TABLE 1.
Time-resolved fluorescence parameters of singly and doubly labeled cTAR derivatives
| T (°C) | α0 | τ1 (ns)* | α1* | τ2 (ns)* | α2* | τ3 (ns)* | α3* | 〈τ〉 (ns)* | |
|---|---|---|---|---|---|---|---|---|---|
| cTAR-3′-Fl | 20 | 2.00(±0.10) | 0.15(±0.01) | 4.28(±0.02) | 0.85(±0.01) | 3.94(±0.02) | |||
| 60 | 2.00(±0.20) | 0.23(±0.04) | 4.00(±0.05) | 0.77(±0.03) | 3.54(±0.01) | ||||
| TMR-5′-cTAR-3′-Fl | 20 | 0.12(±0.01) | 0.56(±0.04) | 1.24(±0.05) | 0.17(±0.03) | 3.85(±0.06) | 0.27(±0.02) | 1.30(±0.10) | |
| 0.80† | 0.12† | 0.03† | 0.05† | ||||||
| 60 | 1.80(±0.10) | 0.27(±0.04) | 3.47(±0.05) | 0.73(±0.04) | 3.02(±0.01) | ||||
| TMR-5′-cTARC12-3′-Fl | 20 | 0.21(±0.02) | 0.21(±0.03) | 1.11(±0.04) | 0.62(±0.07) | 3.50(±0.10) | 0.17(±0.02) | 1.33(±0.02) | |
| 60 | 1.90(±0.30) | 0.18(±0.09) | 3.50(±0.10) | 0.82(±0.10) | 3.21(±0.02) | ||||
| TMR-5′-cTAR-3′-Fl+dTAR‡ | 20 | 0.13(±0.04) | 0.14(±0.04) | 1.60(±0.50) | 0.13(±0.05) | 4.04(±0.08) | 0.73(±0.09) | 3.20(±0.10) | |
| cTAR-3′-Fl + dTAR‡ | 20 | 0.09(±0.01) | 0.16(±0.02) | 1.33(±0.08) | 0.09(±0.01) | 4.19(±0.02) | 0.75(±0.02) | 3.28(±0.06) |
The fluorescence lifetimes, τ1 to τ3 the relatives amplitudes, α1 to α3 and the mean lifetime 〈τ〉, are expressed as means ± standard error of the mean for three experiments. The width of the distribution associated with each lifetime was found to be narrow in each condition, being less than 20% of lifetime value. The mean lifetime was calculated by: 〈τ〉 = Σαiτi.
The relative amplitude, α0 of the dark species in TMR-5′-cTAR-3′-Fl derivative is calculated by: α0 = 1 − 〈τ〉Fl/(〈τ〉mes × R′), where 〈τFl〉 is the mean lifetime of cTAR-3′-Fl at 20°C, 〈τmes〉 is the measured mean lifetime of TMR-5′-cTAR-3′-Fl at 20°C and R′ is the ratio between the steady state fluorescence of cTAR-3′-Fl and TMR-5′-cTAR-3′-Fl. The remaining amplitudes αic were calculated according to: αic = αi(1 − α0).
The duplexes of TMR-5′-cTAR-3′-Fl and cTAR-3′-Fl derivatives with their complementary sequence, dTAR, were mediated by temperature.