Patent ID: 6268132
Filing Date: 2001-07-31
Classification: C07H,C12Q

Abstract:
An improvement in methods of synthesis, amplification, enzymatic digestion, base-pairing, labeling, sequencing, replication, transcription, location, detection, or identification of DNA or RNA oligonucleotides, wherein said improvement comprises the incorporation of inherently fluorescent nucleosides, or structural analogs thereof, said inherently fluorescent nucleosides or structural analogs thereof being fluorescent under physiological conditions, having the following structures: ##STR2##whereinX.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, and X.sub.6.dbd.N, O, C, S, or Si, wherein at least one of X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, or X.sub.6.dbd.N, and wherein X.sub.7 is --CH--;R.sub.4 is a reactive group derivatizable with a detectable label wherein said reactive group is selected from the group consisting of NH.sub.2, SH, .dbd.O, and optionally, a linking moiety selected from the group consisting of an amide, a thioether, a disulfide, a combination of an amide a thioether or a disulfide, R.sub.1 --(CH.sub.2).sub.x --R.sub.2 and R.sub.1 --R.sub.2 --(CH.sub.2).sub.x --R.sub.3 wherein x is an integer from 1 to 25 inclusive, and R.sub.1, R.sub.2, and R.sub.3 are H, OH, alkyl, acyl, amide, thioether, or disulfide, and wherein said detectable label is selected from the group consisting of radioisotopes, fluorescent or chemiluminescent reporter molecules, antibodies, haptens, biotin, photobiotin, digoxigenin, fluorescent aliphatic amino groups, avidin, enzymes, and acridinium; R.sub.5 is H, absent, or part of an etheno linkage with R.sub.4 ;R.sub.6 is H, NH.sub.2, SH, or .dbd.O;R.sub.8 and R.sub.9 are hydrogen, methyl, bromine, fluorine, or iodine; alkyl or aromatic substituents, or an optional linking moiety selected from the group consisting of an amide, a thioether, a disulfide linkage, and a combination thereof;R.sub.10 is hydrogen, an acid-sensitive/base-stable blocking group, or a phosphorous derivative;R.sub.11.dbd.R.sub.13.dbd.H;R.sub.12 is hydrogen, OH, 3 amino, 3-azido, 3-thiol, 3-unsaturated or a 3-phosphorous derivative; andR.sub.14 is H, OH, or OR.sub.3 where R.sub.3 is a reactive group, protecting group, or additional fluorophore;provided that excluded from such structure is any purine-like compound in which:(i) X.sub.1.dbd.X.sub.4.dbd.C; X.sub.2.dbd.X.sub.3.dbd.N; R.sub.4.dbd.NH.sub.2 ; R.sub.5.dbd.R.sub.8 which is absent; R.sub.6.dbd.H; R.sub.9 is H or is absent; R.sub.10.dbd.H; and R.sub.12.dbd.R.sub.14.dbd.OH; or(ii) X.sub.1.dbd.C; X.sub.2.dbd.X.sub.3.dbd.X.sub.4.dbd.N; R.sub.4.dbd.NH.sub.2 or H; R.sub.5.dbd.R.sub.8 which is absent; R.sub.6.dbd.NH.sub.2 ; R.sub.9 is H or is absent; R.sub.10.dbd.H; and R.sub.12.dbd.R.sub.14 OH; or(iii) R.sub.4 and R.sub.5 in combination form an etheno linkage; R.sub.6.dbd.R.sub.8.dbd.H; R.sub.9 is absent; X.sub.1.dbd.X.sub.3.dbd.C; and X.sub.2.dbd.X.sub.4.dbd.N; or(iv) X.sub.1.dbd.X.sub.2.dbd.C; X.sub.3.dbd.X.sub.4.dbd.N; R.sub.4.dbd.halogen or --S(CH.sub.2).sub.n R with n being an integer between 1-6 and R is lower alkoxy, alkylthio, phenoxy, phenylthio, unsubstituted or substituted phenyl, --C.dbd.C--R' wherein R' is unsubstituted or mono-, di- or trisubstituted phenyl; R.sub.9.dbd.R.sub.10.dbd.H; and R.sub.12.dbd.R.sub.14.dbd.acyloxy; or(v) R.sub.4.dbd.NH.sub.2 or OH; R.sub.5 is absent; R.sub.9 is --COOH, --CONH.sub.2, --C(S)NH.sub.2, --C(NH)N.sub.2, or --C(N--NH.sub.2)NH.sub.2 ; X.sub.1.dbd.X.sub.2.dbd.X.sub.3.dbd.C; and X.sub.4.dbd.N;and further provided that R.sub.8 is absent if X.sub.3.dbd.N and R.sub.9 is absent if X.sub.2.dbd.N;and provided that the structure is not a nucleoside which is effectively non-fluorescent under physiological conditions and is selected from the group consisting of adenosine, cytidine, guanine, thymidine, uridine, and inosine.