Probing Variations In The Structural Environment Of A DNA Sequence Using Fluorescence Properties Of The Pteridine Analog Probes, 3MI and 6MI

Abstract

We explored two different microenvironments in the sequence; 5′-actaGagatccctcagacccttttagtcagtGtgga -3′ in single and duplex form using two similar nucleoside analogs. 3MI and 6MI were each investigated in two different environments, one flanked by thymines (PTRT) and the other, by adenines (PTRA)(shown by G's noted above). Each site is equidistant from a terminus. The probes differ only by the position of a methyl group in either the 3- (3MI) position or the 6- (6MI) position. Both time-resolved anisotropies and lifetimes of the probes depend upon local electrostatics which are impacted by duplex formation. 3MI shows less response to structural change as compared to 6MI. Integrals of lifetime curves compared with quantum yields of each sample reveal that each displays a “dark” component which we are unable to detect with TCSPC (e.g.,tau<70ps). For 6MI in the A environment this QSSQ “quasi static quenching” eliminates approximately half the molecules, whether in SS or DS form. 6MI in the T environment displays an unexpected increase in the quantum yield upon duplex formation (0.107 to 0.189) apparently the result of escape from QSSQ which simultaneously declines from 66% to 33%. Escape from the dark state is accompanied by doubling of steady state anisotropy of 6MI in PTRT in the duplex. Only 6MI in the T duplex displays a rotational correlation time over 7 ns. The DS A environment fails to constrain local motion and QSSQ remains the same as in SS; in contrast, the flanking T duplex environment restricts local motion and halves the QSSQ.