Substituted unsymmetrical cyanine dyes with selected permeability

Claim 1

A compound of the formula ##STR109## wherein each R 1 is independently H; or an alkyl group having from 1-6 carbons; an alkoxy group having from 1-6 carbons; or a trifluoromethyl; or a halogen; and t=1-4; R 2 is an alkyl group having 1-6 carbons; X is O, S, Se or NR 15 , where R 15 is H or an alkyl group having 1-6 carbons; or X is CR 16 R 17 where R 16 and R 17 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 16 and R 17 taken in combination complete a five or six membered saturated ring; n=0, 1 or2; Ψ - is a biologically compatible counterion; Q has the formula Q1 or Q2 ##STR110## wherein Y is --CR 3 ═CR 4 --; p and m=0 or 1, such that p+m=1; R 5 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or R 5 is a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 3 , R 4 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; or R 6 and R 7 , taken in combination are --(CH 2 ) v -- where v=3 or 4, or R 6 and R 7 form a fused aromatic ring according to formula Q2; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a TAIL; or --OH, --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, P, O and S, such that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; or single, double, triple or aromatic carbon-carbon bonds; or phosphorus-oxygen, phosphorus-sulfur bonds, nitrogen-nitrogen or nitrogen-oxygen bonds; or aromatic or heteroaromatic bonds; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR111## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 5 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and when R 5 is a TAIL, LINK is a single bond; and where only R 5 is a TAIL, R 3 or R 4 is not hydrogen; and, where more than one of R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL, each TAIL is optionally the same or different.

Claim 40

A method of staining nucleic acids, comprising a) combining a sample that contains or is thought to contain a nucleic acid, with a mixture containing a dye compound of the formula (formula I): ##STR124## or having the formula ##STR125## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, aIkenyl, polyaIkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR126## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; and b) incubating the sample and mixture for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form one or more dye-nucleic acid complexes that give a detectable fluorescent signal.

Claim 41

A method of staining nucleic acids, comprising a) combining a sample that contains or is thought to contain a nucleic acid, with a mixture containing a dye compound of the formula (formula I): ##STR124## or having the formula ##STR125## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, aIkenyl, polyaIkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR126## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; and b) incubating the sample and mixture for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form one or more dye-nucleic acid complexes that give a detectable fluorescent signal.

Claim 49

A method of detecting a biological structure, comprising a) combining a sample that contains or is thought to contain a specific biological structure, with a dye of the formula ##STR127## or having the formula ##STR128## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , (NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - , wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR129## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; wherein said biological structure contains nucleic acids; b) incubating the combined sample and dye compound for a time sufficient for the dye compound to combine with the nucleic acids in the biological structure to form a pattern of dye-nucleic acid complexes having a detectable fluorescent signal that corresponds to the biological structure; and c) detecting the fluorescent signal that corresponds to the biological structure.

Claim 50

A method of detecting a biological structure, comprising a) combining a sample that contains or is thought to contain a specific biological structure, with a dye of the formula ##STR127## or having the formula ##STR128## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , (NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - , wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR129## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; wherein said biological structure contains nucleic acids; b) incubating the combined sample and dye compound for a time sufficient for the dye compound to combine with the nucleic acids in the biological structure to form a pattern of dye-nucleic acid complexes having a detectable fluorescent signal that corresponds to the biological structure; and c) detecting the fluorescent signal that corresponds to the biological structure.

Claim 57

A method of characterizing a sample, comprising: a) combining a sample containing or thought to contain nucleic acid polymers with a dye compound of the formula ##STR130## or having the formula ##STR131## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR132## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; b) incubating the sample for a time sufficient for the dye compound to combine with nucleic acid polymers in the sample to form a staining profile of dye-nucleic acid complexes having detectable fluorescent signals, said staining pattern having a shape, location, spectral property, or intensity that is indicative of a characteristic of the sample; and c) characterizing the sample based on said staining profile.

Claim 58

A method of characterizing a sample, comprising: a) combining a sample containing or thought to contain nucleic acid polymers with a dye compound of the formula ##STR130## or having the formula ##STR131## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR132## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; b) incubating the sample for a time sufficient for the dye compound to combine with nucleic acid polymers in the sample to form a staining profile of dye-nucleic acid complexes having detectable fluorescent signals, said staining pattern having a shape, location, spectral property, or intensity that is indicative of a characteristic of the sample; and c) characterizing the sample based on said staining profile.

Claim 71

A method of determining cell membrane integrity comprising: a) incubating a sample containing one or more cells with a first dye compound of the formula ##STR133## or having the formula ##STR134## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR135## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; for a time sufficient for the dye compound to combine with intracellular nucleic acids to form a first intracellular dye-nucleic acid complex having a detectable fluorescent signal; and c) determining cell membrane integrity of cells in the sample based on presence of the detectable fluorescent signal, where the presence of the detectable fluorescent signal indicates that the cell membrane integrity is compromised and the absence of the detectable fluorescent signal indicates that the cell membrane integrity is intact.

Claim 72

A method of determining cell membrane integrity comprising: a) incubating a sample containing one or more cells with a first dye compound of the formula ##STR133## or having the formula ##STR134## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR135## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; for a time sufficient for the dye compound to combine with intracellular nucleic acids to form a first intracellular dye-nucleic acid complex having a detectable fluorescent signal; and c) determining cell membrane integrity of cells in the sample based on presence of the detectable fluorescent signal, where the presence of the detectable fluorescent signal indicates that the cell membrane integrity is compromised and the absence of the detectable fluorescent signal indicates that the cell membrane integrity is intact.

Claim 77

A method of quantitating nucleic acids in a sample, comprising a) combining an aliquot of a sample, optionally the entire sample, that contains or is thought to contain a nucleic acid, with mixture containing a dye compound of the formula ##STR136## or having the formula ##STR137## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR138## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; b) incubating the aliquot and mixture for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form a dye-nucleic acid complex that gives a detectable fluorescent signal; and c) quantifying the nucleic acid present in said sample based on comparison of the detectable fluorescent signal with a fluorescent standard characteristic of a given amount of nucleic acid.

Claim 78

A method of quantitating nucleic acids in a sample, comprising a) combining an aliquot of a sample, optionally the entire sample, that contains or is thought to contain a nucleic acid, with mixture containing a dye compound of the formula ##STR136## or having the formula ##STR137## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring; and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons, or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR138## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; b) incubating the aliquot and mixture for a time sufficient for the dye compound to combine with the nucleic acid in the sample to form a dye-nucleic acid complex that gives a detectable fluorescent signal; and c) quantifying the nucleic acid present in said sample based on comparison of the detectable fluorescent signal with a fluorescent standard characteristic of a given amount of nucleic acid.

Claim 85

A method of analyzing nucleic acid-analyte interactions, comprising: a) forming a fluorescent complex comprising a nucleic acid polymer and one or more dye molecules of the formula: ##STR139## or having the formula ##STR140## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring, and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons; or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR141## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; wherein the fluorescent complex has a set of characteristic spectral properties; b) combining the fluorescent complex with a sample that contains or is thought to contain an analyte that interacts with the nucleic acid polymer; c) detecting a change in the spectral properties of the fluorescent complex; and d) determining the presence or activity of the analyte in said sample based on comparison of the change in spectral properties of the complex with a fluorescent standard characteristic of analyte activity.

Claim 86

A method of analyzing nucleic acid-analyte interactions, comprising: a) forming a fluorescent complex comprising a nucleic acid polymer and one or more dye molecules of the formula: ##STR139## or having the formula ##STR140## wherein R 2 is ethyl or methyl; X is O or S; n=0, 1 or2; Ψ - is a biologically compatible counterion; R 5 is alkyl having 1-6 carbons; or a cyclic substituent that is a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or R 5 is a TAIL; R 4 is an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; where R 8 and R 9 , which can be the same or different, are independently H; or alkyl groups having 1-6 carbons; or 1-2 alicyclic or aromatic rings; or R 8 and R 9 taken in combination are --(CH 2 ) 4 -- or --(CH 2 ) 5 -- to give a 5 or 6 membered ring, and where R 19 is alkyl having 1-6 carbons, or perfluoroalkyl having 1-6 carbons, or aryl; having 1-6 carbons, or aryl; R 3 , R 6 and R 7 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-10 carbons; or --OR 8 , --SR 8 , --(NR 8 R 9 ); or --OSO 2 R 19 ; or a TAIL; R 11 , R 12 , R 13 , and R 14 , which may be the same or different, are independently H; or an alkyl, alkenyl, polyalkenyl, alkynyl or polyalkynyl group having 1-6 carbons; or a halogen; or a substituted or unsubstituted aryl or heteroaryl; or a substituted or unsubstituted cycloalkyl having 3-8 carbons; or a TAIL; or --OR 8 , --SR 8 , or --(NR 8 R 9 ); TAIL is a heteroatom-containing moiety having the formula LINK-SPACER-CAP; wherein LINK is a single covalent bond, --O--, --S--, or --NR 20 --; where R 20 is H, a linear or branched alkyl having 1-8 carbons; or R 20 is --SPACER'--CAP'; SPACER and SPACER', which may be the same or different, are covalent linkages, linear or branched, cyclic or heterocyclic, saturated or unsaturated, each having 1-16 nonhydrogen atoms selected from the group consisting of C, N, O and S, such that the linkage begins and ends with a carbon atom, and contains any combination of ether, thioether, amine, ester, amide, or aliphatic, olefinic or aromatic carbon-carbon bonds, or aromatic carbon-nitrogen or nitrogen-nitrogen bonds; wherein all heteroatoms in the linear backbone of SPACER are separated by at least two carbon atoms; CAP and CAP', which may be the same or different, are --O--R 21 , --S--R 21 , --NR 21 R 22 , or --N + R 21 R 22 R 23 Ψ - ; wherein R 21 , R 22 , and R 23 are independently H, or a linear or branched alkyl or cycloalkyl having 1-8 carbons, optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, carboxyalkyl having 1-8 carbons, or phenyl, where phenyl is optionally further substituted by halogen, hydroxy, alkoxy having 1-8 carbons, aminoalkyl having 1-8 carbons, or carboxyalkyl having 1-8 carbons; or, one or more of R 21 , R 22 and R 23 , taken in combination with SPACER or SPACER' or R 20 forms a 5- or 6-membered aromatic, heteroaromatic, alicyclic or heteroalicyclic ring, the heteroatoms selected from O, N or S; where Ψ - is a biologically compatible counterion; or CAP and CAP' are independently ##STR141## where R 21 , R 22 and Ψ - are as defined previously; such that at least one of R 3 , R 4 , R 6 , R 7 , R 11 , R 12 , R 13 , and R 14 is a TAIL; and, where more than one substituent is a TAIL, each TAIL is optionally the same or different; wherein the fluorescent complex has a set of characteristic spectral properties; b) combining the fluorescent complex with a sample that contains or is thought to contain an analyte that interacts with the nucleic acid polymer; c) detecting a change in the spectral properties of the fluorescent complex; and d) determining the presence or activity of the analyte in said sample based on comparison of the change in spectral properties of the complex with a fluorescent standard characteristic of analyte activity.