METHODS FOR TAGGING DNA-ENCODED LIBRARIES
The present invention relates to oligonucleotide-encoded libraries and methods of tagging such libraries. In particular, the methods and oligonucleotides can include one or more 2′-substituted nucleotides, such as 2′-O-methyl or 2′-fluoro nucleotides, and other conditions or reagents to enhance enzyme ligation or one or more chemical functionalities to support chemical ligation.
1 . A method of tagging a first library comprising an oligonucleotide-encoded chemical entity, said method comprising:
(i) providing a single-stranded oligonucleotide headpiece having a first functional group and a second functional group;
(ii) binding said first functional group of said headpiece to a first component of said chemical entity, 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 headpiece to a first building block tag to form a complex,
wherein said ligating comprises chemical ligation of one or more chemically co-reactive pairs selected from:
(a) an optionally substituted alkyne and an optionally substituted azido group; or
(b) a phosphorothioate group and an iodo group;
wherein said steps (ii) and (iii) can be performed in any order; and
wherein said first building block tag encodes for the binding reaction of said step (ii),
thereby providing a tagged library.
2 - 75 . (canceled)
76 . The method of claim 1 , wherein said chemically co-reactive pair is an optionally substituted alkynyl group and an optionally substituted azido group.
77 . The method of claim 1 , wherein said chemically co-reactive pair is a phosphorothioate group and an iodo group.
78 . The method of claim 77 , wherein said phosphorothioate group is at the 5′-terminus of an oligonucleotide and said iodo group is at the 3′-terminus of an oligonucleotide.
79 - 94 . (canceled)
95 . The method of claim 77 , wherein said phosphorothioate group is at the 3′-terminus of an oligonucleotide and said iodo group is at the 5′-terminus of an oligonucleotide.
96 . The method of claim 77 , wherein said second functional group of said headpiece is a phosphorothioate group.
97 . The method of claim 76 , wherein said azido group is at the 5′-terminus of an oligonucleotide and said alkyne group is at the 3′-terminus of an oligonucleotide.
98 . The method of claim 76 , wherein said azido group is at the 3′-terminus of an oligonucleotide and said alkyne group is at the 5′-terminus of an oligonucleotide.
99 . The method of claim 76 , wherein said second functional group of said headpiece is an alkyne group.
100 . The method of claim 1 , wherein said chemical ligation further comprises a splint oligonucleotide in the chemical ligation reaction between said chemically co-reactive pair.
101 . The method of claim 1 , wherein said chemically co-reactive pair produces a spacer having a length from about 4 to about 24 atoms.
102 . The method of claim 1 , wherein the method further comprises:
(iv) binding a single-stranded oligonucleotide second building block tag to the 5′-terminus or 3′-terminus of said complex; and
(v) binding a second component of said chemical library to said first component,
wherein said steps (iv) and (v) can be performed in any order;
wherein the second building block tag encodes for the binding reaction of step (v); and
wherein step (iv) is carried out using chemical ligation comprising use of one or more chemically co-reactive pairs selected from:
(a) an optionally substituted alkynyl group and an optionally substituted azido group; or
(b) a phosphorothioate group and an iodo group.
103 . The method of claim 102 , wherein said chemical ligation further comprises a splint oligonucleotide in the binding reaction between said chemically co-reactive pair.
104 . The method of claim 102 , wherein said chemical ligation of said first building block tag and said chemical ligation of said second building block tag comprise orthogonal chemically co-reactive pairs for ligating successive building block tags,
wherein said orthogonal chemically co-reactive pairs comprises (a) an optionally substituted alkynyl group and an optionally substituted azido group; and (b) a phosphorothioate group and an iodo group.
105 . The method of claim 102 , wherein:
(a) said method further comprises separating said complex from any unreacted tag or unreacted headpiece before any one of binding steps (ii)-(v);
(b) said method further comprises purifying said complex before any one of binding steps (ii)-(v); and/or
(c) said method further comprises binding one or more additional building block tags to said complex and binding one or more additional components to said complex.
106 . The method of claim 105 , wherein said method further comprises binding one or more additional building block tags to said complex and binding one or more additional components to said complex,
wherein said binding one or more additional building block tags comprises chemical ligation of one or more additional building block tags using one or more chemically co-reactive pairs for ligating successive building block tags.
107 . The method of claim 106 , wherein said chemical ligation of one or more additional building block tags comprises orthogonal chemically co-reactive pairs for ligating successive building block tags,
wherein said orthogonal chemically co-reactive pairs comprises (a) an optionally substituted alkynyl group and an optionally substituted azido group; and (b) a phosphorothioate group and an iodo group.
108 . The method of claim 102 , wherein said complex, said headpiece, said first building block tag, said second building block tag, and/or said one or more additional building block tags, if present, comprises a modified phosphate group between the terminal nucleotide at the 3′-terminus and the nucleotide adjacent to said terminal nucleotide.
109 . The method of claim 1 , wherein said headpiece comprises a hairpin structure.
110 . The method of claim 1 , wherein:
(a) said headpiece, said first building block tag, said second building block tag, and/or said one or more additional building block tags, if present, comprises from 5 to 20 nucleotides;
(b) said headpiece, said first building block tag, said second building block tag, and/or said one or more additional building block tags, if present, further comprises a first library-identifying sequence;
(c) said method further comprises binding a first library-identifying tag to said complex;
(d) said headpiece, said first building block tag, said second building block tag, and/or said one or more additional building block tags, if present, further comprises a use sequence and/or an origin sequence;
(e) said method further comprises binding a use tag and/or an origin tag to said complex; and/or
(f) said method further comprises binding a tailpiece to said complex.