US6683173B2
Expired / Lapsed
US
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Tm leveling methods
Assignee: DRUG ROYALTY TRUST 9
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Filing: 2001-12-21
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Granted: 2004-01-27
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Expires: 2021-12-21
Abstract
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.
Inventors
- 👤 Robert O. Dempcy
- 👤 Alexander A. Gall
- 👤 Sergey G. Lokhov
- 👤 Irina A. Afonina
- 👤 Michael J. Singer
- 👤 Igor V. Kutyavin
- 👤 Nicolaas M. J. Vermeulen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 23:44
CPC Classifications
C; C07; C07H; C07H21/00; C07H19/00; C07H19/02; C07H19/04; C07H19/06; C07H19/10; C07H19/16; C07H19/20; C07H19/23; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6832; G; G16; G16B; G16B30/00; C07B; C07B2200/00; C07B2200/11; C40; C40B; C40B40/00
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
93 words
A method for designing an oligonucleotide-MGB conjugate having a leveled T m , said method comprising: a) providing an oligonucleotide with a known sequence having n bases and a known T m ; b) determining i.) the sequence dependent thermodynamic contribution of a minor groove binder (MGB) attached to said oligonucleotide, and ii.) the sequence dependent thermodynamic contribution of said oligonucleotide; and c) designing an oligonucleotide-MGB conjugate using said thermodynamic contributions, wherein said oligonucleotide-MGB conjugate has fewer than n bases and a T m that is level with said known T m .
Claim 2
Independent Claim
93 words
A method for designing an oligonucleotide-MGB conjugate having a leveled T m , said method comprising: a) providing an oligonucleotide with a known sequence having n bases and a known T m ; b) determining i.) the sequence dependent thermodynamic contribution of a minor groove binder (MGB) attached to said oligonucleotide, and ii.) the sequence dependent thermodynamic contribution of said oligonucleotide; and c) designing an oligonucleotide-MGB conjugate using said thermodynamic contributions, wherein said oligonucleotide-MGB conjugate has fewer than n bases and a T m that is level with said known T m .
Claim 3
Dependent Claim
44 words
The method of claim 1 , wherein the thermodynamic contribution of said MGB attached to said oligonucleotide is determined by comparing the duplex stability of a first duplex of said oligonucleotide to the stability a second duplex of said oligonucleotide with an attached MGB.
Claim 4
Dependent Claim
17 words
The method of claim 1 , wherein said known T m of said oligonucleotide is empirically determined.
Claim 5
Dependent Claim
20 words
The method of claim 1 , wherein said known T m of said oligonucleotide is determined using nearest neighbor parameters.
Claim 6
Dependent Claim
20 words
The method of claim 1 , wherein said known sequence having n bases contributes to said known T m .
Claim 7
Dependent Claim
13 words
The method of claim 1 , wherein said oligonucleotide is derived from Genbank.
Claim 8
Dependent Claim
125 words
The method of claim 1 , wherein said MGB 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 9
Dependent Claim
18 words
The method of claim 1 , wherein said MGB is attached to the 3′-end of said oligonucleotide-MGB conjugate.
Claim 10
Dependent Claim
18 words
The method of claim 1 , wherein said MGB is attached to the 5′-end of said oligonucleotide-MGB conjugate.
Claim 11
Dependent Claim
22 words
The method of claim 1 , wherein said MGB is attached to other than the 3′-end or 5′-end of said oligonucleotide-MGB conjugate.
Claim 12
Dependent Claim
25 words
The method of claim 1 , wherein said T m of said oligonucleotide-MGB conjugate is within about ±5° C. of said known T m .
Claim 13
Dependent Claim
16 words
The method of claim 1 , wherein said oligonucleotide-MGB conjugate has at least one modified base.
Claim 14
Dependent Claim
41 words
The method of claim 12 , wherein said oligonucleotide-MGB conjugate has at least one modified base wherein said base is selected from the group consisting of a base attached to an amino acid, a locked-nucleic acid base and a universal base.
Claim 15
Dependent Claim
53 words
The method of claim 12 , wherein said at least one modified base is a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH 2 ) 2 PPPA, (NH 2 ) 2 PPPAOH, (NH 2 ) 2 BuPPAOH, (NH 2 ) 2 PPAI, and HOBuPPG.
Claim 16
Dependent Claim
33 words
The method of claim 1 , further comprising repeating step (c) to generate a plurality of different oligonucleotide-MGB conjugates each having a T m that is level with said known T m .
Claim 17
Dependent Claim
23 words
The method of claim 15 , wherein each of said plurality of different oligonucleotide-MGB conjugates are within about 1-2 bases from each other.
Claim 18
Dependent Claim
18 words
The method of claim 15 , wherein said plurality of different oligonucleotide-MGB conjugates are immobilized on a substrate.
Claim 19
Dependent Claim
18 words
The method of claim 15 , wherein said plurality of different oligonucleotide-MGB conjugates are synthesized on a substrate.
Claim 20
Dependent Claim
29 words
The method of claim 17 , wherein said substrate is a member selected from the group consisting of glass, polystyrene, nylon, nitrocellulose, glass, silicon wafers, optical fibers and plastic.
Claim 21
Independent Claim
86 words
A method for designing a modified oligonucleotide having a leveled T m , said method comprising: a) providing an oligonucleotide with a known sequence having n bases and a known T m ; and b) designing a modified oligonucleotide having at least one modified base using the sequence dependent thermodynamic contribution of said at least one modified base determined using nearest neighbor parameters, wherein said modified oligonucleotide has fewer than n bases and has a T m that is level with said known T m .
Claim 22
Independent Claim
86 words
A method for designing a modified oligonucleotide having a leveled T m , said method comprising: a) providing an oligonucleotide with a known sequence having n bases and a known T m ; and b) designing a modified oligonucleotide having at least one modified base using the sequence dependent thermodynamic contribution of said at least one modified base determined using nearest neighbor parameters, wherein said modified oligonucleotide has fewer than n bases and has a T m that is level with said known T m .
Claim 23
Dependent Claim
13 words
The method of claim 20 , wherein said oligonucleotide is derived from Genbank.
Claim 24
Dependent Claim
37 words
The method of claim 20 , wherein said at least one modified base is a member selected from the group consisting of a base attached to an amino acid, a locked-nucleic acid base and a universal base.
Claim 25
Dependent Claim
53 words
The method of claim 20 , wherein said at least one modified base is a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH 2 ) 2 PPPA, (NH 2 ) 2 PPPAOH, (NH 2 ) 2 BuPPAOH, (NH 2 ) 2 PPAI, and HOBuPPG.
Claim 26
Dependent Claim
15 words
The method of claim 20 , wherein said modified oligonucleotide has a MGB attached thereto.
Claim 27
Dependent Claim
125 words
The method of claim 24 , wherein said MGB 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 28
Dependent Claim
18 words
The method of claim 24 , wherein said MGB is attached to the 3′-end of said modified oligonucleotide.
Claim 29
Dependent Claim
18 words
The method of claim 24 , wherein said MGB is attached to the 5′-end of said modified oligonucleotide.
Claim 30
Dependent Claim
22 words
The method of claim 24 , wherein said MGB is attached to other than the 3′-end or 5′-end of said oligonucleotide-MGB conjugate.
Claim 31
Dependent Claim
19 words
The method of claim 20 , wherein said at least one modified base decreases stability of said modified oligonucleotide.
Claim 32
Dependent Claim
19 words
The method of claim 20 , wherein said at least one modified base increases stability of said modified oligonucleotide.
Claim 33
Dependent Claim
33 words
The method of claim 20 , further comprising repeating step (b) to generate a plurality of different modified oligonucleotides each having a T m that is level with said known T m .
Claim 34
Dependent Claim
35 words
The method of claim 31 , wherein said plurality of different modified oligonucleotides each having a T m that is level with said known T m has at least one oligonucleotide without a modified base.
Claim 35
Dependent Claim
17 words
The method of claim 31 , wherein said plurality of different oligonucleotides are immobilized on a substrate.
Claim 36
Dependent Claim
27 words
The method of claim 20 , wherein designing said modified oligonucleotide further comprises using the sequence dependent thermodynamic contribution of the natural bases in said modified oligonucleotide.
Claim 37
Dependent Claim
17 words
The method of claim 20 , wherein said oligonucleotide and said modified oligonucleotide have similar hybridization properties.
Claim 38
Dependent Claim
18 words
The method of claim 31 , wherein said plurality of different modified oligonucleotides have MGB conjugates attached thereto.
Claim 39
Dependent Claim
19 words
The method of claim 36 , wherein said plurality of different modified oligonucleotide-MGB conjugates are synthesized on a substrate.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2007-01-26 | 2007-07-28 | 2008-01-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2011-01-26 | 2011-07-28 | 2012-01-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2015-01-26 | 2015-07-28 | 2016-01-27 | $7700.00 | Window Closed / Lapsed |