Associate Professor Marcus Wilhelmsson, Co-director for Area of Advance Nanoscience and Nanotechnology, Chalmers University of Technology, Gothenburg, Sweden

Designing, Developing and Applying Fluorophores for Nucleic Acids Investigation

Fluorescent base analogues is a class of molecules that is rapidly increasing in importance for investigating systems containing DNA and RNA in biology and nanotechnology.[1] We use quantum chemical calculations in designing novel fluorescent base analogues with new or improved properties. The aim is to decrease the time and synthetic effort needed to find promising new candidate molecules and with these to develop a fluorescent genetic alphabet that can be used in Förster resonance energy transfer (FRET)-studies. Recently we have used this approach on a class of quadracyclic adenines, qAs,[2] and we are currently investigating their possible use as donors and acceptors in FRET measurements.

We also utilize our previously developed family of molecules called tricyclic cytosines, tC, tCO, and tCnitro. In contrast to other reported fluorescent base analogues tCO, for example, has i) a high quantum yield (f0.2) in duplex that is virtually insensitive to neighboring base combination, ii) an emission after incorporation into DNA being characterized by a single exponential decay in double stranded systems, and iii) an average luminescence brightness of the base analogues in duplex DNA being among the highest reported so far and up to 50 times higher than the most commonly used fluorescent base analogue 2-aminopurine.[3] Importantly, we have recently utilized tCO as a donor and developed tCnitro as an acceptor and, thus, established the first nucleic acid base analogue FRET-pair.[4]

As a consequence of the exact and rigid positioning, this FRET-pair enables high control of the orientation factor (k²). To allow optimized use of this kind of FRET-pair we have developed a freeware called FRETmatrix[5], suited for rigidly positioned probes, that globally fit a set of time-resolved FRET-data to obtain the best overall structure/dynamics of the nucleic acid structure under investigation. Recently we have successfully utilized our FRET-pair in studies on DNA structural changes[6] and have ongoing investigations using our FRET-pair and FRETmatrix to study both DNA and RNA conformations. We envision our method, possibly in combination with single-molecule FRET on longer distances, to be a powerful complement for techniques like NMR, where molecular size is a problem, as well as X-ray crystallography, where solution structures and dynamics are impossible to monitor.

References:

[1] Wilhelmsson, L.M. Q. Rev. Biophys. 2010, 43, 159.
[2] Dumat, B. et al. Chem. Eur. J. 2015, 21, 4039.
[3] Sandin, P. et al. Nucleic Acids Res. 2008, 36, 157.
[4] Börjesson, K. et al. J. Am. Chem. Soc. 2009, 131, 4288. [5] Preus, S. et al. Nucleic Acids Res. 2013, 41, e18.
[6] Shi, Y. et al. P.N.A.S. 2012, 109, 16510.