Modified oligonucleotides for mismatch discrimination

Claim 2

A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, a covalently attached minor groove binder, at least one covalently attached reporter group, wherein said reporter group is a fluorophore, and an attached quencher.

Claim 3

A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

Claim 4

A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

Claim 5

A modified oligonucleotide of claim 1 comprising from 4 to 70 bases.

Claim 6

A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, wherein at least one base of said at least two bases is an unsubstituted pyrazolo[3,4-d]pyrimidine base having the formula: and at least one of said at least two bases is a 3-substituted pyrazolo[3,4-d]pyrimidine base having the formula: wherein each of said X 1 and X 2 groups is a member independently selected from the group consisting of H, OH, NH 2 and a protected amino group; and each of said R 1 groups is a member independently selected from the group consisting of (C 3 –C 7 ) alkynyl, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, —O—(C 1 –C 12 )alkyl, —O—(C 2 –C 12 )alkenyl, —O—(C 2 –C 12 )alkynyl, —S—(C 1 –C 12 )alkyl, —S—(C 2 –C 12 )alkenyl, —S—(C 2 –C 12 )alkynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl, aryl(C 2 –C 12 )alkenyl, aryl(C 2 –C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN, —CONH 2 and protected forms thereof.

Claim 7

A modified oligonucleotide comprising at least two bases selected from the group consisting of unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidine bases, wherein at least one base of said at least two bases is an unsubstituted pyrazolo[3,4-d]pyrimidine base having the formula: and at least one of said at least two bases is a 3-substituted pyrazolo[3,4-d]pyrimidine base having the formula: wherein each of said X 1 and X 2 groups is a member independently selected from the group consisting of H, OH, NH 2 and a protected amino group; and each of said R 1 groups is a member independently selected from the group consisting of (C 3 –C 7 ) alkynyl, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, —O—(C 1 –C 12 )alkyl, —O—(C 2 –C 12 )alkenyl, —O—(C 2 –C 12 )alkynyl, —S—(C 1 –C 12 )alkyl, —S—(C 2 –C 12 )alkenyl, —S—(C 2 –C 12 )alkynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl, aryl(C 2 –C 12 )alkenyl, aryl(C 2 –C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN, —CONH 2 and protected forms thereof.

Claim 8

A modified oligonucleotide of claim 1 , having the formula: R 2 represents a first end of said modified oligonucleotide; R 1 represents a second end of said modified oligonucleotide; the subscript n is an integer of from 4 to 70; each B is a member independently selected from the group consisting of adenine, thymine, cytosine, guanine, uracil, a pyrazolo[3,4-d]pyrimidine and a 3-substituted pyrazolo[3,4-d]pyrimidine; and each M is a member selected from the group consisting of an oligomer-forming sugar and a peptide-nucleic acid-forming amino acid.

Claim 9

A modified oligonucleotide of claim 5 , wherein at least one M is a non-natural oligomer-forming sugar.

Claim 10

A modified oligonucleotide comprising at least one 5-substituted pyrimidine base and at least one unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine base, providing that the pyrimidine base is other than a thiopyrimidine.

Claim 11

A modified oligonucleotide comprising at least one 5-substituted pyrimidine base and at least one unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine base, providing that the pyrimidine base is other than a thiopyrimidine.

Claim 12

A modified oligonucleotide of claim 7 , further comprising a covalently attached minor groove binder.

Claim 13

A modified oligonucleotide of claim 7 , further comprising at least one covalently attached reporter group.

Claim 14

A modified oligonucleotide of claim 7 , further comprising at least one covalently attached quencher.

Claim 15

A modified oligonucleotide of claim 9 , wherein said reporter group is a fluorophore.

Claim 16

A modified oligonucleotide of claim 9 , wherein said reporter group is a fluorophore and said modified oligonucleotide further comprises an attached quencher.

Claim 17

A modified oligonucleotide of claim 7 , comprising from 4 to 70 bases.

Claim 18

A modified oligonucleotide of claim 7 , comprising from 4 to 70 bases and further comprising an attached minor groove binder.

Claim 19

A modified oligonucleotide of claim 7 , comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

Claim 20

A modified oligonucleotide of claim 8 comprising from 4 to 70 bases and further comprising an attached fluorophore and a quencher.

Claim 21

A modified oligonucleotide of claim 7 , wherein said at least one 5-substituted pyrimidine base is selected from the group consisting of: and said at least one unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine base is selected from the group consisting of: wherein each of said X 1 , X 2 and X 3 groups is a member independently selected from the group consisting of H, OH, NH 2 and a protected amino group; and each of said R 1 and R 4 groups is a member independently selected from the group consisting of (C 3 –C 7 ) alkynyl, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, —O—(C 1 –C 12 )alkyl, —O—(C 2 –C 12 )alkenyl, —O—(C 2 –C 12 )alkynyl, —S—(C 1 –C 12 )alkyl, —S—(C 2 –C 12 )alkenyl, —S—(C 2 –C 12 )alkynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl, aryl(C 2 –C 12 )alkenyl, aryl(C 2 –C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN, —CONH 2 and protected forms thereof.

Claim 22

A modified oligonucleotide of claim 7 , having the formula: R 2 represents a first end of said modified oligonucleotide; R 3 represents a second end of said modified oligonucleotide; the subscript n is an integer of from 4 to 70; each B is a member independently selected from the group consisting of adenine, thymine, cytosine, guanine, uracil, a 5-substituted pyrimidine and a 3-substituted pyrazolo[3,4-d]pyrimidine; and each M is a member selected from the group consisting of an oligomer-forming sugar and a peptide-nucleic acid-forming amino acid.

Claim 23

A modified oligonucleotide of claim 18 , wherein at least one M is a locked oligomer-forming sugar.

Claim 24

A modified oligonucleotide comprising from about 4 to about 70 bases and an attached minor groove binder, wherein at least one of said bases is replaced by a 5-substituted pyrimidine.

Claim 25

A modified oligonucleotide comprising from about 4 to about 70 bases and an attached minor groove binder, wherein at least one of said bases is replaced by a 5-substituted pyrimidine.

Claim 26

A modified oligonucleotide of claim 20 , further comprising an attached reporter group.

Claim 27

A modified oligonucleotide of claim 21 , wherein said reporter group is a fluorophore and said modified oligonucleotide further comprises an attached quencher.

Claim 28

A modified oligonucleotide of claim 22 , comprising of from about 4 to about 20 bases.

Claim 29

A modified oligonucleotide of claim 23 , wherein said fluorophore is attached at the 5′-terminus and said quencher is attached at the 3′-terminus.

Claim 30

A modified oligonucleotide of claim 23 , wherein said fluorophore is attached at the 3′-terminus and said quencher is attached at the 5′-terminus.

Claim 31

A modified oligonucleotide of claim 20 , wherein said minor groove binder has a formula selected from the group consisting of: wherein the subscript m is an integer of from 2 to 5; the subscript r is an integer of from 2 to 10; and each R a and R b is independently a linking group to said modified oligonucleotide, H, —OR c , —NR c R d , —COOR c and —CONR c R d wherein each R c and R d is selected from the group consisting of H, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, (C 1 –C 12 )alkyl, (C 2 –C 12 )alkenyl, (C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl and aryl.

Claim 32

A modified oligonucleotide of claim 21 , wherein said reporter group is selected from the group consisting of a resorufin dye, a coumarin dye, a rhodamine dye, a cyanine dye, a BODIPY dye, a fluorescein dye and a pyrene.

Claim 33

A modified oligonucleotide of claim 22 , wherein said reporter group is selected from the group consisting of resorufin dye, a coumarin dye, a rhodamine dye, a cyanine dye, a BODIPY dye, a fluorescein dye and a pyrene, and said quencher is selected from the group consisting of non-fluorescent quenchers, 1-aza-1,2-diphenylethene derivatives and rhodamine derivatives.

Claim 34

A modified oligonucleotide of claim 20 , wherein said modified base is selected from the group consisting of wherein X 1 and X 2 are each independently selected from the group consisting of H, NH 2 , OH and SH; and R 5 is a member selected from the group consisting of (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, —O—(C 1 –C 12 )alkyl, —O—(C 2 –C 12 )alkenyl, —O—(C 2 –C 12 )alkynyl, —S—(C 1 –C 12 )alkyl, —S—(C 2 –C 12 )alkenyl, —S—(C 2 –C 12 )alkynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl, aryl(C 2 –C 12 )alkenyl, aryl(C 2 –C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN, —CONH 2 and protected forms thereof.

Claim 35

A modified oligonucleotide of claim 29 , wherein said heterocyclyl and aryl groups are selected from the group consisting of phenyl, tolyl, pyridyl, thiazolyl, imidazolyl, furanyl, oxazolyl, thienyl, pyrrolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, indolyl, triazinyl, pyrimidinyl and naphthyl.

Claim 36

A modified oligonucleotide comprising from about 4 to about 70 bases and an attached minor groove binder, wherein at least one of said bases is replaced by a modified base selected from the group consisting of wherein each of said X 1 and X 2 groups is a member independently selected from the group consisting of H, OH, NH 2 and a protected amino group; and R 1 is a member selected from the group consisting of (C 3 –C 7 ) alkynyl, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, —O—(C 1 –C 12 )alkyl, —O—(C 2 –C 12 )alkenyl, —O—(C 2 –C 12 )alkynyl, —S—(C 1 –C 12 )alkyl, —S—(C 2 –C 12 )alkenyl, —S—(C 2 –C 12 )alkynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl, aryl(C 2 –C 12 )alkenyl, aryl(C 2 –C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN, —CONH 2 and protected forms thereof.

Claim 37

A modified oligonucleotide comprising from about 4 to about 70 bases and an attached minor groove binder, wherein at least one of said bases is replaced by a modified base selected from the group consisting of wherein each of said X 1 and X 2 groups is a member independently selected from the group consisting of H, OH, NH 2 and a protected amino group; and R 1 is a member selected from the group consisting of (C 3 –C 7 ) alkynyl, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, —O—(C 1 –C 12 )alkyl, —O—(C 2 –C 12 )alkenyl, —O—(C 2 –C 12 )alkynyl, —S—(C 1 –C 12 )alkyl, —S—(C 2 –C 12 )alkenyl, —S—(C 2 –C 12 )alkynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl, aryl(C 2 –C 12 )alkenyl, aryl(C 2 –C 12 )alkynyl, aryl, heterocyclyl, halogen, —CN, —CONH 2 and protected forms thereof.

Claim 38

A modified oligonucleotide of claim 31 , wherein said heterocyclyl and aryl groups are selected from the group consisting of phenyl, tolyl, pyridyl, thiazolyl, imidazolyl, furanyl, oxazolyl, thienyl, pyrrolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, indolyl, triazinyl, pyrimidinyl and naphthyl.

Claim 39

A modified oligonucleotide of claim 30 , wherein said minor groove binder has a formula selected from the group consisting of: wherein the subscript m is an integer of from 2 to 5; the subscript r is an integer of from 2 to 10; and each R a and R b is independently a linking group to said modified oligonucleotide, H, —OR c , —NR c R d , —COOR c and —CONR c R d wherein each R c and R d is selected from the group consisting of H, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, (C 1 –C 12 )alkyl, (C 2 –C 12 )alkenyl, (C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl and aryl.

Claim 40

A modified oligonucleotide of claim 32 , wherein said minor groove binder has a formula selected from the group consisting of: wherein the subscript n is an integer of from 2 to 5; the subscript r is an integer of from 2 to 10; and each R a and R b is independently a linking group to said modified oligonucleotide, H, —OR c , —NR c R d , —COOR c and —CONR c R d wherein each R c and R d is selected from the group consisting of H, (C 1 –C 12 )heteroalkyl, (C 2 –C 12 )heteroalkenyl, (C 2 –C 12 )heteroalkynyl, (C 1 –C 12 )alkyl, (C 2 –C 12 )alkenyl, (C 2 –C 12 )alkynyl, aryl(C 1 –C 12 )alkyl and aryl.

Claim 41

A compound having the formula: wherein Z 1 is a member selected from the group consisting of H, F and OR a wherein R a is a member selected from the group consisting of H, (C 1 –C 8 )alkyl and a hydroxy protecting group; Z 2 is a member selected from the group consisting of H and (C 1 –C 8 )alkyl, or optionally Z 2 is combined with Z 1 for form a five- to seven-membered ring; Y 1 is a member selected from the group consisting of OH, a protected hydroxy group and O—P 1 , wherein P 1 is a phosphoramidite or H-phosphonate group; Y 2 is a member selected from the group consisting of OH, a protected hydroxy group and O—P 2 , wherein P 2 is a phosphoramidite, H-phosphonate, monophosphate, diphosphate or triphosphate, provided that Z 1 and Y 1 are not both OH; and B is a modified nucleotide selected from the group consisting of: wherein X 11 and X 12 are each independently selected from the group consisting of H, NH 2 and a protected amino group; each R 11 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl; and each R 12 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl, 3-(hydroxymethyl)-4-hydroxy-1-butynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl and heterocyclyl, with the proviso that R 12 is other than 2-pyridylethynyl.

Claim 42

A compound having the formula: wherein Z 1 is a member selected from the group consisting of H, F and OR a wherein R a is a member selected from the group consisting of H, (C 1 –C 8 )alkyl and a hydroxy protecting group; Z 2 is a member selected from the group consisting of H and (C 1 –C 8 )alkyl, or optionally Z 2 is combined with Z 1 for form a five- to seven-membered ring; Y 1 is a member selected from the group consisting of OH, a protected hydroxy group and O—P 1 , wherein P 1 is a phosphoramidite or H-phosphonate group; Y 2 is a member selected from the group consisting of OH, a protected hydroxy group and O—P 2 , wherein P 2 is a phosphoramidite, H-phosphonate, monophosphate, diphosphate or triphosphate, provided that Z 1 and Y 1 are not both OH; and B is a modified nucleotide selected from the group consisting of: wherein X 11 and X 12 are each independently selected from the group consisting of H, NH 2 and a protected amino group; each R 11 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl; and each R 12 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl, 3-(hydroxymethyl)-4-hydroxy-1-butynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl and heterocyclyl, with the proviso that R 12 is other than 2-pyridylethynyl.

Claim 43

A compound of claim 35 , wherein B is

Claim 44

A compound of claim 35 , wherein B is

Claim 45

A compound of claim 35 , wherein B is

Claim 46

A compound of claim 35 , wherein B is

Claim 47

A compound of claim 36 , wherein X 11 and X 12 are each NH 2 .

Claim 48

A compound of claim 40 , wherein Y 1 is O—P 1 , Y 2 is a protected hydroxy, Z 1 is H, R 12 is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

Claim 49

A compound of claim 41 , wherein Y 1 is —O—[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y 2 is —O— (4,4′-dimethoxytrityl).

Claim 50

A compound of claim 36 , wherein X 11 is NH 2 and X 12 is H.

Claim 51

A compound of claim 43 , wherein Y 1 is O—P 1 , Y 2 is a protected hydroxy, Z 1 is H, R 12 is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

Claim 52

A compound of claim 44 , wherein Y 1 is —O-[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y 2 is —O-(4,4′-dimethoxytrityl).

Claim 53

A compound of claim 37 , wherein X 12 is H or NH 2 .

Claim 54

A compound of claim 46 , wherein Y 1 is O—P 1 , Y 2 is a protected hydroxy, Z 1 is H, R 12 is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

Claim 55

A compound of claim 47 , wherein X 1 is —O—[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and X 2 is —O— (4,4′-dimethoxytrityl).

Claim 56

A compound of claim 38 , wherein X 11 is NH 2 .

Claim 57

A compound of claim 49 , wherein Y 1 is O—P 1 , Y 2 is a protected hydroxy, Z 1 is H, R 11 is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

Claim 58

A compound of claim 50 , wherein Y 1 is —O—[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y 2 is —O—(4,4′-dimethoxytrityl).

Claim 59

A compound of claim 39 , wherein Y 1 is O—P 1 , Y 2 is a protected hydroxy, Z 1 is H, and R 11 is selected from the group consisting of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl.

Claim 60

A compound of claim 52 , wherein Y 1 is —O—[(2-cyanoethyl) N,N-diisopropylphosphoramidite] and Y 2 is —O— (4,4′-dimethoxytrityl).

Claim 61

An oligonucleotide having the formula: wherein each Z 1 a member independently selected from the group consisting of H, F and OR a wherein R a is a member selected from the group consisting of H, (C 1 –C 8 )alkyl and a hydroxy protecting group; each Z 2 is a member selected from the group consisting of H and (C 1 –C 8 )alkyl, or optionally Z 2 and Z 1 on one or more of the same furanose rings are combined to form a five- to seven-membered ring; each Z 3 is selected from the group consisting of O, S and NH; each Y is a member independently selected from the group consisting of P(O)OH, P(S)OH and P(O)CH 3 ; the subscript n is an integer of from 1 to 98; W 1 and W 2 are each independently selected from the group consisting of H, a monophosphate, a diphosphate, a triphosphate and a minor groove binder-linking group moiety having an optionally attached reporter group; and each B is a member independently selected from the group consisting of adenine, guanine, cytosine, uridine and modified bases of the formula: wherein X 11 and X 12 are each independently selected from the group consisting of H, NH 2 and a protected amino group; each R 11 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl; and each R 12 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl, 3-(hydroxymethyl)-4-hydroxy-1-butynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl and heterocyclyl, with the proviso that R 12 is other than 2-pyridylethynyl; and with the further proviso that at least one of said Bs is selected from said modified bases, and optionally, one or more of said Bs has an attached minor groove binder-linking group moiety, reporter group or a combination thereof.

Claim 62

An oligonucleotide having the formula: wherein each Z 1 a member independently selected from the group consisting of H, F and OR a wherein R a is a member selected from the group consisting of H, (C 1 –C 8 )alkyl and a hydroxy protecting group; each Z 2 is a member selected from the group consisting of H and (C 1 –C 8 )alkyl, or optionally Z 2 and Z 1 on one or more of the same furanose rings are combined to form a five- to seven-membered ring; each Z 3 is selected from the group consisting of O, S and NH; each Y is a member independently selected from the group consisting of P(O)OH, P(S)OH and P(O)CH 3 ; the subscript n is an integer of from 1 to 98; W 1 and W 2 are each independently selected from the group consisting of H, a monophosphate, a diphosphate, a triphosphate and a minor groove binder-linking group moiety having an optionally attached reporter group; and each B is a member independently selected from the group consisting of adenine, guanine, cytosine, uridine and modified bases of the formula: wherein X 11 and X 12 are each independently selected from the group consisting of H, NH 2 and a protected amino group; each R 11 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl and 3-(hydroxymethyl)-4-hydroxy-1-butynyl; and each R 12 is independently selected from the group consisting of protected or unprotected forms of 3-hydroxyprop-1-ynyl, 3-aminoprop-1-ynyl, 3-methoxyprop-1-ynyl, 4-hydroxy-1-butynyl, 3-(hydroxymethyl)-4-hydroxy-1-butynyl, heterocyclyl(C 1 –C 12 )alkyl, heterocyclyl(C 2 –C 12 )alkenyl, heterocyclyl(C 2 –C 12 )alkynyl and heterocyclyl, with the proviso that R 12 is other than 2-pyridylethynyl; and with the further proviso that at least one of said Bs is selected from said modified bases, and optionally, one or more of said Bs has an attached minor groove binder-linking group moiety, reporter group or a combination thereof.

Claim 63

An oligonucleotide of claim 54 , wherein n is an integer of from 4 to 30.

Claim 64

An oligonucleotide of claim 54 , wherein at least one B is

Claim 65

An oligonucleotide of claim 54 , wherein at least one B is

Claim 66

An oligonucleotide of claim 54 , wherein at least one B is

Claim 67

An oligonucleotide of claim 54 , wherein at least one B is

Claim 68

An oligonucleotide of claim 54 , wherein W 1 is a minor groove binder-linking group moiety.

Claim 69

An oligonucleotide of claim 54 , wherein W 1 is a minor groove binder-linking group moiety having an attached reporter group.

Claim 70

An oligonucleotide of claim 54 , wherein W 2 is a minor groove binder-linking group moiety.

Claim 71

An oligonucleotide of claim 54 , wherein W 2 is a minor groove binder-linking group moiety having an attached reporter group.

Claim 72

An oligonucleotide of claim 54 , wherein at least one Z 3 is NH.

Claim 73

A modified oligonucleotide array, said array comprising a solid support and a plurality of attached oligonucleotides, wherein at least 50% of the oligonucleotides in said array contain a modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines.

Claim 74

A modified oligonucleotide array, said array comprising a solid support and a plurality of attached oligonucleotides, wherein at least 50% of the oligonucleotides in said array contain a modified base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines.

Claim 75

A modified oligonucleotide array of claim 65 , wherein said attached oligonucleotides have T m s within about 2° C. of each other and basepair lengths within about 2 bases of each other.

Claim 76

A modified oligonucleotide array of claim 65 , wherein said attached oligonucleotides have T m s within about 1° C. of each other and basepair lengths within about 2 bases of each other.

Claim 77

A modified oligonucleotide array of claim 65 , wherein said array comprises from about 10 to about 10,000 attached oligonucleotides, each having T m s within about 2° C. of each other and basepair lengths within about 2 bases of each other.

Claim 78

A modified oligonucleotide array of claim 65 , wherein said array comprises from about 10 to about 10,000 attached oligonucleotides, a portion of said attached oligonucleotides having a covalently attached minor groove binder.

Claim 79

A composition comprising a plurality of modified oligonucleotides having at least one base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and further comprising a minor groove binder and an attached fluorophore wherein each of said plurality of oligonucleotides comprises an attached fluorophore and an attached quencher.

Claim 80

A composition comprising a plurality of modified oligonucleotides having at least one base selected from the group consisting of unsubstituted pyrazolo[3,4-d]pyrimidines, 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and further comprising a minor groove binder and an attached fluorophore wherein each of said plurality of oligonucleotides comprises an attached fluorophore and an attached quencher.

Claim 81

A composition of claim 70 , wherein each of said modified oligonucleotides are from 4 to 30 bases in length and have T m s that are within about 2° C. of each other.

Claim 82

A composition of claim 71 , wherein said plurality is from about 6 to about 100.

Claim 83

A compound of claim 35 wherein one of Y 1 and Y 2 is a phosphoramidite group.

Claim 84

A compound of claim 73 wherein the other of Y 1 and Y 2 is a protected hydroxy group.

Claim 85

A compound of claim 74 wherein the protected hydroxy group is a 4,4′-dimethoxytrityloxy group.

Claim 86

A compound of claim 73 wherein the phosphoramidite group is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

Claim 87

A compound of claim 75 wherein the phosphoramidite group is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

Claim 88

A compound of claim 73 wherein B is

Claim 89

A compound of claim 78 wherein X 11 and X 12 are protected amino groups.

Claim 90

A compound of claim 79 wherein one of Y 1 and Y 2 is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite and the other of Y 1 and Y 2 is a 4,4′-dimethoxytrityloxy group.

Claim 91

A compound of claim 79 wherein one or both of the protected amino groups comprises a benzoyl analogue protecting group.

Claim 92

A compound of claim 73 wherein B is

Claim 93

A compound of claim 82 wherein X 12 is a protected amino group.

Claim 94

A compound of claim 83 wherein X 12 is —N═CH—N(CH 3 ) 2 .

Claim 95

A compound of claim 84 wherein one of Y 1 and Y 2 is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite and the other of Y 1 and Y 2 is a 4,4–dimethoxytrityloxy group.

Claim 96

A compound of claim 73 wherein B is

Claim 97

A compound of claim 86 wherein the phosphoramidite group is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

Claim 98

A compound of claim 73 wherein B is

Claim 99

A compound of claim 88 wherein the phosphoramidite group is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

Claim 100

A compound of claim 73 wherein B is and X 11 and X 12 are protected amino groups.

Claim 101

A compound of claim 90 wherein the other of Y 1 and Y 2 is a protected hydroxy group.

Claim 102

A compound of claim 91 wherein the phosphoramidite group is —O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite.

Claim 103

A compound of claim 92 wherein the other of Y 1 and Y 2 is a 4,4′-dimethoxytrityloxy group.

Claim 104

A compound of claim 91 wherein one or both of the protected amino groups comprises a benzoyl analogue protecting group.

Claim 105

A compound of claim 73 wherein Z 1 is hydrogen, Y 1 is OH and Y 2 is OH.

Claim 106

A compound of claim 73 wherein Z 1 and Z 2 are combined to form a five-to-seven-membered ring.

Claim 107

An oligonucleotide of claim 54 wherein Z 1 and Z 2 on one or more of the same furanose rings are combined to form a five-to seven-membered ring.

Claim 108

A modified oligonucleotide of claim 29 wherein X 1 and X 2 are each independently selected from the group consisting of H, NH 2 , and OH.