece.

Generally, the headpiece includes a non-complementary sequence on the 5’- or 3’- terminus that allows for binding an oligonucleotide tag by polymerization, enzymatic on, or chemical reaction. In Figure 4E, the exemplary headpiece allows for ligation of oligonucleotide tags (labeled 1-4), and the method includes purification and phosphorylation steps. After the addition of tag 4, an additional adapter sequence can be added to the 5’-terminus of tag 4. Exemplary adapter sequences include a primer binding sequence or a sequence having a label (e.g., biotin). In cases where many building blocks and corresponding tags are used (e.g., 100 tags), a mix-and-split strategy may be ed during the oligonucleotide synthesis step to create the necessary number of tags. Such mix-and-split strategies for DNA sis are known in the art. The resultant library members can be amplified by PCR following selection for binding entities versus a target(s) of interest.

The headpiece or the complex can optionally include one or more primer binding sequences. For e, the headpiece has a sequence in the loop region of the hairpin that serves as a primer binding region for amplification, where the primer binding region has a higher melting ature for its complementary primer (e.g., which can include flanking identifier regions) than for a sequence in the headpiece. In other ments, the complex includes two primer binding sequences (e.g., to enable a PCR reaction) on either side of one or more tags that encode one or more building blocks. atively, the ece may n one primer g sequence on the 5’- or 3’-terminus. In other embodiments, the headpiece is a hairpin, and the loop region forms a primer g site or the primer binding site is introduced through hybridization of an ucleotide to the headpiece on the 3’ side of the loop. A primer oligonucleotide, containing a region homologous to the 3’-terminus of the headpiece and carrying a primer binding region on its 5’-terminus (e.g., to enable a PCR reaction) may be hybridized to the headpiece and may contain a tag that encodes a building block or the addition of a building block. The primer oligonucleotide may contain additional information, such as a region of randomized nucleotides, e.g., 2 to 16 nucleotides in length, which is included for bioinformatics analysis.

The headpiece can optionally include a hairpin structure, where this structure can be ed by any useful method. For example, the headpiece can e complementary bases that form intermolecular base pairing rs, such as by -Crick DNA base pairing (e.g., adenine-thymine and guanine-cytosine) and/or by wobble base pairing (e.g., guanine-uracil, inosine-uracil, inosineadenine , and inosine-cytosine). In r example, the headpiece can include modified or substituted nucleotides that can form higher affinity duplex formations compared to unmodified nucleotides, such modified or substituted nucleotides being known in the art. In yet another example, the ece includes one or more crosslinked bases to form the hairpin structure. For example, bases within a single strand or bases in different double strands can be crosslinked, e.g., by using psoralen.

The headpiece or complex can ally include one or more labels that allow for detection. For example, the headpiece, one or more oligonucleotide tags, and/or one or more primer ces can include an e, a radioimaging agent, a marker, a tracer, a fluorescent label (e.g., rhodamine or fluorescein), a chemiluminescent label, a quantum dot, and a reporter molecule (e.g., biotin or a his-tag).

In other embodiments, the headpiece or tag may be modified to support solubility in semi-, reduced-, or non-aqueous (e.g., organic) conditions. Nucleotide bases of the headpiece or tag can be rendered more hobic by modifying, for example, the C5 positions of T or C bases with aliphatic chains without significantly disrupting their ability to hydrogen bond to their complementary bases. ary modified or substituted nucleotides are 5’-dimethoxytrityl-N4-diisobutylaminomethylidene (1-propynyl)-2’-deoxycytidine,3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 5’- dimethoxytrityl(1-propynyl)-2’-deoxyuridine,3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; ’-dimethoxytritylfluoro-2’-deoxyuridine,3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; and ’-dimethoxytrityl(pyrenyl-ethynyl)-2’-deoxyuridine, or 3’-[(2-cyanoethyl)-(N,N-diisopropyl)]- oramidite.

In addition, the headpiece oligonucleotide can be interspersed with modifications that promote solubility in c solvents. For e, azobenzene phosphoramidite can introduce a hydrophobic moiety into the headpiece design. Such insertions of hydrophobic amidites into the ece can occur anywhere in the molecule. However, the insertion cannot interfere with subsequent tagging using additional DNA tags during the library synthesis or ensuing PCR once a selection is complete or microarray analysis, if used for tag deconvolution. Such additions to the headpiece design described herein would render the headpiece soluble in, for example, 15%, 25%, 30%, 50%, 75%, 90 %, 95%, 98%, 99%, or 100% organic solvent. Thus, addition of hydrophobic residues into the headpiece design allows for improved solubility in semi- or non-aqueous (e.g., organic) conditions, while rendering the headpiece competent for oligonucleotide tagging. Furthermore, DNA tags that are subsequently introduced into the library can also be modified at the C5 position of T or C bases such that they also render the library more hydrophobic and soluble in c ts for subsequent steps of library synthesis.

In particular ments, the headpiece and the first building block tag can be the same entity, i.e., a plurality of headpiece-tag entities can be constructed that all share common parts (e.g., a primer g region) and all differ in another part (e.g., encoding region). These may be ed in the “split” step and pooled after the event they are encoding has occurred.

In particular embodiments, the headpiece can encode information, e.g., by including a sequence that encodes the first split(s) step or a ce that encodes the identity of the library, such as by using a particular sequence related to a specific y.

Enzymatic ligation and chemical ligation ques Various ligation techniques can be used to add scaffolds, building blocks, linkers, building block tags, and/or the headpiece to produce a complex. Accordingly, any of the binding steps described herein can include any useful ligation techniques, such as enzyme on and/or chemical ligation. These binding steps can include the addition of one or more building block tags to the headpiece or complex; the addition of a linker to the headpiece; and the addition of one or more scaffolds or building blocks to the headpiece or complex. In particular embodiments, the ligation techniques used for any oligonucleotide provide a resultant product that can be transcribed and/or reverse ribed to allow for decoding of the library or for template-dependent polymerization with one or more DNA or RNA polymerases.

Generally, enzyme ligation produces an oligonucleotide having a native phosphodiester bond that can be transcribed and/or reverse transcribed. Exemplary methods of enzyme ligation are provided herein and include the use of one or more RNA or DNA ligases, such as T4 RNA ligase, T4 DNA ligase, CircLigaseTM ssDNA ligase, CircLigaseTM II ssDNA ligase, and ThermoPhageTM ssDNA ligase (Prokazyme Ltd., Reykjavik, Iceland). al ligation can also be used to produce ucleotides capable of being transcribed or reverse transcribed. One benefit of chemical on is that solid phase sis of such oligonucleotides can be optimized to support efficient ligation yield. However, the efficacy of a chemical ligation technique to provide oligonucleotides e of being transcribed or reverse transcribed may need to be tested. This efficacy can be tested by any useful method, such as liquid chromatography-mass spectrometry, RT-PCR analysis, and/or PCR analysis. Examples of these methods are ed in Example 5.

In particular embodiments, chemical ligation includes the use of one or more chemically coreactive pairs to provide a spacer that can be transcribed or reverse transcribed. In particular, reactions suitable for chemically co-reactive pairs are preferred candidates for the cyclization process (Kolb et al., Angew. Chem. Int. Ed., 40:2004-2021 (2001); Van der Eycken et al., QSAR Comb. Sci., 26:1115-1326 (2007)). Exemplary chemically co-reactive pairs are a pair including an optionally substituted l group and an optionally substituted azido group to form a triazole spacer via a Huisgen 1,3-dipolar cycloaddition reaction; an optionally substituted diene having a 4π electron system (e.g., an optionally substituted 1,3-unsaturated compound, such as optionally substituted 1,3-butadiene, 1-methoxy hylsilyloxy-1,3-butadiene, cyclopentadiene, exadiene, or furan) and an optionally substituted dienophile or an optionally substituted heterodienophile having a 2π electron system (e.g., an ally tuted alkenyl group or an optionally substituted l group) to form a cycloalkenyl spacer via a Diels-Alder reaction; a nucleophile (e.g., an optionally substituted amine or an optionally substituted thiol) with a strained heterocyclyl electrophile (e.g., optionally substituted epoxide, aziridine, aziridinium ion, or episulfonium ion) to form a heteroalkyl spacer via a ring opening reaction; a phosphorothioate group with an iodo group, such as in a splinted ligation of an oligonucleotide containing 5’-iodo dT with a 3’-phosphorothioate oligonucleotide; and an aldehyde group and an amino group, such as a reaction of a 3’-aldehyde-modified oligonucleotide, which can optionally be obtained by oxidizing a commercially available 3’-glyceryl-modified oligonucleotide, with 5’-amino oligonucleotide (i.e., in a reductive amination reaction) or a razido oligonucleotide.

In other embodiments, chemical ligation includes introducing an analog of the phosphodiester bond, e.g., for post-selection PCR analysis and sequencing. Exemplary analogs of a phosphodiester e a phosphorothioate linkage (e.g., as introduced by use of a phosphorothioate group and a leaving group, such as an iodo group), a oramide linkage, or a phosphorodithioate linkage (e.g., as introduced by use of a phosphorodithioate group and a g group, such as an iodo group).

Reaction conditions to promote enzymatic ligation or chemical ligation Also described herein are one or more reaction conditions that promote enzymatic or al on between the headpiece and a tag or between two tags. These reaction conditions include using modified nucleotides within the tag, as described herein; using donor tags and acceptor tags having different lengths and varying the concentration of the tags; using different types of ligases, as well as combinations thereof (e.g., CircLigaseTM DNA ligase and/or T4 RNA ligase), and varying their concentration; using poly ethylene glycols (PEGs) having different molecular weights and varying their tration; use of non-PEG crowding agents (e.g., betaine or bovine serum albumin); varying the temperature and duration for on; varying the concentration of various agents, ing ATP, Co(NH3)6Cl3, and yeast inorganic pyrophosphate; using enzymatically or chemically phosphorylated ucleotide tags; using 3’-protected tags; and using preadenylated tags. These reaction conditions also include chemical ligations.

The headpiece and/or tags can include one or more modified or substituted nucleotides. In preferred embodiments, the headpiece and/or tags include one or more modified or substituted nucleotides that promote enzymatic ligation, such as 2’-O-methyl nucleotides (e.g., 2’-O-methyl guanine or 2’-O-methyl ), 2’-fluoro nucleotides, or any other modified nucleotides that are utilized as a substrate for on. atively, the headpiece and/or tags are modified to include one or more chemically reactive groups to t chemical ligation (e.g. an optionally substituted alkynyl group and an optionally substituted azido group). ally, the tag ucleotides are functionalized at both termini with chemically reactive groups, and, optionally, one of these termini is protected, such that the groups may be addressed independently and eactions may be reduced (e.g., reduced polymerization side-reactions).

Enzymatic ligation can include one or more ligases. Exemplary ligases include CircLigaseTM ssDNA ligase (EPICENTRE Biotechnologies, Madison, WI), CircLigaseTM II ssDNA ligase (also from EPICENTRE Biotechnologies), ThermoPhageTM ssDNA ligase (Prokazyme Ltd., Reykjavik, d), T4 RNA , and T4 DNA . In preferred embodiments, ligation includes the use of an RNA ligase or a combination of an RNA ligase and a DNA ligase. Ligation can further include one or more soluble multivalent cations, such as Co(NH3)6Cl3, in combination with one or more ligases.

Before or after the ligation step, the complex can be purified for three s. First, the complex can be purified to remove unreacted headpiece or tags that may result in cross-reactions and introduce “noise” into the encoding process. Second, the complex can be purified to remove any reagents or unreacted starting material that can inhibit or lower the on activity of a ligase. For example, phosphate may result in lowered ligation ty. Third, entities that are introduced into a chemical or ligation step may need to be removed to enable the subsequent chemical or ligation step. Methods of purifying the complex are described herein. tic and chemical ligation can include poly ethylene glycol having an average molecular weight of more than 300 Daltons (e.g., more than 600 Daltons, 3,000 Daltons, 4,000 Daltons, or 4,500 Daltons). In particular embodiments, the poly ethylene glycol has an average molecular weight from about 3,000 Daltons to 9,000 Daltons (e.g., from 3,000 Daltons to 8,000 Daltons, from 3,000 Daltons to 7,000 Daltons, from 3,000 Daltons to 6,000 Daltons, and from 3,000 s to 5,000 Daltons). In preferred embodiments, the poly ethylene glycol has an average molecular weight from about 3,000 Daltons to about 6,000 s (e.g., from 3,300 Daltons to 4,500 Daltons, from 3,300 Daltons to 5,000 Daltons, from 3,300 s to 5,500 Daltons, from 3,300 Daltons to 6,000 Daltons, from 3,500 Daltons to 4,500 s, from 3,500 Daltons to 5,000 Daltons, from 3,500 Daltons to 5,500 s, and from 3,500 Daltons to 6,000 Daltons, such as 4,600 s). Poly ethylene glycol can be present in any useful amount, such as from about 25% (w/v) to about 35% (w/v), such as 30% (w/v).

In a preferred embodiment of this invention, the building block tags are installed by ligation of a single-stranded oligonucleotide to a single-stranded oligonucleotide using the ligation protocol outlined below: Headpiece: 25 μM (5’ terminus: 5’-monophospho/2’-OMe G, intervening nucleotides: 2’-deoxy, and 3’ terminus: 2’- blocked/3’-blocked) Building Block Tag: 25 μM (5’-terminus: 2’-OMe/5’-OH G, ening nucleotides: 2’-deoxy, and 3’-terminus: 3’-OH/2’-OMe) Co(NH3)6Cl3: 1 mM PEG 4600: 30% (w/v) T4 RNA Ligase (Promega): 1.5 units/μl Yeast Inorganic Pyrophosphatase: 0.0025 μl Tris: 50 mM MgCl2: 10 mM ATP: 1 mM pH: 7.5 Water: e In further embodiments, the protocol includes incubation at 37°C for 20 hours. For the purposes of actual library construction, higher concentration of ece, tags, and/or ligase may be used, and such modifications to these trations would be nt to those skilled in the art.

Methods for encoding chemical entities within a library The methods of the invention can be used to synthesize a library having a diverse number of chemical entities that are encoded by oligonucleotide tags. Examples of building blocks and encoding DNA tags are found in U.S. Patent Application Publication No. 2007/0224607, hereby incorporated by reference.

Each chemical entity is formed from one or more building blocks and optionally a scaffold. The scaffold serves to provide one or more ity nodes in a particular geometry (e.g., a triazine to provide three nodes spatially arranged around a heteroaryl ring or a linear geometry).

The ng blocks and their encoding tags can be added directly or indirectly (e.g., via a linker) to the headpiece to form a complex. When the headpiece includes a linker, the ng block or scaffold is added to the end of the linker. When the linker is absent, the building block can be added directly to the headpiece or the building block itself can include a linker that reacts with a onal group of the headpiece. Exemplary linkers and headpieces are described herein.

The scaffold can be added in any useful way. For example, the scaffold can be added to the end of the linker or the headpiece, and successive building blocks can be added to the available diversity nodes of the scaffold. In another example, building block An is first added to the linker or the headpiece, and then the diversity node of scaffold S is reacted with a functional group in building block An.

Oligonucleotide tags encoding a particular scaffold can optionally be added to the headpiece or the complex. For e, Sn is added to the complex in n on vessels, where n is an integer more than one, and tag Sn (i.e., tag S1, S2, … Sn-1, Sn) is bound to the functional group of the complex.

Building blocks can be added in multiple, synthetic steps. For e, an aliquot of the headpiece, optionally having an attached linker, is separated into n reaction vessels, where n is an integer of two or greater. In the first step, ng block An is added to each n reaction vessel (i.e., building block A1, A2, … An-1, An is added to reaction vessel 1, 2, … n-1, n), where n is an integer and each building block An is unique. In the second step, scaffold S is added to each reaction vessel to form an An- S x. Optionally, scaffold Sn can be added to each reaction vessel to from an An-Sn complex, where n is an integer of more than two, and each scaffold Sn can be unique. In the third step, building block Bn is to each n reaction vessel containing the An-S complex (i.e., building block B1, B2, … Bn-1, Bn is added to reaction vessel 1, 2, … n-1, n containing the A1-S, A2-S, … AnS, An-S complex), where each building block Bn is . In further steps, building block Cn can be added to each n reaction vessel containing the Bn-An-S complex (i.e., building block C1, C2, … Cn-1, Cn is added to reaction vessel 1, 2, … n-1, n containing the B1-A1-S … Bn-An-S complex), where each building block Cn is unique. The resulting library will have n3 number of complexes having n3 tags. In this manner, additional synthetic steps can be used to bind additional ng blocks to further ify the y.

After forming the library, the resultant xes can optionally be purified and subjected to a polymerization or ligation reaction using one or more primers. This l strategy can be expanded to include additional diversity nodes and building blocks (e.g., D, E, F, etc.). For example, the first diversity node is reacted with building blocks and/or S and d by an oligonucleotide tag. Then, additional building blocks are reacted with the resultant complex, and the subsequent diversity node is derivatized by additional building blocks, which is d by the primer used for the polymerization or ligation reaction To form an encoded library, oligonucleotide tags are added to the complex after or before each synthetic step. For example, before or after the addition of building block An to each reaction vessel, tag An is bound to the functional group of the headpiece (i.e., tag A1, A2, … An-1, An is added to reaction vessel 1, 2, … n-1, n containing the ece). Each tag An has a distinct ce that ates with each unique building block An, and determining the sequence of tag An es the chemical structure of building block An. In this manner, onal tags are used to encode for additional building blocks or additional lds.

Furthermore, the last tag added to the x can either include a primer sequence or provide a functional group to allow for binding (e.g., by ligation) of a primer sequence. The primer sequence can be used for amplifying and/or sequencing the oligonucleotides tags of the complex. Exemplary methods for amplifying and for sequencing include polymerase chain reaction (PCR), linear chain ication (LCR), rolling circle amplification (RCA), or any other method known in the art to amplify or determine nucleic acid sequences.

Using these methods, large libraries can be formed having a large number of encoded chemical entities. For example, a headpiece is reacted with a linker and building block An, which includes 1,000 different variants (i.e., n = 1,000). For each building block An, a DNA tag An is ligated or primer extended to the headpiece. These reactions may be performed in a 1,000-well plate or 10 x 100 well plates. All ons may be pooled, optionally ed, and split into a second set of plates. Next, the same procedure may be performed with building block Bn, which also include 1,000 different variants. A DNA tag Bn may be ligated to the An-headpiece complex, and all reactions may be pooled. The resultant library includes 1,000 x 1,000 combinations of An x Bn (i.e., 1,000,000 compounds) tagged by 1,000,000 different combinations of tags. The same approach may be extended to add building blocks Cn, Dn, En, etc. The generated library may then be used to identify compounds that bind to the target. The structure of the chemical entities that bind to the library can optionally be assessed by PCR and sequencing of the DNA tags to identify the compounds that were enriched.

This method can be modified to avoid tagging after the addition of each ng block or to avoid pooling (or mixing). For example, the method can be ed by adding building block An to n reaction vessels, where n is an integer of more than one, and adding the identical building block B1 to each reaction well. Here, B1 is identical for each chemical entity, and, ore, an oligonucleotide tag encoding this building block is not needed. After adding a ng block, the complexes may be pooled or not pooled. For example, the library is not pooled following the final step of building block addition, and the pools are screened individually to identify nd(s) that bind to a target. To avoid pooling all of the reactions after synthesis, a BIND® Reader (from SRU tems, Inc.), for example, may be used to monitor binding on a sensor surface in high throughput format (e.g., 384 well plates and 1,536 well ). For example, building block An may be encoded with DNA tag An, and building block Bn may be encoded by its position within the well plate. Candidate compounds can then be identified by using a binding assay (e.g., using a BIND® Biosensor, also available by SRU Biosystems, Inc., or using an ELISA assay) and by analyzing the An tags by sequencing, microarray is and/or restriction digest analysis. This analysis allows for the identification of ations of building blocks An and Bn that produce the desired molecules.

The method of amplifying can ally include forming a water-in-oil on to create a plurality of aqueous microreactors. The reaction conditions (e.g., concentration of complex and size of microreactors) can be adjusted to provide, on average, a microreactor having at least one member of a library of compounds. Each microreactor can also contain the target, a single bead capable of binding to a complex or a portion of the complex (e.g., one or more tags) and/or binding the target, and an amplification reaction solution having one or more necessary reagents to perform nucleic acid amplification. After amplifying the tag in the microreactors, the amplified copies of the tag will bind to the beads in the microreactors, and the coated beads can be identified by any useful method.

Once the ng blocks from the first library that bind to the target of interest have been identified, a second library may be prepared in an iterative fashion. For example, one or two additional nodes of diversity can be added, and the second library is created and d, as described herein. This process can be repeated as many times as necessary to create molecules with desired molecular and pharmaceutical properties.

Various ligation techniques can be used to add the scaffold, building blocks, linkers, and building block tags. Accordingly, any of the binding steps described herein can include any useful ligation technique or techniques. Exemplary ligation techniques include enzymatic ligation, such as use of one of more RNA ligases and/or DNA ligases, as described herein; and chemical on, such as use of chemically co-reactive pairs, as bed herein.

Scaffold and building blocks The scaffold S can be a single atom or a molecular scaffold. Exemplary single atom scaffolds include a carbon atom, a boron atom, a nitrogen atom, or a phosphorus atom, etc. Exemplary polyatomic scaffolds include a cycloalkyl group, a cycloalkenyl group, a heterocycloalkyl group, a heterocycloalkenyl group, an aryl group, or a heteroaryl group. Particular embodiments of a heteroaryl scaffold include a triazine, such as 1,3,5-triazine, 1,2,3-triazine, or 1,2,4-triazine; a dine; a pyrazine; a pyridazine; a furan; a e; a pyrrolline; a idine; an oxazole; a pyrazole; an isoxazole; a pyran; a pyridine; an ; an indazole; or a purine.

The scaffold S can be operatively linked to the tag by any useful method. In one example, S is a triazine that is linked directly to the headpiece. To obtain this exemplary scaffold, trichlorotriazine (i.e., a nated precursor of triazine having three chlorines) is reacted with a nucleophilic group of the headpiece. Using this method, S has three positions having chlorine that are available for substitution, where two positions are available diversity nodes and one position is attached to the headpiece. Next, building block An is added to a diversity node of the ld, and tag An encoding for building block An (“tag An”) is d to the headpiece, where these two steps can be performed in any order. Then, building block Bn is added to the remaining diversity node, and tag Bn encoding for building block Bn is ligated to the end of tag An. In another example, S is a triazine that is operatively linked to the linker of a tag, where trichlorotriazine is reacted with a philic group (e.g., an amino group) of a PEG, aliphatic, or ic linker of a tag. Building blocks and associated tags can be added, as described above.

In yet another example, S is a ne that is operatively linked to building block An. To obtain this scaffold, building block An having two diversity nodes (e.g., an electrophilic group and a philic group, such as an Fmoc-amino acid) is reacted with the nucleophilic group of a linker (e.g., the terminal group of a PEG, tic, or aromatic linker, which is attached to a headpiece). Then, trichlorotriazine is reacted with a nucleophilic group of building block An. Using this method, all three chlorine positions of S are used as diversity nodes for building blocks. As described herein, additional building blocks and tags can be added, and onal scaffolds Sn can be added.

Exemplary building block An’s include, e.g., amino acids (e.g., alpha-, beta-, gamma-, , and epsilon- amino acids, as well as derivatives of l and unnatural amino , chemically co-reactive reactants (e.g., azide or alkyne chains) with an amine, or a thiol nt, or combinations thereof. The choice of building block An depends on, for example, the nature of the reactive group used in the linker, the nature of a scaffold moiety, and the solvent used for the chemical synthesis.

Exemplary building block Bn’s and Cn’s include any useful structural unit of a chemical entity, such as optionally substituted aromatic groups (e.g., optionally substituted phenyl or benzyl), optionally substituted heterocyclyl groups (e.g., ally substituted quinolinyl, isoquinolinyl, indolyl, isoindolyl, azaindolyl, benzimidazolyl, azabenzimidazolyl, benzisoxazolyl, pyridinyl, piperidyl, or pyrrolidinyl), optionally substituted alkyl groups (e.g., optionally tuted linear or branched C1-6 alkyl groups or optionally substituted C1-6 aminoalkyl groups), or optionally substituted carbocyclyl groups (e.g., ally substituted cyclopropyl, cyclohexyl, or cyclohexenyl). Particularly useful building block Bn’s and Cn’s include those with one or more reactive groups, such as an optionally substituted group (e.g., any described herein) having one or optional substituents that are reactive groups or can be chemically ed to form reactive groups. Exemplary reactive groups include one or more of amine (-NR2, where each R is, independently, H or an optionally substituted C1-6 alkyl), hydroxy, alkoxy (-OR, where R is an optionally substituted C1-6 alkyl, such as methoxy), carboxy (-COOH), amide, or chemically co-reactive substituents. A restriction site may be introduced, for example, in tag Bn or Cn, where a complex can be identified by performing PCR and restriction digest with one of the corresponding ction enzymes.

Linkers The bifunctional linker between the headpiece and the chemical entity can be varied to provide an appropriate spacer and/or to increase the solubility of the headpiece in organic solvent. A wide variety of linkers are cially available that can couple the headpiece with the small molecule library. The linker typically consists of linear or branched chains and may include a C1-10 alkyl, a heteroalkyl of 1 to atoms, a C2-10 alkenyl, a C2-10 alkynyl, C5-10 aryl, a cyclic or polycyclic system of 3 to 20 atoms, a phosphodiester, a e, an oligosaccharide, an oligonucleotide, an oligomer, a polymer, or a poly alkyl glycol (e.g., a poly ethylene glycol, such as –(CH2CH2O)nCH2CH2-, where n is an integer from 1 to 50), or ations thereof.

The tional linker may provide an appropriate spacer between the headpiece and a chemical entity of the y. In certain embodiments, the bifunctional linker includes three parts. Part 1 may be a reactive group, which forms a covalent bond with DNA, such as, e.g., a ylic acid, preferably ted by a N-hydroxy succinimide (NHS) ester to react with an amino group on the DNA (e.g., amino-modified dT), an amidite to modify the 5’ or 3’-terminus of a single-stranded headpiece (achieved by means of standard oligonucleotide chemistry), chemically co-reactive pairs (e.g., azido-alkyne cycloaddition in the presence of Cu(I) catalyst, or any described herein), or thiol reactive groups. Part 2 may also be a ve group, which forms a covalent bond with the chemical entity, either building block An or a scaffold. Such a reactive group could be, e.g., an amine, a thiol, an azide, or an alkyne. Part 3 may be a chemically inert spacer of variable , introduced between Part 1 and 2. Such a spacer can be a chain of ethylene glycol units (e.g., PEGs of different lengths), an alkane, an alkene, a polyene chain, or a peptide chain. The linker can contain branches or s with hydrophobic es (such as, e.g., benzene rings) to improve solubility of the headpiece in organic solvents, as well as fluorescent moieties (e.g. scein or Cy-3) used for library detection purposes. Hydrophobic residues in the headpiece design may be varied with the linker design to facilitate library synthesis in organic solvents. For example, the headpiece and linker combination is designed to have riate residues wherein the octanol:water coefficient (Poct) is from, e.g., 1.0 to 2.5.

Linkers can be empirically ed for a given small le library design, such that the library can be synthesized in organic solvent, for example, in 15%, 25%, 30%, 50%, 75%, 90%, 95%, 98%, 99%, or 100% organic solvent. The linker can be varied using model reactions prior to library synthesis to select the appropriate chain length that solubilizes the headpiece in an organic solvent.

Exemplary linkers include those having sed alkyl chain length, increased poly ethylene glycol units, branched species with positive charges (to neutralize the negative phosphate charges on the headpiece), or increased amounts of hydrophobicity (for example, addition of benzene ring ures).

Examples of commercially available linkers include amino-carboxylic linkers, such as those being peptides (e.g., Gly-Gly-Osu (N-alpha-benzyloxycarbonyl-(Glycine)3-N-succinimidyl ester) or Z-Gly-Gly-Gly-Gly-Gly-Gly-Osu (N-alpha-benzyloxycarbonyl-(Glycine)6-N-succinimidyl ester, SEQ ID NO: 3)), PEG (e.g., Fmoc-aminoPEG2000-NHS or amino-PEG (12-24)-NHS), or alkane acid chains (e.g., Boc-ε-aminocaproic acid-Osu); chemically co-reactive pair linkers, such as those chemically co- reactive pairs described herein in combination with a peptide moiety (e.g., azidohomoalanine-Gly-Gly- Gly-OSu (SEQ ID NO: 4) or propargylglycine-Gly-Gly-Gly-OSu (SEQ ID NO: 5)), PEG (e.g., azido- PEG-NHS), or an alkane acid chain moiety (e.g., 5-azidopentanoic acid, (S)(azidomethyl)Bocpyrrolidine , 4-azidoaniline, or 4-azido-butanoic acid N-hydroxysuccinimide ester); thiol-reactive linkers, such as those being PEG (e.g., )n G-maleimide), alkane chains (e.g., 3-(pyridin- 2-yldisulfanyl)-propionic acid-Osu or sulfosuccinimidyl 6-(3’-[2-pyridyldithio]- propionamido)hexanoate)); and es for oligonucleotide synthesis, such as amino modifiers (e.g., 6- (trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), thiol ers (e.g., S- tritylmercaptohexyl[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or ally co-reactive pair modifiers (e.g., 6-hexynyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 3- dimethoxytrityloxy(3-(3-propargyloxypropanamido)propanamido)propylO-succinoyl, long chain alkylamino CPG, or 4-azido-butanoic acid N-hydroxysuccinimide ester)). Additional linkers are known in the art, and those that can be used during library synthesis include, but are not limited to, 5’-O- dimethoxytrityl-1’,2’-dideoxyribose-3’-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 9-O- dimethoxytrityl-triethylene glycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; 3-(4,4’- dimethoxytrityloxy)propyl[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite; and 18-O- dimethoxytrityl hexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. Any of the s herein can be added in tandem to one r in different combinations to generate linkers of different desired lengths.

Linkers may also be branched, where branched linkers are well known in the art and examples can consist of symmetric or asymmetric doublers or a symmetric r. See, for e, Newcome et al., Dendritic Molecules: Concepts, Synthesis, Perspectives, VCH Publishers (1996); Boussif et al., Proc.

Natl. Acad. Sci. USA 92:7297-7301 (1995); and Jansen et al., Science 266:1226 (1994).

Example 1 General gy to improve single-stranded ligation of DNA tags Various reaction conditions were explored to improve single-stranded ligation of tags to form an encoded y. These reaction conditions included using modified nucleotides within the tag (e.g., use of one or more nucleotides having a 2’-OMe group to form a MNA/DNA tag, where “MNA” refers to an oligonucleotide having at least one 2’-O-methyl nucleotide); using donor tags and acceptor tags having ent lengths and varying the concentration of the tags; using different types of s, as well as combinations thereof (e.g., CircLigaseTM ssDNA ligase and/or T4 RNA ligase), and varying their concentration; purifying the complex by removing unreacted starting materials; using poly ethylene glycols (PEGs) having ent molecular weights and varying their concentration; varying the temperature and duration for reaction, such as ligation; varying the concentration of various , including ATP, Co(NH3)6Cl3, and yeast inorganic pyrophosphate; using enzymatically or ally phosphorylated ucleotide tags; using 3’-protected tags; and using 5’-chemically adenylated tags.

After a thorough analysis of different conditions, optimal combinations of ters that provided up to 90% ligation efficiency (e.g., Figure 5C), as ined by the fraction of ligated final product to un-ligated starting reactant (“fraction ligated”), were found. A scheme of the ligation reaction using ligase is shown in Figure 5A, and a typical ring polyacrylamide gel electrophoresis is shown in Figure 5B. The donor oligonucleotide was labeled at the 3’-terminus and could be detected on a gel by scanning at 450 nm excitation on a StormTM 800 PhosphorImager. The gel depicts an unligated donor (or starting material) and the ligated product. In particular, the adenylated donor can be resolved and distinguished from the starting material on this gel.

Table 2 es ligation efficiencies measured as a function of the ition of the oligonucleotide (i.e., oligonucleotides with all DNA nucleotides versus oligonucleotides with at least one 2’-O-methyl nucleotide, labeled “MNA”) and the type of ligase (i.e., RNA ligase versus ssDNA ligase).

These ligation experiments included the following tags: an A donor having the sequence of 5’-PGCT GTG CAG GTA GAG TGCFAM-3’ (SEQ ID NO: 6); a 5’-MNA-DNA donor having the sequence of 5’-P-mGCT GTG CAG GTA GAG TGCFAM-3’ (SEQ ID NO: 7); an all-MNA donor having the sequence of 5’-P-mGmUmG mCmAmG mGmUmA mGmAmG mUmGmCFAM-3’ (SEQ ID NO: 8); a DNA-3’MNA acceptor having the sequence of 5’-HO-TAC GTA TAC GAC TGmG-OH-3’ (SEQ ID NO: 9); an all-DNA acceptor having the sequence of 5’-HO-GCA GAC TAC GTA TAC GAC TGG-OH-3’ (SEQ ID NO: 10); and an A or having the sequence of 5’-HO-mUmAmC mGmUmA mUmAmC mGmAmC mUmGmG-OH-3’ (SEQ ID NO: 11), where “m” indicates a 2’-OMe base, “P” indicates a phosphorylated nucleotide, and “FAM” indicates fluorescein.

Ligation encies were calculated from gel densitometry data as the ratio between the intensity from the ligation product and the sum of the intensity from the ligation product and the unligated starting material. The reaction ions for T4 RNA ligase included the following: 5 μM each of donor and acceptor oligonucleotides (15-18 nucleotides (nts) long) in a buffer solution containing 50 mM Tris HCl, mM MgCl2, 1 mM hexamine cobalt chloride, 1 mM ATP, 25% PEG4600, and 5 units of T4 RNA ligase (NEB- new units) at pH 7.5. The ons were incubated at 37°C for 16 hours. The on conditions for CircLigase™ included the following: 5 μM each of donor and acceptor oligonucleotides (length 15 or 18 nts) incubated in a buffer containing 50 mM MOPS (pH 7.5), 10 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.05 mM ATP, 2.5 mM MnCl2, and 25% (w/v) PEG 8000 with 20 units of CircLigase™ (Epicentre) at 50°C for 16 hours. The reactions were resolved on 8M urea/15% PAAG, followed by ometry using excitation at 450 nm.

Table 2 Donor Acceptor T4 RNA ligase CircLigase™ All-DNA All-DNA 9 % 89% A A 14% 68% All-DNA DNA-3’MNA 46% 85% All-MNA All-DNA 11% 84% All-MNA All-MNA 20% 29% All-MNA DNA-3’MNA 32% 73% -DNA All-DNA 29% 90% ’-MNA-DNA All-MNA 16 % 46% -DNA DNA-3’MNA 69% 81% Generally, CircLigase™ produced higher ligation yields than T4 RNA ligase (Table 2). When both donor and acceptor were A hybrid oligonucleotides, efficient ligation was achieved with T4 RNA .

Figure 5C shows high yield ligation achieved for T4 RNA ligase at high enzyme and oligonucleotide concentrations. The reaction conditions included following: 250 μM each of donor and acceptor oligonucleotides in a buffer containing 50 mM Tris HCl, 10 mM MgCl2, 1 mM hexamine cobalt chloride, 2.5 mM ATP, 30% (w/v) PEG4600, pH 7.5, different amounts of T4 RNA ligase at 40 units/ μL (NEB- new units), and 0.1 unit of yeast inorganic pyrophosphatase. The reactions were incubated at 37°C for 5 and 20 hours and resolved on 8M urea/15% PAAG, followed by densitometry using excitation at 450 nm.

Overall, these data suggest that enzymatic ligation can be optimized by including one or more modified 2’-nucleotides and/or by using an RNA or DNA ligase. Further details for several other tested ions, such as PEG or tag length, that can contribute to on efficiency are discussed below.

Example 2 Effect of PEG on single-stranded ligation To ine the effect of PEG molecular weight (MW) on ligation, single-stranded tags were ligated with 25% (w/v) of PEG having a MW from 300 to 20,000 Daltons. As shown in Figure 6A, 80% or greater ligation was observed for PEG having a MW of 3,350, 4,000, 6,000, 8,000, and 20,000. These ligation ments included the following tags: a 15mer donor having the sequence of 5’-P-mGTG CAG GTA GAG TGCFAM-3’ (SEQ ID NO: 12) and a 15mer acceptor having the ce of 5’-HO- mUAC GTA TAC GAC TGmG-OH-3’ (SEQ ID NO: 13). These oligonucleotide tags were DNA sequences with one or two terminal 2’O-methyl (2’-OMe) RNA bases (e.g., 2’-OMe-U (mU) or 2’-OMe- G (mG)).

Experiments were also conducted to determine the effect of PEG concentration. Single-stranded tags were ligated with various concentration of PEG having a MW of 4,600 Daltons (PEG4600). As shown in Figure 6B, 70% or greater ligation, on average, was observed for 25% (w/v) to 35% (w/v) PEG4600.

Example 3 Effect of tag length on single-stranded ligation To determine the effect of tag length on ligation, acceptor and donor tags of various lengths were constructed. For CircLigase™ experiments, a 15mer donor having the sequence 5’-P-mGTG CAG GTA GAG FAM-3’ (SEQ ID NO: 12) was used and paired with 10, 12, 14, 16, and 18mer DNA or oligonucleotides. For T4 RNA ligase experiments, the tags included one or more 2’-OMe-bases (designated as being MNA/DNA tags). Table 3 es the sequence for the three donor tags (15mer, 8mer, and 5mer) and the three acceptor tags (15mer, 8mer, and 5mer).

Table 3 Oligonucleotide tag Sequence* 15mer donor 5’-P-mGTG CAG GTA GAG TGCFAM-3’ (SEQ ID NO: 12) 15mer acceptor 5’-HO-mUAC GTA TAC GAC TGmG-OH-3’ (SEQ ID NO: 13) 8mer donor 5’-P-mGT GAG TGCFAM-3’ (SEQ ID NO: 14) 8mer acceptor 5’-HO-C A GAC TGmG-OH-3’(SEQ ID NO: 15) 5mer donor 5’-P-mGT FAM-3’ (SEQ ID NO: 16) 5mer acceptor 5’-HO-mAC H-3’ (SEQ ID NO: 17) * “m” indicates a 2’-OMe base, “P” indicates a phosphorylated nucleotide, and “FAM” indicates fluorescein.

The extent of ligation was analyzed by densitometry of electrophoretic gels (Figures 7A-7B).

The results of the gase™ reactions indicate a strong dependence of on yield on the length of the acceptor oligonucleotide (Figure 7A). The highest ligation yield was observed with an 18mer acceptor (62%), while ligation yield with a 10mer acceptor was lower than 10%. The s of the T4 RNA ligase reactions indicate that the combination of an 8mer acceptor with an 8mer donor provided the highest yield and that combinations having a 15mer donor with any of the tested acceptors provided yields greater than 75% e 7B). If a library includes r tags (i.e., about 10mer or shorter), then T4 RNA ligase may be preferred for tag ligation. In other cases, ligation can be further optimized by using CircLigase™ or a combination of T4 RNA ligase and CircLigaseTM.

Effect of purification on single-stranded ligation To determine the effect of purification on ligation, single-stranded tags were ligated to imitate the library synthetic process. For these experiments, the tags included 15mer donor and 15mer acceptor tags, as provided above in Table 3. The chemical entity was bound to the 3’-terminus of the library, where the chemical entity was fluorescein in this e to aid in visualization. As shown in Figure 9 (right), successive tags were ligated to the 5’-OH group of the complex after phosphorylation by T4 PNK.

Experiments were also conducted by purifying the ligated product (i.e., the complex) prior to the PNK reaction, where particular agents useful in the ligation reaction (e.g., phosphate, cobalt, and/or unreacted tags) can inhibit the phosphorylation on with PNK or reduce ligation yield. As shown in Figure 9 (left), purifying the x (i.e., minimal precipitation) prior to the PNK reaction increased ligation (see data marked with *, ting purification). s 8A-8B show LC-MS spectra for a 15mer MNA/DNA tag before and after phosphorylation. The presence or absence of DTT had no effect on phosphorylation.

Example 5 Chemically co-reactive pair on and reverse transcription of junctions The methods described herein can further include chemically co-reactive pair ligation techniques, as well as enzyme ligation ques. Accordingly, as an example of chemical ligation, an ary chemically co-reactive pair (i.e., an alkyne and an azido pair in a cycloaddition reaction) in two variants: a short chemically co-reactive pair and a long chemically co-reactive pair, was used.

Materials In a first variant, a short chemically ctive pair (Figure 10A) was used. The pair included (i) an oligonucleotide having the sequence 5’-GCG TGA ACA TGC ATC TCC CGT ATG CGT ACA GTC CAT T/propargylG/-3’ (“5end3propargyl,” SEQ ID NO: 18) and (ii) an oligonucleotide having the sequence 5’-/azidoT/ATA GCG CGA TAT ACA CAC TGG CGA GCT TGC GTA CTG-3’ (“3end5azido,” SEQ ID NO: 19). This pair of oligonucleotides was prepared by TriLink BioTechnologies, Inc. (San Diego, CA). These oligonucleotides were designed to produce a short spacer between two oligonucleotides upon ligation, where the linker would be 5 atoms long (counting from the sition of the 5end3propargyl oligonucleotide to the C5’-position of the 3end5azido oligonucleotide). In addition, the 5’-azido oligonucleotide azido) was prepared by converting the iodo group in the ponding 5’-iodo ucleotide into an azido group.

In a second variant, a long chemically co-reactive pair (Figure 10B) was used. The pair included (i) an oligonucleotide having the sequence 5’-GCG TGA ACA TGC ATC TCC CGT ATG CGT ACA GTC CAT TG/spacer7-azide/-3’ (“5end3azide,” SEQ ID NO: 20) and (ii) an oligonucleotide having the sequence 5’-/hexynyl/TA GCG CGA TAT ACA CAC TGG CGA GCT TGC GTA CTG-3’ (“3end5hexynyl,” SEQ ID NO: 21). This pair of ucleotides was prepared by Integrated DNA Technologies, Inc. (IDT DNA, San Diego, CA, and Coralville, IA). The 5end3azide oligonucleotide was prepared by reacting an azidobutyrate N-hydroxysuccinimide ester with a 3’-amino-modifier C7 (2- dimethoxytrityl oxymethylfluorenylmethoxycarbonylamino-hexanesuccinoyl-long chain alkylamino), which was introduced during oligonucleotide column synthesis. This pair was designed to produce a 24 atom long spacer between the oligonucleotides (counting from the C3’-position of the 5end3azide oligonucleotide to the C5’-position of the 3end5hexynyl oligonucleotide).

For reverse transcription (as shown by the tic in Figure 11A), the primers and tes included the following: a reverse transcription primer having the sequence of 5’-/Cy5/ CAG TAC GCA AGC TCG-3’ (“Cy5s_primer15,” SEQ ID NO: 22); a control template having the sequence of 5’-GCG TGA ACA TGC ATC TCC CGT ATG CGT ACA GTC CAT TGT ATA GCG CGA TAT ACA CAC TGG CGA GCT TGC GTA CTG-3’ (“templ75,” SEQ ID NO: 23); a 5’-PCR primer having the sequence of 5’-GCG TGA ACA TGC ATC TCC-3’ (SEQ ID NO: 24); and a 3’-PCR primer having the sequence of 5’-CAG TAC GCA AGC TCG CC-3’ (SEQ ID NO: 25), where these sequences were obtained from IDT DNA. A Cy5-labeled DNA primer was used for the experiments to enable separate detection of the reverse transcription products by LC.

Experimental Conditions For the ally co-reactive pair ligations, 1 mM solutions of chemically co-reactive pairs, such as ropargyl+3end5azido (short) or 5end3azide+3end5hexynyl (long), were incubated for 12 hours in the presence of 100 equivalents of TBTA ligand (tris-[(1-benzyl-1H-1,2,3-triazol yl)methyl]amine) and 50 equivalents of CuBr in a water/dimethyl acetate mixture. ing the reaction, an excess of EDTA was added, and the reaction mixtures were desalted using Zeba Spin Desalting Columns (Invitrogen Corp., ad, CA) and then l precipitated. For the e transcription reactions, the templates were purified on a 15% polyacrylamide gel containing 8M urea.

Liquid chromatography-mass ometry (LC-MS) was performed on a Thermo Scientific LCQ Fleet using an ACE 3 C18-300 (50 x 2.1 mm) column and a 5 minute gradient of 5-35% of buffer B using buffer A (1% hexafluoroisopropanol (HFIP), 0.1% di-isopropylethyl amine (DIEA), 10μM EDTA in water) and buffer B (0.075% HFIP, 0.0375% DIEA, 10 μM EDTA, 65% acetonitrile/35% water). LC was monitored at 260 nm and 650 nm. MS was detected in the negative mode, and mass peak deconvolution was med using ProMass software.

Reverse transcription reactions were performed using ThermoScriptTM RT (Invitrogen Corp.), according to the manufacturer’s protocol, at 50°C for 1-2 hours. The results were analyzed by LC-MS and by PCR. PCR was performed using Platinum® SuperMix and resolved on 4% agarose E-Gels (both from Invitrogen Corp.). Eleven and eighteen cycles of PCR were performed with or without a ing RT reaction. The 75mer template was not reverse ribed and used directly for the PCR amplification.

Results and discussion In both the ligations forming a short spacer and a long spacer, reaction yields were high, close to quantitative, as analyzed by LC-MS. Accordingly, chemical ligation provides a high yield technique to bind or operatively associate a headpiece to one or more building block tags.

For a viable chemical on strategy to produce DNA-encoded ies, the resultant complex should be capable of oing PCR or RT-PCR for further sequencing applications. While PCR and RT-PCR may not be an issue with enzymatically ligated tags, such as described above, unnatural chemical linkers may be difficult to process by RNA or DNA polymerases. The data provided in Figures. 11B-11E suggest that oligonucleotides having a spacer of particular lengths can be ribed and/or reverse transcribed.

In the case of a chemically co-reactive pair linker resulting in a triazole-linked oligonucleotide, a dependence on the length of the linker was observed. For the short chemically co-reactive pair, the resultant template was reverse transcribed and analyzed by LC-MS. LC is revealed three major absorption peaks at 2.79 min., 3.47 min., and 3.62 min. for 260 nm, where the peaks at 3.47 min. and 3.62 min. also provided absorption peaks at 650 nm. MS analysis of the peak at 3.47 min. showed only the presence of the template 23097.3 d 23098.8), and the peak at 3.62 min. contained a template (23098.0) and a fully extended primer (23670.8, calc’d: 6) at an approximately 1.7:1 ratio, suggesting a 50-60% yield for this RT reaction (Figure 11C). For comparison, reverse transcription (RT) of the control having an all-DNA template produced the extended primer (peak 9) in an amount roughly equivalent to the template .7), suggesting close to a 100% yield (Figure 11B).

For the long chemically co-reactive pair, LC of the RT on showed two absorption peaks at 2.77 min and 3.43 min for 260 nm, where the peak at 3.43 min also provided absorption peaks at 650 nm, i.e., ned a Cy5 labeled material, which is the ed RT product. MS analysis of the peak at 3.43 min. revealed the te (observed 23526.6, calc’d: 23534.1), as well as the Cy5 primer extended to the linker (11569.1). No full length t was observed by LC-MS, indicating that the RT reaction did not occur in a measureable amount (Figure 11D).

RT-PCR was performed with the tes described above and revealed that only the short linker yielded reverse transcription product, albeit at 5-10 lower efficiency (Figure 11E). Efficiency of the RT was estimated to be about 2-fold lower than the template (templ75). For example, the PCR product of the short ligated template around 2-fold lower after RT and around 5-10 times lower t RT, as compared to the PCR product of the all-DNA template 75 (templ75). Accordingly, these data provide support for the use of chemical ligation to produce a complex that can be reverse transcribed and/or transcribed, and ally ligated headpieces and/or tags can be used in any of the binding steps described herein to produce encoded libraries.

Example 6 Ligation of 3’-phosphorothioate oligonucleotides with 5’-iodo oligonucleotides To determine the flexibility of the methods described herein, the ligation efficiency of oligonucleotides having other modifications were determined. In particular, analogs of the natural phosphodiester linkage (e.g., a phosphorothioate analog) could provide an alternative moiety for postselection PCR analysis and sequencing.

The following oligonucleotides were synthesized by TriLink hnologies, Inc. (San Diego, CA): (i) 5’-/Cy5/ CGA TAT ACA CAC TGG CGA GCT/thiophosphate/-3’ (“CCy5,” SEQ ID NO: 26), (ii) 5’-/IododT/ GC GTA CTG AGC/6-FAM/-3’ (“CFL,” SEQ ID NO: 27), as shown in Figure 12A, and (iii) a splint ucleotide having the sequence of CAG TAC GCA AGC TCG CC (“spl,” SEQ ID NO: 28). Ligation reactions were performed with 100 μM of each reactant oligonucleotide in a buffer containing 50mM Tris HCl (pH 7.0), 100 mM NaCl, and 10 mM MgCl2 (“ligation buffer”) at room temperature. The on reactions were supplemented by either of the ing: 100 μM of the splint oligonucleotide, 10 mM Co(NH3)6Cl3, 40% (w/v) of PEG4000, or 80% (w/v) of PEG300. The reaction was allowed to progress for up to 48 hours. Ligation products were ed by LC-MS using detection at 260 nm, 495 nm, and 650 nm, as well as by an 8M urea/ 15% polyacrylamide gel (PAAG) that was further scanned at 450 and 635 nm excitation on a StormTM 800 PhosphorImager.

In the absence of the splint ucleotide, no ligation was observed (Figure 12B, lanes labeled “-spl”). In the presence of the splint oligonucleotide, ligation occurred and reached around 60% of fraction ligated after 48 hours (Figures 12B-12C). LC-MS revealed several peaks in the chromatogram, with a peak at 3.00 min absorbing at 260 nm, 495 nm, and 650 nm. MS of this peak showed mostly the product of ligation at 11539.6 Da (calc’d 11540) with less than 10% of CCy5 oligonucleotide at 7329.8 Da (calc’d 7329.1). Low levels of ligation were detected in the presence of PEGs and hexamine cobalt, where hexamine cobalt caused precipitation of the beled oligonucleotide. These data suggest that headpieces and/or tags having modified phosphate groups (e.g., modified phosphodiester linkages, such as phosphorothioate linkages) can be used in any of the binding steps bed herein to produce d libraries.

In order to further study the iodo-phosphorothioate ligation reaction, the ligation of 5’-I dT-oligo- 3’-FAM (CFL) and 5’-Cy5-oligo-3’-PS (CCy5) was performed in the absence and presence of a splint under different reaction conditions.

In a first set of conditions, ligation experiments were conducted with incubation for seven to eight days. These experiments were performed in the same ligation buffer as above with 50 µM of each oligonucleotide and incubated for a week at room temperature. Figure 12D shows LC-MS analysis of the ligation of CFL and CCy5 in the absence (top) and ce (bottom) of a splint (positive control), where on reactions were ted for seven days. Three LC traces were recorded for each reaction at 260 nm (to detect all nucleic acids), at 495 nm (to detect the CFL oligonucleotide and the ligation product), and at 650 nm (to detect the CCy5 oligonucleotide and the on product).

In the absence of the splint, no ligation occurred, and only starting materials CFL (4339 Da) and CCy5 (7329 Da) were detected (Figure 12D, top). When the splint oligonucleotide was present for seven days, a characteristic peak was observed in 495 nm channel with a ion time of 2.98 min, which corresponds to the ligated product (11542 Da) (Figure 12D, bottom). This peak overlapped with that for the CCy5 oligonucleotide ed at the 650 nm channel and, thus, was indistinguishable from CCy5 at 650 nm.

Figure 12E shows the LC-MS analysis of CFL and CCy5 in the absence of a splint, where ligation reactions were incubated for eight days at 400 µM of each oligonucleotide. No ligation product was detected. Peak 1 (at 495 nm) contained CFL ng material (4339 Da), as well as traces of the loss of iodine product (4211 Da) and an n degradation product (4271 Da, possibly ethyl mercaptane displacement). Peak 2 (at 650 nm) contained CCy5 starting al (7329 Da) and oxidized CCy5 ucleotide (7317 Da). Peak 3 (at 650 nm) contained dimerized CCy5 (14663 Da).

In a second set of conditions, iodine displacement reactions were conducted in the presence of piperdine and at a pH higher than 7.0. Figure 12F shows MS analysis for a reaction of CFL ucleotide with piperidine, where this reaction was intended to displace the al iodine present in CFL. One reaction condition included oligonucleotides at 100 µM, dine at 40 mM (400 equivalents) in 100 mM borate buffer, pH 9.5, for 20 hrs at room temperature (data shown in left panel of Figure 12F); and another reaction condition included oligonucleotides at 400 µM, piperidine at 2 M (4,000 equivalents) in 200 mM borate buffer, pH 9.5, for 2 hrs at 65°C (data shown in right panel of Figure 12F).

In the on condition including 40 mM of piperidine (Figure 12F, left), no piperidine displacement was observed, and a small amount of hydrolysis product was detected (4229 Da). In addition, traces of the loss of iodine (4211 Da) and unknown degradation product (4271 Da) were observed. In the on condition including 2 M of piperidine (Figure 12F, right), piperidine displacement of iodine was observed (4296 Da), and the amount of starting material was substantially diminished (4339 Da). In addition, peaks corresponding to hydrolysis of iodine (by displacement of OH) or ty (4229 Da) and loss of iodine (4214 Da) were also observed. These data show that the ce of an amine (e.g., as part of chemical library synthesis) will not detrimentally effect the oligonucleotide portion of the library members and/or interfere with this on strategy.

In a third set of ions, splint ligation reactions were conducted in the presence of piperdine and at a pH higher than 7.0. Figure 12G shows a splint ligation reaction of CFL and CCy5 oligonucleotides at 50 µM performed in the presence of 400 equivalents of piperidine in 100 mM borate buffer, pH 9.5, for 20 hrs at room temperature. The characteristic peak detected in the LC trace (at 495 nm) contained predominantly the product of ligation at 11541.3 Da (calc’d 11540 Da). Based on these results, it can be concluded that that piperidine does not impair enzymatic ligation and that the presence of other amines (e.g., as part of chemical library synthesis) will likely not interfere with this ligation strategy.

Taking together, these data indicate that this ligation strategy can be performed under various on conditions that are suitable for a broad range of chemical ormations, including extended tion times, elevated pH conditions, and/or presence of one or more amines. Thus, the present methods can be useful for developing library members with diverse reaction conditions and precluding the necessity of buffer exchange, such as itation or other resource-intensive methods.

Example 7 Minimization of ing with modified nucleotides During single-stranded enzymatic ligation with T4 RNA ligase, low to moderate extent of terminal nucleotide shuffling can occur. Shuffling can result in the ion or excision of a nucleotide, where the final product or complex includes or excludes a nucleotide compared to the expected ligated ce (i.e., a sequence having the te sequence for both the acceptor and donor oligonucleotides).

Though low levels of shuffling can be tolerated, shuffling can be minimized by including a modified phosphate group. In particular, the modified phosphate group is a phosphorothioate linkage between the al nucleotide at the 3’-terminus of an acceptor oligonucleotide and the tide adjacent to the terminal nucleotide. By using such a phosphorothioate linkage, ing was greatly reduced. Only residual shuffling was detected by mass spectrometry, where shuffling likely arose due to incomplete conversion of the native phosphodiester e into the phosphorothioate linkage or to low levels of oxidation of the phosphorothioate linkage followed by conversion into the native phosphodiester e. Taking together this data and the ligation data in Example 6, one or more modified phosphate groups (e.g., a phosphorothioate or a hosphoramidite linkage) could be included in any oligonucleotide sequence described herein (e.g., between the terminal nucleotide at the 3’-terminus of a headpiece, a complex, a building block tag, or any tag described herein, and the nucleotide adjacent to the terminal nucleotide) to minimize shuffling during single-stranded ligation.

A single stranded headpiece (ssHP, 3636 Da) was phosphorylated at the 5’-terminus and ed with a hexylamine linker at the 3’-terminus to provide the sequence of 5’-P- mCGAGTCACGTC/Aminohex/-3’ (SEQ ID NO: 29). The headpiece was ligated to a tag (tag 15, XTAGSS000015, 2469 Da) having the ce of 5’-mCAGTGTCmA-3’ (SEQ ID NO: 30), where mC and mA indicate 2’-O methyl nucleotides. LC-MS analysis (Figure 13A) revealed that the ligation product peak contained up to three species, which was partially separated by LC and had the following molecular weights: 6089 Da (expected), 5769 Da (-320 Da from expected) and 6409 Da (+320 Da from expected). This mass difference of 320 Da corresponds exactly to either removal or addition of an extra O-Me C nucleotide (“terminal nucleotide shuffling”).

Experiments with other terminal O-Me nucleotides, as well as terminal 2’-fluoro nucleotides, confirmed that shuffling likely occurs by cleavage of the 5’-terminal nucleotide of the donor oligonucleotide, probably after adenylation of the . The ism of this event is unknown. t being limited by mechanism, Figure 13B illustrates a possible scheme for nucleotide reshuffling during T4 RNA ligase reaction n a headpiece and a tag, where one of skill in the art would understand that this reaction could occur n any donor and acceptor oligonucleotides (e.g., between two tags, where one tag is the donor oligonucleotide and the other tag is the acceptor oligonucleotide).

Generally, the majority of the ligation reaction with T4 RNA ligase (T4Rnl1) provides the expected l) ligation product having the combined sequence of both the donor and acceptor oligonucleotides (Figure 13B-1, reaction on left). A small minority of the reaction provides aberrant ligation products (Figure 13B-1, reaction on right), where these nt products include those having the removal or addition of a terminal nucleotide (“Product -1 nt” and “Product + 1 nt,” tively, in Figure 13B-2).

Without being d by mechanism, cleavage of the donor oligonucleotide (“headpiece” or “HP” in Figure 13B-1) may occur by ng with the 3’-OH group of the acceptor (“tag”), thereby providing a 5’-phosphorylated donor lacking one nucleotide (“HP-1 nt”) and an adenylated nucleotide with an accessible 3’-OH group (“1 nt”). Figure 13B-2 shows two exemplary schemes for the reaction between the headpiece (HP), tag, HP-1 nt, and 1 nt. To provide a product with an excised terminal nucleotide e 13B-2, left), the sphorylated donor g one nucleotide (HP-1 nt) acts a substrate for the ligation event. This HP-1 nt headpiece is re-adenylated by T4 RNA ligase (to provide “Adenylated HP-1 nt” in Figure 13B-2) and ligated to the tag, resulting in a ligation product minus one tide (“Product-1 nt”). To e a product with an additional terminal nucleotide (Figure 13B-2, left), the ated nucleotide (1 nt) likely serves as a substrate for ligation to the tag, thereby producing an oligonucleotide having one nucleotide longer than the acceptor (“Tag+1 nt”). This Tag+1 nt oligonucleotide likely serves as an acceptor for the unaltered headpiece, where this reaction provides a ligation product having an onal nucleotide (“Product+1 nt”). LC-MS analyses of “Product”, “Product-1 nt”, and “Product+1 nt” were performed (Figure . When an aberrant tag and an aberrant headpiece (i.e., Tag+1 nt and HP-1 nt, respectively) recombine, then the resultant ligation product is indistinguishable from the expected product.

To further study the mechanism of terminal nucleotide fling, a headpiece (HP-PS) having the ce of 5’P-mC*GAGTCACGTC/Aminohex/-3’ (SEQ ID NO: 31) was prepared. Headpiece HP-PS has the same sequence as ssHP but contains one modification, namely the first phosphodiester linkage between 5’-terminal nucleotide mC and the following G was synthesized as a phosphorothioate linkage (one idging phosphate oxygen was substituted by a sulfur). LC-MS analysis of the HP-PS ligation to tag 15 revealed that shuffling was almost completely inhibited (Figure 13C). Traces of +/- 320 peaks likely correspond to the oxidative sion of the orothioate linkage into native phosphodiester linkages or lete sulfurization.

Example 8 Size exclusion chromatography of library members Libraries of chemical entities that are ted using short, single-stranded oligonucleotides as encoding ts are well suited for the enrichment of binders via size exclusion chromatography (SEC). SEC is chromatographic technique that separates molecules on the basis of size, where larger molecules having higher molecular weight flow through the column faster than smaller molecules having lower molecular weight.

Complexes of proteins and ssDNA library members can be readily separated from d library members using SEC. Figure 14 is an ultraviolet trace from an SEC experiment in which a small molecule covalently attached to short ssDNA (a range of oligonucleotides with defined lengths in the 20- 50 mer range) was mixed with a protein target known to bind the small molecule. The peaks that elute first from the column, in the 11-13 minute time range, represent target-associated library members. The later peaks, eluting from 14-17 minutes, represent d library members. The ratio of protein target to library molecule was 2:1, so approximately 50% of the library molecules should associate with the protein in the early eluting fraction, as observed in Figure 14. Libraries with larger, double-stranded oligonucleotide coding regions cannot be selected using this method since the unbound library members co-migrate with the bound library members on SEC. Thus, small molecule libraries attached to encoding single-stranded oligonucleotides in the er length range enable the use of a powerful separation technique that has the potential to significantly increase the signal-to-noise ratio required for the effective ion of small molecule binders to one or more s, e.g., novel n targets that are optionally untagged and/or wild-type protein. In particular, these approaches allow for fying target-binding chemical entities in d combinatorially-generated libraries without the need for tagging or immobilizing the target (e.g., a protein target).

Example 9 ng with chemically ligated DNA tags using the same chemistry for each ligation step Encoding DNA tags can be ligated enzymatically or chemically. A general approach to chemical DNA tag ligation is illustrated in Figure 15A. Each tag bears co-complementary reactive groups on its 5’ and 3’ ends. In order to prevent polymerization or cyclization of the tags, either (i) tion of one or both reactive groups (Figure 15A), e.g., in case of rotected 3’ alkynes, or (ii) -dependent ligation chemistry (Figure 15B), e.g., in the case of 5’-iodo/3’-phosphorothioate ligation, is used. For (i), unligated tags can be removed or capped after each library cycle to t mistagging or polymerization of the deprotected tag. This step may be optional for (ii), but may still be included. Primer extension reactions, using polymerase s that are capable of reading through chemically ligated junctions, can also be performed to demonstrate that ligated tags are readable and therefore the encoded information is recoverable by election amplification and sequencing (Figure 15C).

A library tagging strategy that implements ligation of the tags using “click-chemistry” (Cu(I) catalyzed azide/alkyne cycloaddition) is shown in Figure 16A. The implementation of this strategy relies on the ability of precise successive ligation of the tags, avoiding mistagging, and tag polymerizations, as well as the ability to copy the chemically ligated DNA into amplifiable natural DNA (cDNA) for lection amplification and sequencing (Figure 16C).

To achieve accurate tag ligation triisopropylsilyl (TIPS)-protected 3’ propargyl nucleotides, (synthesized from propargyl U in the form of a CPG matrix used for ucleotide synthesis) was used (Figure 16B). The TIPS protecting group can be specifically removed by treatment with tetrabutylammonium de (TBAF) in DMF at 60°C for 1-4 hours. As a result, the ligation during library synthesis includes a 5’-azido/3’-TIPS-propargyl nucleotide (Tag A) reacting with the 3’-propargyl of the ece through a click reaction. After purification, the previous cycle is treated with TBAF to remove TIPS and generate the reactive alkyne which in turn reacts with the next cycle tag. The procedure is repeated for as many cycles as it is necessary to produce 2, 3 or 4 or more successively installed encoding tags (Figure 16A).

Materials and methods Oligos: The following oligos were synthesized by Trilink Biotechnologies, San Diego CA: ss- HP-alkyne: 5’- NH2-TCG AAT GAC TCC GAT AT (3’-Propargyl G)-3’(SEQ ID NO: 32); ss-azido-TP: ’-azido dT ATA GCG CGA TAT ACA CAC TGG CGA GCT TGC GTA CTG -3’(SEQ ID NO: 33); and B-azido: 5’ azido dT ACA CAC TGG CGA GCT TGC GTA CTG -3’ (SEQ ID NO: 34). ag-TIPS: 5’-azdido dT AT GCG TAC AGT CC (propargyl )-3’ (SEQ ID NO: ) and 5’Dimethoxytrityl cinyl 3’-O-(triisopropyl silyl) Propargyl uridine cpg were synthesized by Prime Organics, Woburn MA.

The following oligos were sized by IDT DNA technologies, Coralville, IA: FAM-clickprimer : (5’FAM) CAG TAC GCA AGC TCG CC -3’ (SEQ ID NO: 36) and Cy5-click-primer: (5’- Cy5) CAG TAC GCA AGC TCG CC -3’ (SEQ ID NO: 37).

DNA55-control: /5’Biotin-TEG//ispC3//ispC3/-TCGAATGACTCCGATATGT ATA GCG CGA TAT ACA CAC TGG CGA GCT TGC GTA CTG -3’ (SEQ ID NO: 38). rDNA55-control: TEG//ispC3//ispC3/-TCGAATGACTCCGATAT(riboG)T ATA GCG CGA TAT ACA CAC TGG CGA GCT TGC GTA CTG -3’ (SEQ ID NO: 39) Synthesis of the tes: In the following examples, the phrase “chemically ligated tags”, or control sequences related to them, are referred to as “templates” because the subsequent step (“reading”) utilizes them as templates for template-dependent polymerization.

Tag ligation: To a solution of 1 equivalent (1 mM) of lkyne and 1 equivalent (1 mM) of ss-azidoTP in 500 mM pH 7.0 ate buffer, was added a solution of pre-mixed 2 eq of Cu(II)Acetate (to a final concentration of 2 mM), 4 eq of sodium ate (to a final concentration of 4 mM), 1 eq TBTA (to a final concentration of 1 mM) in DMF/water. The mixture was incubated at room temperature overnight. After LC-MS confirmation of the completion of the reaction, the reaction was precipitated using salt/ethanol.

“Single click” templates Y55 and Y185 were synthesized by the reaction of ss-HP-alkyne with ss-azido-TP and B-azido, respectively. Double and triple click templates (YDC and YTC) were synthesized by click ligation of ss-HP-alkyne with ClickTag-TIPS, followed by deprotection of TIPS using TBAF (tetrabutylammonium fluoride) in DMF at 60°C for an hour, followed by click ligation with ss-azido TP. For triple click template (YTC), the ligation and deprotection of ClickTag-TIPS was repeated twice.

The tes were reacted with biotin-(EG)4-NHS and desalted (Figure 17A). The final products were purified by RP HPLC and/or on a 15-20% polyacryl amide gel/ 8M urea and analyzed by LC-MS.

Enzymes: The following DNA polymerases with their reaction buffers were purchased from New England Biolabs: Klenow fragment of E. coli DNA polymerase I, Klenow fragment (exo-), E. coli DNA polymerase I, nator™, 9°N™, Superscript III™ .

Streptavidin magnetic Dynabeads® M280 were purchased from Invitrogen.

Template-dependent polymerization ment: Each template (5 μM) was incubated with 1 equivalent of either Cy5 or FAM Click-primer in 40 to 50 μL of the corresponding 1x on buffer and each enzyme, using reaction conditions according to the manufacturer’s guidelines for 1 hour. Certain reactions (such as SSII or SSIII transcriptions) were additionally supplemented with 1 mM MnCl2. The product of the on was loaded on 125 μL of pre-washed SA beads for 30 minutes with shaking. The beads were then collected, and the flowthrough was discarded. Beads were washed with 1 mL of Trisbuffered saline (pH 7.0) and eluted with 35 μL of 100 mM NaOH. The eluate was immediately neutralized by adding 10 μL of 1 M Tris HCl, pH 7.0. The products were analyzed using LC-MS.

Results and discussion Template Preparation: Each template, Y55, Y185 (Figures 17B and 17C), YDC and YTC (Figure 19) was synthesized and purified to greater than 85% purity (the major impurity being inylated template). LC-MS revealed the following MWs for the templates: Y55 17,624 (calculated 17,619) Da; YDC 22,228 (calculated ) Da; and YTC 26,832 (calculated 26,837) Da.

The single click templates Y55 and Y185 (Figures 17B and 17C) were synthesized from oligonucleotides that bear only one click chemistry functionality (alkyne or azide). The efficiency of the click reaction (chemical on) was over 90% in an overnight reaction using Cu(I) catalyst ted in situ. tes YDC and YTC (Figures 19A-19D) serve to demonstrate successive chemical ligations. Both YDC and YTC use individual tags which aneously contain both azido and TIPS- protected alkyne functionalities. Template YTC demonstrates three successive cycles of tagging as may be used to encode three steps of chemical library generation.

All of the above tes were tested for primer extension h and beyond the ligation linkages to demonstrate that ligated tags are readable, and therefore that encoded information is recoverable.

Template-dependent rization using “single-click” template Y55: A large set of polymerases was tested to read through a le click linkage (Figure 18A). Initial experiments were performed using Cy5-click-primer. In later experiments FAM-click-primer was used. The fluorophore had no effect on the copying of the template, i.e., the results were equivalent using either primer. As a control template DNA55-control and rDNA55-control were used (to test the effect of a single cleotide in the template, since propargyl-G used for a click ligation is a ribonucleotide derivative). ed full length products in all three templates have the same molecular weight, which is 17446 (FAM primer) (Figure 18B) or 17443 (Cy5 primer). A small amount of the product which corresponds to primer extension up to, but stopping at, the click ligation linkage (11880 Da) was also observed for some polymerases.

A set of polymerases that can produce substantial degree of read-through of the click linkage (production of full-length cDNA) were discovered and are tabulated below.

Full-length cDNA yields of over 50% Klenow fragment of E. coli DNA polymerase I Klenow fragment (exo-) E. coli DNA polymerase I Therminator™ 9°N™ cript III™ supplemented with 1 mM MnCl2 The highest yields (over 80% read-through at a single click junction) were achieved when using Klenow fragment with incubation at 37°C (Figure 18B). Somewhat lower yield was observed using E. coli DNA polymerase I. 50% yields with Therminator™ and 9°N™ polymerases, as well as Klenow fragment exo- were achieved.

Superscript III™ reverse transcriptase produced about 50% yield of cDNA when the buffer was mented with 1 mM MnCl2. However, manganese caused the mis-incorporation of nucleotides which was observed by MS, i.e., polymerization ty was reduced.

Template-dependent polymerization using “single-click” template Y185: Template Y185 features the same primer binding site as all templates used in this example, except, due to a different tailpiece B- azido, the distance between the last nucleotide of the primer binding site to the click linkage is 8 nucleotides, as compared to 20 nucleotides in Y55 and all other tes. The template was used to test whether transcription of a click e was still possible when the enzyme was in initiation-early elongation conformation. Klenow was capable of copying the Y185 template with similar efficiency to Y55, opening the possibility of reducing the length of the click-ligated encoding tags (Figure 18C).

Template-dependent polymerization using double and triple click-ligated templates YDC and YTC: After establishing that the Klenow nt was the most efficient enzyme to read through the click ligation linkages under the assay condition employed, cDNA using YDC and YTC templates (Figures C) were also generated. Primer extension reactions with both YDC and YTC templates produced full length products. Other observed products, which composed around 10-15% of total reaction output, corresponded to partially ed primer, stalled at each click junction, such as e.g., 11880 Da and 16236 Da. The yields were measured by LC-MS analysis in the presence of the internal standard and were about 80-90% per junction (i.e., around 85% for 1 click, 55% for 2-click and 50% for 3-click templates, see Figure 21).

The t of YDC transcription lacked 1 dA nucleotide (calculated 22110, observed 27197 Da; -313 dA Figure 20B) and the product of YTC transcription lacked 2 dA nucleotides (calculated 26773, observed 26147; -626 2xdA) (Figure 20C). This correlates with the number of propargyl U nucleotides in the template. Without wishing to be limited by ism, it can be hypothesized that Klenow skipped over those U’s in the context of zole-U junction. In contrast, the propargyl G nucleotide in the 1st click junction was correctly . e 10 Use of sphorothioate/5’-iodo tags to chemically ligate a succession of encoding DNA tags that encode a chemical y covalently installed upon the 5’-terminus Protection of 3’-phosphorothioate on tag: As shown in Figure 24A, a 5’-iodo-3’- phosphorothioate tag (1 eq.) was dissolved in water to give a final concentration of 5 mM. Subsequently, vinyl methyl sulfone (20 eq.) was added and the reaction was incubated at room temperature overnight.

Upon completion of the reaction, the product was itated by ethanol.

Library synthesis (Figure 24B) Cycle A: To each well in the split was added single-stranded DNA ece (1 eq., 1 mM solution in 500 mM pH 9.5 borate buffer), one cycle A protected tag (1.5 eq.), and splint (1.2 eq.). The chemical ligation was ted at room temperature overnight. To each well (in the split) was then added one Fmoc amino acid (100 eq.), ed by 4-(4,6-dimethoxy-1,3,5-triazinyl) methylmorpholinium chloride (100 eq.). The chemical reaction was incubated at room temperature overnight. Upon tion, all wells were pooled and the products precipitated using ethanol. The cycle A pool was purified using LC and lized to dryness, and then dissolved in water to give a 1 mM final concentration and piperidine (10% v/v) was added to perform the deprotection of cycle A tag (60°C, 2h). The deprotected product was precipitated again using ethanol.

Cycle B: The deprotected cycle A pool was dissolved in 500 mM, pH 9.5, borate buffer to give a 1mM concentration and then split into te on wells (1 eq. of cycle A product in each well). To each well was added one cycle B protected tag (1.5 eq.), and splint (1.2 eq.). The chemical ligation was incubated at room temperature overnight. To each well (in the split) was added a mixture of one formyl acid (100 eq.), diisopropyl carbodiimide (100 eq.) and 1-hydroxyaza-benzotriazole (100 eq.). The chemical reaction was incubated at room ature overnight. Upon completion, all wells were pooled and the products precipitated using ethanol. The cycle B pool was purified using LC and lyophilized to dryness, and then dissolved in water to give a 1 mM final concentration and piperidine (10% v/v) was added to perform the deprotection of cycle B tag (60°C, 2h). The ected product was precipitated again using ethanol.

Cycle C: The deprotected cycle B pool was dissolved in 500 mM pH 5.5 phosphate buffer to give a 1 mM concentration and then split into separate reaction wells (1 eq. of cycle B product in each well). To each well was added one cycle C tag (1.5 eq.) and splint (1.2 eq.). The chemical on was incubated at room temperature overnight. To each well (in the split) was added an amine (80 eq.) and sodium cyanoborohydride (80 eq.). The chemical reaction was incubated at 60°C for 16h. Upon tion, all wells were pooled and the products precipitated using ethanol. The cycle C pool was purified using LC and lyophilized to dryness.

Example 11 Encoding with chemically ligated DNA tags using a pair of onal chemistries for each successive tag ligation step Another approach for generation of chemically ligated encoding DNA tags is the use of a pair of orthogonal chemistries for successive ligations (Figure 22A). Tags that bear orthogonal reactive groups at their ends will not tag polymerize or cyclize, and the orthogonal nature of successive ligation steps will reduce the frequency of ging events. Such approaches require (i) having at least two orthogonal chemistries available for oligonucleotide conjugation, and (ii) available read-through strategy for each of the junctions thus created (Figures 22B and 22C). This approach may also obviate the need for the use of protection groups or capping steps, thereby simplifying the tag ligation process.

Orthogonal chemical ligation gy utilizing 5’-Azido/3’-Alkynyl and 5’-Iodo/3’- Phosphorothioate ligation for successive steps: An example of the use of two orthogonal chemistries tag ligation is the combination of 5’-azido/3’-alkynyl and 5’-iodo/3’-phosphorothioate ligations. Figure 23 shows an exemplary schematic of the synthesis of a 3-cycle orthogonal chemical ligation tagging strategy using these successive ligation chemistries. Figures 25A-25B show an example of the use of 3’- phosphorothioate/5’-azido and 3’-propargyl/5’-iodo tags to chemically ligate a succession of onal encoding DNA tags that encode a chemical library covalently installed upon the minus.

Protection of 3’-phosphorothioate on tags: As shown in Figure 25A, a 5’-azido-3’- phosphorothioate tag (1 eq.) was dissolved in water to give a final concentration of 5 mM. Subsequently, vinyl methyl sulfone (20 eq.) was added and the reaction was incubated at room temperature overnight.

Upon completion of the reaction, the product was precipitated by l.

Library synthesis (Figure 25B) Cycle A: To each well in the split was added single ed DNA headpiece (1 eq., 1 mM on in 500 mM pH 9.5 borate buffer), one cycle A tag (1.5 eq.), and splint (1.2 eq.). The chemical ligation was incubated at room temperature overnight. To each well (in the split) was then added one Fmoc amino acid (100 eq.), ed by 4-(4,6-dimethoxy-1,3,5-triazinyl)methylmorpholinium chloride (100 eq.). The chemical reaction was incubated at room temperature ght. Upon completion, all wells were pooled and the products precipitated using ethanol. The cycle A pool was purified using LC and lyophilized to dryness. Fmoc deprotection was performed on cycle A pool by treating the pool (1mM in water) with piperidine (10% v/v) for 2h at room temperature. The deprotected product was precipitated again using ethanol.

Cycle B: The purified cycle A pool was dissolved in 500 mM, pH 7.0 phosphate buffer to give a 1 mM concentration and then split into separate reaction wells (1 eq. of cycle A product in each well). To each well was added one cycle B protected tag (1.2 eq.), copper (II) acetate (2 eq.), sodium ascorbate (4 eq.), and tris-(benzyltriazolylmethyl)amine (1 eq.). The chemical ligation was incubated at room temperature overnight. Upon tion, the products were precipitated (in the split) using ethanol and then diluted to a 1 mM concentration using 500 mM, pH 9.5 borate buffer. To each well (in the split) was then added a mixture of one formyl acid (100 eq.), diisopropyl carbodiimide (100 eq.), and oxy aza-benzotriazole (100 eq.). The al reaction was incubated at room temperature overnight. Upon completion, all wells were pooled and the products itated using ethanol. The cycle B pool was then dissolved in water to give a 1 mM final concentration, and piperidine (10% v/v) was added to perform the ection of cycle B tag (room temperature, 18h). The deprotected t was precipitated again using ethanol. The ected Cycle B pool was purified using LC and lyophilized to dryness.

Cycle C: The purified cycle B pool was dissolved in 500 mM, pH 5.5 phosphate buffer to give a 1 mM concentration and then split into separate reaction wells (1 eq. of cycle B product in each well). To each well was added one cycle C tag (1.5 eq.) and splint (1.2 eq.). The chemical on was incubated at room temperature overnight. To each well (in the split) was added an amine (80 eq.) and sodium cyanoborohydride (80 eq.). The chemical reaction was incubated at 60°C for 16h. Upon completion, all wells were pooled and the products precipitated using ethanol. The cycle C pool was purified using LC and lyophilized to dryness.

Other ments All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

Various cations and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific d embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, pharmacology, or related fields are intended to be within the scope of the invention.

In this specification where reference has been made to patent ications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the es of the invention. Unless specifically stated ise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any iction, are prior art, or form part of the common general knowledge in the art.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification, and claims which e the term “comprising”, it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

Claims (18)

Claims

1. A method of tagging a library sing an oligonucleotide-encoded small molecule or peptide, said method comprising: (i) providing an oligonucleotide ece having a first functional group and a second functional group; (ii) binding said first functional group of said oligonucleotide headpiece to a first component of said small molecule or peptide, wherein said headpiece is directly connected to said first component or said headpiece is indirectly connected to said first component by a bifunctional ; and (iii) ligating said second functional group of said oligonucleotide headpiece to a first building block tag to form a x, wherein said ligating comprises chemical ligation which results in the formation of a linkage that does not comprise a phosphodiester or a phosphorothioate, n steps (ii) and (iii) can be performed in any order, wherein said oligonucleotide ece does not comprise a 2'-substituted nucleotide at the 5'-terminus and/or the 3'-terminus.

2. The method of claim 1, wherein said oligonucleotide headpiece is indirectly connected to said first ent by a bifunctional linker.

3. The method of claim 1 or 2, wherein said method comprises separating the complex from any unreacted tag or unreacted headpiece prior to steps (ii) or (iii).

4. The method of any one of claims 1 to 3, wherein said method comprises purifying the complex prior to steps (ii) or (iii).

5. The method of any one of claims 1 to 4, wherein the method further comprises: (iv) ligating one or more additional building block tags to the 5’-terminus or 3’-terminus of the complex; and (v) binding one or more additional components of said small le or peptide, wherein steps (iv) and (v) can be performed in any order.

6. The method of claim 5, wherein said ligating comprises chemical ligation.

7. The method of claim 5 or 6, wherein said method comprises separating the complex from any unreacted tag or unreacted headpiece prior to steps (ii) or (iii).

8. The method of any one of claims 5 to 7, n said method comprises ing the complex prior to steps (ii) or (iii).

9. The method of any one of claims 1 to 8, wherein the chemical ligation results in the ion of an ral chemical linkage.

10. The method of any one of claims 1 to 9, wherein said chemical ligation comprises the use of one or more chemically co-reactive pairs.

11. The method of claim 10, wherein said one or more chemically co-reactive pairs is an optionally substituted alkynyl group and an optionally substituted azido group; an optionally substituted diene having a 4 π-electron system and an optionally substituted dienophile or an optionally substituted heterodienophile having a 2 π-electron ; a nucleophile and a strained heterocyclyl electrophile; or an amino group and an aldehyde.

12. The method of any one of claims 1 to 11, wherein the oligonucleotide headpiece comprises a hairpin oligonucleotide.

13. The method of any one of claims 1 to 12, n the oligonucleotide headpiece or a building block tag encodes for the identity of the library.

14. The method of any one of claims 1 to 11, wherein the oligonucleotide headpiece or a building block tag s for the use of the member of the library.

15. The method of any one of claims 1 to 14, wherein the method further comprises: (vi) binding an oligonucleotide tailpiece to the complex.

16. The method of claim 15, wherein the oligonucleotide tailpiece encodes the identity of the library, the use of the member of the library, or the origin of the member of the y.

17. The method of any one of claims 1 to 16, wherein the complex ses a modification that supports solubility in organic conditions.

18. A method as d in any one of claims 1 to 17, substantially as herein described with or without reference to any example thereof.