Abstract
2-aminopurine (2AP) is a responsive fluorescent base analogue that is used widely as a probe of the local molecular environment in DNA. The ability of 2AP to report changes in local conformation and base-stacking interactions arises from the efficient quenching of its fluorescence by the natural DNA bases. However, the mechanism of this inter-base quenching remains imperfectly understood. Two previous studies of the collisional quenching of 2AP by the natural bases, in different buffer solutions, showed that dynamic quenching efficiency depends on the identity of the natural base, but disagreed on the relative quenching efficiencies of the bases. We report a comprehensive investigation of inter-base quenching of 2AP by the natural nucleoside monophosphates (NMPs), replicating the buffer conditions used in the previous studies. Using time-resolved fluorescence measurements to distinguish between dynamic and static quenching, we find that the dynamic quenching rate constants of the different bases show a consistent trend across both buffers, and this is in line with a charge-transfer mechanism. Time-resolved measurements also provide insight into static quenching, revealing formation of 2AP-NMP ground-state complexes in which 2AP displays a very short fluorescence lifetime, comparable to that seen in oligonucleotides. In these complexes, the dependence of the rate of quenching on the partner base also supports a charge-transfer mechanism.
Highlights
DNA is increasingly recognised as playing new and unforeseen roles in biochemical processes, not just as a store of genetic information, and undergoing repair and epigenetic modification, suggesting a more complex web of interactions rather than the traditional linear transcription-translation model
We report a comprehensive investigation of interbase quenching of 2AP by the natural nucleoside monophosphates (NMPs), replicating the buffer conditions used in the previous studies
Using time-resolved fluorescence measurements to distinguish between dynamic and static quenching, we find that the dynamic quenching rate constants of the different bases show a consistent trend across both buffers, and this is in line with a chargetransfer mechanism
Summary
DNA is increasingly recognised as playing new and unforeseen roles in biochemical processes, not just as a store of genetic information, and undergoing repair and epigenetic modification, suggesting a more complex web of interactions rather than the traditional linear transcription-translation model. Understanding these processes in more detail is often achieved using fluorescent probes inserted at specific sites in DNA strands, exploiting the high sensitivity and noninvasive nature of fluorescence spectroscopy. Structures of 2-aminopurine (2AP) and the DNA nucleotides, cytidine-5′-monophosphate (CMP), deoxythymidine-5′monophosphate (dTMP), guanosine-5′-monophosphate (GMP) and adenosine-5′-monophosphate (AMP)
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