Expanding the alphabet of fluorescent nucleic acid base analogues: Characterization of new members

Licentiate thesis, 2011

Fluorescence has proven to be a powerful tool for studying structure, dynamics and interactions of biomolecules such as proteins, RNA and DNA. However, since the natural DNA/RNA nucleobases are virtually non-fluorescent, there is a need for developing fluorescent nucleic acid markers. Many dyes have been designed to be covalently attached by linkers as external probes to the DNA. Nevertheless, there are some important limitations with these markers, such as perturbations caused to the duplex structure and the lack of site-specific information. Therefore there is an increasing focus on the development of fluorescent nucleic acid base analogues (FBAs). These are artificial DNA bases, which are significantly fluorescent, resemble the structure of one of the natural nucleobases, have some hydrogen bonding capacity to the opposite base and preserve the B-DNA helical structure. In this thesis the photophysical and base-mimicking properties of two new fluorescent adenine analogues, triazole adenine (AT) and quadracyclic adenine (qA) are presented. Both analogues proved to be very promising compared to the widely used commercially available 2-aminopurine (2-AP). AT shows a high fluorescence both as a monomer and in DNA. Quantum yields in single-strands and duplexes reach values up to ten and five times higher, respectively, than maximum values reported for 2-AP. Furthermore, as for 2-AP and most other FBAs, AT causes minor perturbations to the B-DNA duplex structure. Interestingly, we found AT to be capable of forming base-pairs both with thymine and adenine. The second fluorescent adenine analogue discussed in this work, qA, is moderately fluorescent as a monomer. However, inside single-and double-stranded DNA, quantum yields reach values which are four times higher or comparable, to corresponding values for 2-AP. Importantly, qA causes no perturbations to the DNA duplex and even stabilizes it depending on the surrounding sequence. Furthermore, in contrast to 2-AP, qA shows specific base-pairing with thymine. These properties of qA are unprecedented for fluorescent adenine analogues. The above mentioned qualities make AT and qA attractive alternatives for 2-AP in future applications. Finally, these two new fluorescent adenine analogues may help to form a better understanding of the relationship between the structural and fluorescence properties of FBAs.