Characterization of Nucleobase Analogue FRET Acceptor tCnitro

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  • Søren Preus
  • Karl Börjesson
  • Kristine Kilså
  • Bo Albinsson
  • L. Marcus Wilhelmsson
The fluorescent nucleobase analogues of the tricyclic cytosine (tC) family, tC and tCO, possess high fluorescence quantum yields and single fluorescence lifetimes, even after incorporation into double-stranded DNA, which make these base analogues particularly useful as fluorescence resonance energy transfer (FRET) probes. Recently, we reported the first all-nucleobase FRET pair consisting of tCO as the donor and the novel tCnitro as the acceptor. The rigid and well-defined position of this FRET pair inside the DNA double helix, and consequently excellent control of the orientation factor in the FRET efficiency, are very promising features for future studies of nucleic acid structures. Here, we provide the necessary spectroscopic and photophysical characterization of tCnitro needed in order to utilize this probe as a FRET acceptor in nucleic acids. The lowest energy absorption band from 375 to 525 nm is shown to be the result of a single in-plane polarized electronic transition oriented 27° from the molecular long axis. This band overlaps the emission bands of both tC and tCO, and the Frster characteristics of these donor-acceptor pairs are calculated for double-stranded DNA scenarios. In addition, the UV-vis absorption of tCnitro is monitored in a broad pH range and the neutral form is found to be totally predominant under physiological conditions with a pKa of 11.1. The structure and electronic spectrum of tCnitrois further characterized by density functional theory calculations.
OriginalsprogEngelsk
TidsskriftJournal of Physical Chemistry B
Vol/bind114
Udgave nummer2
Sider (fra-til)1050-1056
Antal sider7
ISSN1520-6106
DOI
StatusUdgivet - 2010

ID: 17083927