Tmleveling methods
AssigneeEpoch Biosciences, Inc.
InventorsRobert O. Dempcy, Alexander A. Gall, Sergey G. Lokhov, Irina A. Afonina, Michael J. Singer, Igor V. Kutyavin, Nicolaas M.J. Vermeulen
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Abstract
Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.
Claims109
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 modified oligonucleotide comprising at least one 5-substituted pyrimidine base and at least one unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine base.
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 5-substituted pyrimidines and unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines.
A compound having the formula:
- 48
- wherein
- Z1 is a member selected from the group consisting of H, F and ORa wherein Ra is a member selected from the group consisting of H, (C1-C8)alkyl and a hydroxy protecting group;
- Z2 is a member selected from the group consisting of H and (C1-C8)alkyl, or optionally Z2 is combined with Z1 for form a five- to seven-membered ring;
- Y1 is a member selected from the group consisting of OH, a protected hydroxy group and O—P1, wherein P1 is a phosphoramidite or H-phosphonate group;
- Y2 is a member selected from the group consisting of OH, a protected hydroxy group and O—P2, wherein P2 is a phosphoramidite, H-phosphonate, monophosphate, diphosphate or triphosphate;
- and
- B is a modified nucleotide selected from the group consisting of:
- 49
- wherein
- X11 and X12 are each independently selected from the group consisting of H, NH2 and a protected amino group;
- each R11 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 R12 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(C1-C12)alkyl, heterocyclyl(C2-C12)alkenyl, heterocyclyl(C2-C12)alkynyl and heterocyclyl, with the proviso that R12 is other than 2-pyridylethynyl.
An oligonucleotide having the formula:
- 54
- wherein
- each Z1 a member independently selected from the group consisting of H, F and ORa wherein Ra is a member selected from the group consisting of H, (C1-C8)alkyl and a hydroxy protecting group;
- each Z is a member selected from the group consisting of H and (C1-C8)alkyl, or optionally Z2 and Z1 on one or more of the same furanose rings are combined to form a five- to seven-membered ring;
- each Z3 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)CH3;
- the subscript n is an integer of from 1 to 98;
- W1 and W2 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:
- 55
- wherein
- X11 and X12 are each independently selected from the group consisting of H, NH2 and a protected amino group;
- each R11 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 R12 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(C1-C12)alkyl, heterocyclyl(C2-C12)alkenyl, heterocyclyl(C2-C12)alkynyl and heterocyclyl, with the proviso that R12 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.
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.
A composition comprising a plurality of modified oligonucleotides having at l east on e 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 having an attached fluorophore.
A method for distinguishing polynucleotides with related sequences, the method comprising:
- (a) contacting a modified oligonucleotide having a defined sequence comprising at least one 3-substituted pyrazolo[3,4-d]pyrimidine or 5-substituted pyrimidine in place of a purine or pyrimidine base with at least two polynucleotides, wherein one of the polynucleotides has a target sequence that is perfectly complementary to the modified oligonucleotide and at least one of the other polynucleotides has a target sequence with at least one base mismatch;
- and
- (b) determining the degree of hybridization between the modified oligonucleotide and each of the polynucleotides.
A method for detecting the presence of a target sequence in a polynucleotide, the method comprising:
- (a) incubating a polynucleotide to be tested for the presence of the target sequence with a modified oligonucleotide having a sequence that is substantially complementary to the target sequence under hybridization conditions;
- and
- (b) identifying hybridized nucleic acids;
- wherein said modified oligonucleotide comprises at least one 3-substituted pyrazolo[3,4-d]pyrimidine in place of a purine residue.
A method for primer extension, the method comprising incubating a polynucleotide containing a target sequence with one or more oligonucleotide primers complementary to the target sequence, in the presence of a polymerizing enzyme and nucleotide substrates under conditions favorable for polymerization;
- wherein at least one of the oligonucleotide primers contains a modified base selected from the group consisting of an unsubstituted pyrazolo[3,4-d]pyrimidine, a 3-substituted pyrazolo[3,4-d]pyrimidine and a 5-substituted pyrimidine base, in place of a purine or pyrimidine base.
A method for determining the nucleotide sequence of a polynucleotide, the method comprising:
- (a) incubating the polynucleotide with a modified oligonucleotide array under hybridization conditions;
- and
- (b) determining to which of the modified oligonucleotides in the array the polynucleotide hybridizes;
- wherein a plurality of the modified oligonucleotides comprise at least one 3-substituted pyrazolo[3,4-d]pyrimidine in place of a purine base.
A method in accordance with claim101, wherein said array comprises from 10 to 100,000 different modified oligonucleotides.
A method for determining the nucleotide sequence of a target sequence in a polynucleotide, the method comprising:
- (a) contacting a polynucleotide comprising the target sequence with at least two oligonucleotides of known sequence wherein one or more purine residues of the oligonucleotides are replaced by a 3-substituted pyrazolo[3,4-d]pyrimidine, and wherein one of the at least two oligonucleotides has a sequence that is perfectly complementary to the target sequence and at least one other of the oligonucleotides has a related target sequence and incubating each of the oligonucleotides with the polynucleotide under hybridization conditions;
- and
- (b) determining the degree of hybridization between each of the oligonucleotides and the polynucleotide.
A method for examining gene expression in a cell, the method comprising:
- (a) incubating a population of polynucleotides representative of the genes expressed in the cell with an oligonucleotide array comprising a plurality of modified oligonucleotides of different sequences under hybridization conditions, and
- (b) determining which of the modified oligonucleotides in the array become hybridized to polynucleotides;
- wherein said modified oligonucleotides comprise at least one 3-substituted pyrazolo[3,4-d]pyrimidine in place of a purine.
A method for identifying a mutation in a target sequence of a gene of interest, the method comprising:
- (a) incubating a polynucleotide comprising the target sequence with an array of oligonucleotides of different sequences, wherein the different sequences include the wild-type target sequence and different mutant target sequences, under hybridization conditions;
- and
- (b) determining which of the oligonucleotides in the array become hybridized to the polynucleotide;
- wherein one or more purine residues in each of the oligonucleotides are replaced with a 3-substituted pyrazolo[3,4-d]pyrimidine.
Dependencies are inferred from the claim text.