A method of determining presence or absence of nucleic acid in a sample, the method comprising:
- exposing the nucleic acid to a fluorescent nucleic acid dye having the formula:
- wherein BRIDGE is a substantially aliphatic linker comprising from about 8 to about 150 non-hydrogen atoms, inclusive, and wherein the linker comprises no more than one positive charge;
- Q1 is a fluorescent nucleic acid dye constituent;
- Q2 is a fluorescent nucleic acid dye constituent;
- and
- Q1 and Q2 may be the same or different, wherein
- (i) when Q1 and/or Q2 is a phenanthridinium dye, at least one of Q1 and Q2 is a phenanthridinium dye has the structure of Formula I:
- R1 represents where BRIDGE attaches to the structure;
- and Ψ is an anion;
- or
- (ii) when each of Q1 and Q2 is an asymmetric cyanine dye, each of the Q1 and Q2 dye constituents has the structure of Formula II:
- wherein R1′ of Formula II is H;
- alkyl or alkenyl having 1 carbon to 6 carbons, inclusive;
- a halogen;
- —OR9;
- —SR10;
- —NR11R12;
- —CN;
- —NH(C═O)R13;
- —NHS(═O)2R14;
- —C(═O)NHR15;
- or a substituent associated with minor groove binding;
- or represents where BRIDGE attaches to the structure;
- when R1′ of Formula II comprises at least one of R9,R10, R11, R12, R13, R14 and R15, any said one of R9,R10, R11, R12, R13, R14 and R15, independently, is H or alkyl having 1 carbon to 12 carbons, inclusive, optionally incorporating 1 to 2 nitrogen(s), inclusive, or an aryl;
- when R1′ of Formula II comprises R11 and R12, R11 and R12 may in combination form a 5- or 6-membered, saturated or unsaturated ring, which optionally comprises at least one hetero atom selected from N and O;
- X of Formula II is selected from O and S;
- n of Formula II is selected from 0, 1, and 2;
- R6 of Formula II is H;
- alkyl or alkenyl having 1 carbon to 10 carbons, inclusive, optionally comprising at least one hetero atom selected from N, O, and S;
- a halogen;
- —OR16;
- —SR16;
- —NR16R17;
- or a substituted or an unsubstituted aryl, optionally comprising 1 to 3 hetero atom(s), inclusive, selected from N, O, and S;
- or represents where BRIDGE attaches to the structure;
- R7 of Formula II is H;
- alkyl or alkenyl having 1 carbon to 10 carbons, inclusive, optionally comprising an aryl and at least one hetero atom selected from N, O, and S;
- or a substituted or an unsubstituted aryl optionally comprising 1 to 3 hetero atom(s), inclusive, selected from N, O, and S;
- or represents where BRIDGE attaches to the structure;
- R8 and R8′ of Formula II in combination form a fused aromatic ring, which may be further substituted 1 to 4 time(s), inclusive, independently, by C1-C2, inclusive, alkyl, C1-C2, inclusive, alkoxy, C1-C2, inclusive, alkylmercapto, or a halogen;
- each of R16 and R17 independently is H;
- alkyl having 1 carbon to 12 carbons, inclusive, optionally incorporating 1 to 2 nitrogen(s) or an aryl;
- or
- R16 and R17 may in combination form a 5- or 6-membered saturated or unsaturated ring, which optionally comprises at least one hetero atom selected from N and O;
- only one of R1′, R6, and R7 of Formula II represents where BRIDGE attaches to the structure;
- and
- Ψ of Formula II is an anion;
- or
- (iii) when either Q1 or Q2 is an acridine dye, at least one dye constituent of the Q1 and Q2 dye constituents has the structure of Formula III:
- wherein each R1 of Formula III is independently, is H or a C1-C2, inclusive, alkyl;
- one of R2 and R3 of Formula III represents where BRIDGE attaches to the structure;
- when R2 of Formula III represents where BRIDGE attaches to the structure, R3 is H or —CH3;
- when R3 of Formula III represents where BRIDGE attaches to the structure, R2 is selected from H, —CH3, —NH2, —NHCH3, —CN, and —C(═O)NH2;
- each R6 of Formula III independently, is H or a C1-C2, inclusive, alkyl;
- each R7 of Formula III independently, is H or a C1-C2, inclusive, alkyl;
- for each pair of adjacent R6 or R7 and R1, independently, R6 or R7 and R1 may in combination form a 5- or 6-membered, saturated or unsaturated ring;
- and
- Ψ of Formula III is an anion;
- such that, if nucleic acid is present in the sample, a complex of the fluorescent nucleic acid dye and the nucleic acid is formed;
- and
- detecting fluorescence associated with the complex or a lack thereof.