Non-aggregating non-quenching oligomers comprising nucleotide analogs; methods of synthesis and use thereof
The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.
1 . A composition comprising:
wherein
R 1 and R 2 are independently —H, —OH, —OR 6 , —SH, —NH 2 or —NHR 7 ;
R 3 is —H—CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH;
R 6 is —H, —C 6 H 5 (benzyl) or a diphenylcarbamoyl (DPC) group;
R 7 is a protecting group selected from the group consisting of 2-N-dimethylvinyl, benzyloxycarbonyl (Cbz), monomethoxytrityl (MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyl, acetyl, and anisoyl (An) groups; and
X is ═CH— or N—.
2 . A composition comprising
wherein
R 1 and R 2 are independently —H, —OH, —OR 6 , —SH, —NH 2 or —NHR 7
R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1-12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH;
X is ═CH— or ═N—;
R 4 is —H or a protecting, group selected from the group consisting of tert-butyloxycarbonyl (tBoc), 4-methoxyphenyldiphenylmethyl (MMTr), isobutyryl (iBu) and 9-fluoronylmethyloxycarbonyl (Fmoc);
R 5 is —H or —C 6 F 4 H (TFP);
R 6 is —H, —C 6 H 5 (benzyl) or a diphenylcarbamoyl (DPC) group; and
R 7 is a protecting group selected from the group consisting of 2-N-dimethylvinyl, benzyloxycarbonyl (Cbz), monomethoxytrityl (MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyi, acetyl, and anisoyl (An) groups.
3 . The composition of claim 2 wherein:
R 1 is —OH;
R 2 is —NHCOCH(CH 3 ) 2 ;
R 3 is —H;
R 4 is MMTr; and
R 5 is —H.
4 . The composition of claim 2 wherein:
R 1 is —OH;
R 2 , is —N═CH—N(CH 3 ) 2 ;
R 3 is —H;
R 4 is MMTr; and
R 5 is —H.
5 . The composition of claim 2 wherein:
R 1 is —NHAn;
R 2 is —H;
R 3 is —H;
R 4 is MMTr; and
R 5 is —H.
6 . The composition of claim 2 wherein: R 1 is —N═CH—N(CH 3 ) 2 ;
R 2 is —H;
R 3 is —H;
R 4 is MMTr; and
R 5 is —H.
7 . The composition of claim 2 wherein: R 1 is —OH;
R 2 is —H;
R 3 is —H;
R 4 is MMTr; and
R 5 is —H.
8 . A method for synthesizing an oligomer, wherein the oligomer comprises one or more peptide nucleic acid residues, wherein the method comprises stepwise cycles of monomer addition to a growing oligomer chain that is optionally attached to a solid support, wherein he growing oligomer chain contains a blocked growing end, and wherein one or more of the monomer addition cycles comprises the following steps:
a) treatment of the growing chain under, conditions that de-block the growing end;
b) treatment of the composition of claim 2 with an activating agent, if R 5 is —H;
c) condensation of the products of steps (a) and (b);
d) optionally repeating the cycle of steps (a)-(c); and
e) at the completion of synthesis of the oligomer, exposure of the completed oligomer to conditions that remove all blocking groups and, if necessary, cleave the completed oligomer from the support.
9 . The method of claim 8 , wherein the oligomer is a peptide nucleic acid.
10 . The method of claim 8 , wherein the oligomer is a PNA/DNA chimera.
11 . An oligomer synthesized according. to the method of claim 8 .
12 . An oligomer according to claim 11 , wherein the oligomer is a peptide nucleic acid.
13 . An oligomer according to claim 11 , wherein the oligomer, is a PNA/DNA chimera.
14 - 26 . (canceled)
27 . A method for detecting a target sequence in a polynucleotide wherein the improvement comprises:
use of a probe, complementary to the target sequence, wherein one or more residues in the probe comprises abase analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and deazapurines,
such that the base analogues retain the base-pairing specificity of the bases for which they are substituted and reduce the tendency of the probe to self-associate.
28 . The method of claim 27 wherein the probe comprises DNA.
29 . The method of claim 27 wherein the probe comprises peptide nucleic acid (PNA).
30 . The method of claim 27 wherein the probe comprises a PNA/DNA chimera.
31 . The method of claim 27 , wherein the base analogue has the structure
wherein
R 1 and R 2 are independently —H, —OH, —SH, or —NH 2 ;
R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; and
X is ═CH— or N—.
32 . The method of claim 31 wherein one or more guanine bases in the probe are substituted by PPG.
33 . The method of claim 31 wherein one or more adenine bases in the probe are substituted by PPA.
34 . The method of claim 27 , wherein the probe further comprises a minor groove binder.
35 . The method of claim 27 , wherein the probe further comprises a fluorophore.
36 . The method of claim 35 , wherein the probe further comprises a fluorescence quencher.
37 . The method of claim 35 , wherein the probe further comprises a minor groove binder.
38 . The method of claim 36 , wherein the probe further comprises a minor groove binder.
39 . The method of claim 29 , wherein the probe further comprises a minor groove binder.
40 . The method of claim 29 , wherein the probe further comprises a fluorophore.
41 . The method of claim 40 , wherein the probe further comprises a fluorescence quencher.
42 . The method of claim 40 , wherein the probe further comprises a minor groove binder.
43 . The method of claim 41 , wherein the probe further comprises a minor groove binder.
44 . A method for the detection of a target sequence in a polynucleotide using a probe comprising a polymeric portion and a fluorescent portion, wherein the polymeric portion comprises a plurality of monomers; the improvement comprising
one or more of the monomers in the probe comprises a base analogue, wherein the base analogue is selected from the group consisting of pyrazolopyrimidines and deazapurines;
such that the base analogues retain the base-pairing specificity of the bases f which they. are substituted and reduce the tendency of the polymeric portion of the probe to quench emission from the fluorescent portion.
45 . The method of claim 44 wherein the polymeric portion of the probe comprises DNA.
46 . The method of claim 44 wherein the polymeric portion of the probe comprises peptide nucleic acid (PNA).
47 . The method of claim 44 wherein the polymeric portion of the probe comprises a PNA/DNA chimera.
48 . The method of claim 44 , wherein the base analogue has the structure
wherein
R 1 and R 2 are independently —H, —OH, —SH, or —NH 2 ;
R 3 is —H, —CN, halogen (F, Cl, Br or I), or —R 12 —Y, wherein R 12 is C 1 -C 12 alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH 2 or —SH; and
X is ═CH— or ═N—.
49 . The method of claim 48 wherein one or more guanine bases in the polymeric portion of the probe are substituted by PPG.
50 . The method of claim 49 wherein three or more consecutive guanine bases in the polymeric portion of the probe are substituted by PPG.
51 . The method of claim 49 , wherein a PPG residue is adjacent to the fluorescent portion of the probe.
52 . The method of claim 44 , wherein the probe: further comprises a quenching portion.
53 . The method of claim 51 , wherein the probe further comprises a quenching portion.
54 . The method of claim 44 , wherein the probe further comprises a minor groove binder.
55 . The method of claim 53 , wherein the probe further comprises a minor groove binder.
56 - 71 . (canceled)
72 . A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:
the use of the composition of claim 56 as a probe.
73 . A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:
the use of the composition of claim 58 as a probe.
74 . A method for detecting a target sequence in an amplification reaction (hydrolyzable probe assay) wherein the improvement comprises:
the use of the composition of claim 59 as a probe.
75 . An oligomer microarray, comprising one or more oligomers according to claim 11 .
76 . An oligomer microarray, comprising one or more oligomers according to claim 14 .
77 . A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:
use of the oligomer of claim 11 as a probe.
78 . A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:
use of the oligomer of claim 14 as a probe.
79 . A method for detecting a target sequence in a polynucleotide by hybridization, wherein the improvement comprises:
use of the composition of claim 56 as a probe.
80 . A method for detecting a target sequence. in a polynucleotide by hybridization, wherein the improvement comprises:
use of the composition of claim 57 as a probe.
81 . The method of any of claims 77 - 80 wherein the target sequence is distinguished from a-related sequence having a single-nucleotide mismatch with respect to the target sequence