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New Biological Frontiers Illuminated by Molecular Sensors and Actuators
Poster Abstracts
47-POS
Board 47
Sensing and Structure Investigations of Nucleic Acid Systems Using Fluorescent Base
Analogue FRET-probes
Marcus Wilhelmsson
.
Chalmers University of Technology, Gothenburg, Sweden.
Utilization of the tricyclic cytosine family, tC/tCO/tCnitro, is increasing in nucleic acid
applications and the fluorescent members of this family have unique properties among
fluorescent base analogues.[1] In contrast to other fluorescent base analogues tCO, for example,
has i) a high quantum yield in duplex being virtually insensitive to neighboring base
combination, ii) an emission in DNA being characterized by a single-exponential decay in
duplexes, and iii) an average brightness in duplex being among the highest reported and up to 50
times higher than 2-aminopurine.[2] Furthermore, the tricyclic bases form stable base pairs with
guanine and give minimal perturbations to DNA native structure.[1-3] Importantly, we have
utilized tCO as a donor and developed tCnitro as an acceptor and, thus, established the first base
analogue förster resonance energy transfer (FRET)-pair.[3] As a consequence of the exact and
rigid positioning, this FRET-pair enables high control of orientation factor. DNA strands
containing the FRET-pair will, thus, make it possible to accurately distinguish distance- from
orientation-changes. To enable optimized use of our probes we have developed the freeware
FRETmatrix[4], globally fitting FRET-data to obtain the best overall structure/dynamics of the
nucleic acid under investigation. Recently we successfully utilized our FRET-pair in studies on
DNA structural changes[5] and have ongoing investigations using it in combination with
FRETmatrix. We envision our method, possibly in combination with single-molecule FRET on
longer distances, to be a powerful complement for techniques like NMR and X-ray
crystallography. References: [1] Wilhelmsson Q. Rev. Biophys. 2010, 43, 159. [2] Sandin et al.
Nucleic Acids Res. 2008, 36, 157. [3] Börjesson et al. J. Am. Chem. Soc. 2009, 131, 4288. [4]
Preus et al. Nucleic Acids Res. 2013, 41, e18. [5] Shi et al. P.N.A.S. 2012, 109, 16510.