Phosphotriester-modified oligonucleotide primers for nucleic acid amplification
View Patent ↗The present invention provides methods and compositions for nucleic acid amplification. These methods involve the use of oligonucleotide primers in temperature dependent nucleic acid amplification reactions. In certain aspects, the methods are accomplished by use of certain modified oligonucleotide primers which provide utility in nucleic acid amplification. In preferred embodiments, the oligonucleotide primers are modified with particular chemical groups such as esters.
1. A method of amplifying nucleic acids, said method comprising:
amplifying nucleic acid using a modified oligonucleotide primer,
wherein said modified oligonucleotide primer comprises a modification group at one or two internucleotide linkages;
wherein said modification groups are at the n, n-1, n-2, or n-3 positions, wherein n is the 3′ terminal internucleotide linkage;
wherein the presence of said modification group impairs nucleic acid polymerase extension of said modified oligonucleotide primer;
wherein said modification group can be non-reversibly thermally dissociated; and
wherein said modified oligonucleotide primer has structure I as follows
wherein:
Nuc is a nucleoside within the primer sequence;
U and Z are independently O, S, Se, NR 9 , or CR 9 R 10 ;
R 9 and R 10 are each independently hydrogen or optionally substituted straight or branched hydrocarbyl having from 1-20 carbon atoms, wherein each may independently include at least one substituent selected from halo, oxo, hydroxyl, alkoxy, aryloxy, amino, amido or a detectable label;
Y is O, S or Se;
W is O, S, S(O), S(O) 2 , Se, C(O), C(S), C(O)NH, NH or NR 9 ; and
Q is a modification group comprising one or more thermally cleavable groups.
2. The method of claim 1 , wherein said modification group, Q, comprises one or more modification groups selected from the group consisting of
-L-X—R 1 (Formula I)
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms;
X is O, S, S(O), S(O) 2 , C(O), C(S) or C(O)NH; and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy and heteroaryl;
-L-S(O) k —R 1 (Formula Ib)
wherein:
k is an integer from 0-2;
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
Each R 1 is independently hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl;
-L-R 2 (Formula II)
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 2 is hydrogen, cyano, or an optionally substituted carbocycle, heterocycle, aryl or heteroaryl having between
5-10 atoms;
-L a -A-L b -B (Formula III)
wherein:
L a and L b are each independently selected from a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;
A is O, S, S(O), S(O) 2 , Se, CR 3 R 4 , NR 3 , C(O), C(S) or CNR 3 ;
B is C(O)R 3 , C(S)R 3 , C(O)NR 3 R 4 , OR 3 or SR 3 ; and
R 3 and R 4 are each independently hydrogen or straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl; and
-L a -D-L b -E-L c -F (Formula IV)
wherein:
L a , L b and L c are each independently selected from a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;
D is O, S, S(O), S(O) 2 , CR 5 R 6 , and NR 5 ;
E is O, S, S(O), S(O) 2 , CR 5 R 6 , and NR 6 ;
F is hydrogen, C(O)R 7 , C(S)R 7 , C(O)NR 7 R 8 , OR 7 and SR 7 ;
R 5 and R 6 can each independently be hydrogen, aryl, alkyl, halo, oxo, hydroxyl, alkoxy, aryloxy or amino, or R 5 and R 6 can cooperate to form a mono or bicyclic ring consisting 5-10 atoms and including D, R 5 , R 6 , E and L b , provided that when R 5 and R 6 cooperate to form a ring, n is from 0-2; and
R 7 and R 8 are each independently selected from the group consisting of aryl, alkyl, halo, oxo, hydroxyl, alkoxy, aryloxy, amino, amido, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aryloxy, and optionally substituted heteroaryl.
3. The method of claim 1 , wherein said modification group, Q, comprises:
-L-X—R 1 (Formula I)
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms;
X is O, S, S(O), S(O) 2 , C(O), C(S) or C(O)NH; and
R′ is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.
4. The method of claim 1 , wherein said modification group, Q, comprises:
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms;
and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.
5. The method of claim 1 , wherein said modification group, Q, comprises:
-L-S(O) k —R 1 (Formula Ib)
wherein:
k is an integer from 0-2;
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms,
which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.
6. The method of claim 1 , wherein said modification group, Q, comprises:
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
Each R 1 is independently hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.
7. The method of claim 1 , wherein said modification group, Q, comprises:
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 1 is hydrogen or a straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.
8. The method of claim 1 , wherein said modification group, Q, comprises:
-L-R 2 (Formula II)
wherein:
L is a straight or branched hydrocarbylene group having between 1-10 carbon atoms; and
R 2 is hydrogen, cyano, or an optionally substituted carbocycle, heterocycle, aryl or heteroaryl having between
5-10 atoms.
9. The method of claim 1 , wherein said modification group, Q, comprises:
-L a -A-L b -B (Formula III)
wherein:
L a and L b are each independently a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;
A is O, S, S(O), S(O) 2 , Se, CR 3 R 4 , NR 3 , C(O), C(S) or CNR 3 ;
B is C(O)R 3 , C(S)R 3 , C(O)NR 3 R 4 , OR 3 or SR 3 ; and
R 3 and R 4 are each independently hydrogen or straight or branched hydrocarbylene group having from 1-20 carbon atoms, which may optionally include at least one substituent selected from the group consisting of hydrogen, aryl, alkyl, halo, oxo, hydroxyl, alkoxy, amino, amido, cycloalkyl, heterocycloalkyl, aryl, aryloxy, and heteroaryl.
10. The method of claim 1 , wherein said modification group, Q, comprises:
-L a -D-L b -E-L c -F (Formula IV)
wherein:
L a , L b and L c are each independently selected from a bond or a straight or branched hydrocarbylene group having between 1-8 carbon atoms;
D is O, S, S(O), S(O) 2 , CR 5 R 6 , and NR 5 ;
E is O, S, S(O), S(O) 2 , CR 5 R 6 , and NR 6 ;
F is hydrogen, C(O)R 7 , C(S)R 7 , C(O)NR 7 R 8 , OR 7 and SR 7 ;
R 5 and R 6 can each independently be hydrogen, alkyl, aryl, halo, oxo, hydroxyl, alkoxy, aryloxy or amino, or R 5 and R 6 can cooperate to form a mono or bicyclic ring consisting 5-10 atoms and including D, R 5 , R 6 , E and L b , provided that when R 5 and R 6 cooperate to form a ring, n is from 0-2; and
R 7 and R 8 are each independently selected from the group consisting of hydrogen, alkyl, aryl, halo, oxo, hydroxyl, alkoxy, aryloxy, amino, amido, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aryloxy, and optionally substituted heteroaryl.
11. The method of claim 1 , wherein said modification group, Q, comprises one or more chemical moieties selected from the group consisting of 4-oxo-1-hexyl, 4-oxo-1-pentyl, 5-oxo-1-hexyl, 6-oxo-1-heptyl, 1-methyl-4-oxo-pentyl, 4-methylthio-1-butyl, 5-methyl-4-oxo-hexyl, 1-ethyl-4-oxo-pentyl, N-(2-hydroxyethyl)phthalimido, 2-(N-acetyl-N-methyl)aminoethyl, 2-(N-formyl-N-methyl)aminoethyl, 2-methyl-5-oxo-hexyl, 1,1-dimethyl-4-oxo-pentyl, 4-oxo-1-octyl, 4-oxo-1-tetradecyl, 4-oxo-1-eicosamyl, and 3-(N-tertbutylcarboxamido)-1-propyl.
12. The method according to claim 1 , wherein said modification group dissociates during the initial denaturation step of said amplification.
13. The method of claim 1 , wherein said modification group partially or entirely dissociates at a temperature between about 37° C.-95° C.
14. The method of claim 1 , wherein said modified oligonucleotide primer comprises a phosphotriester internucleotide linkage.
15. The method of claim 1 , wherein said modification group impairs hybridization of the oligonucleotide primer to a nucleic acid sequence.
16. The method of claim 1 , wherein t 1/2 of dissociation of said modification group is between about 0.1-120 minutes at 95° C.
17. The method of claim 1 , wherein the modification group dissociates between 50-95° C.
18. The method of claim 1 , wherein said nucleic acid amplification comprises a hot start polymerase.
19. The method of claim 1 wherein said modified oligonucleotide primer is at least 90% chirally pure.
20. The method of claim 1 , wherein said oligonucleotide primer comprises a detectable label.
21. The method of claim 1 , wherein said amplifying comprises polymerase chain reaction (PCR).
22. The method of claim 21 , wherein said PCR comprises multiplex PCR.
23. The method of claim 22 , wherein a first target comprises amplifying using unmodified primers and a second target is amplified using modified primers.
24. The method of claim 22 , wherein a first target comprises amplifying using one or more primers with a first modification group and a second target is amplified using one or more modified primers with a second modification group, wherein said first and second modification groups are different.
25. The method of claim 1 , wherein said amplifying comprises reverse transcriptase (RT).
26. The method of claim 25 , wherein said modification group dissociates at about 37-70° C.
27. The method of claim 25 , wherein said modification group dissociates at 42° C.
28. The method of claim 1 , wherein said amplifying comprises subsequent enzymatic reactions in a single tube.
29. The method of claim 28 , wherein said subsequent enzymatic reactions comprise reverse transcriptase (RT) as a first enzymatic reaction and polymerase chain reaction (PCR) as a second enzymatic reaction.
30. The method of claim 29 , wherein said RT reaction comprises amplifying with one or more unmodified primers and said PCR reaction comprises amplifying with one or more modified primers.
31. The method of claim 29 , wherein said RT reaction comprises amplifying with one or more of a first modified primer and said PCR reaction comprises amplifying with one or more of a second modified primer.
32. The method of claim 31 , wherein said one or more of a first modified primer comprises a modification group that dissociates at about 42° C., and wherein said one or more of a second modified primer comprises a modification group that dissociates at about 95° C.
33. The method of claim 1 , wherein said modified oligonucleotide primer comprises a modification group at the 3′ terminal internucleotide linkage.
34. The method of claim 33 , wherein said modified oligonucleotide primer further comprises a modification groups at the n-1 position; wherein n is the 3′ terminal internucleotide linkage.