DNA and RNA base analogue FRET - from fluorophore design to biochemical applications
This thesis focuses on the development and use of fluorescent base analogues (FBAs). They are important tools in research concerning nucleic acids structure, dynamics and interactions. FBAs are fluorescent molecules that are structurally similar to the natural nucleobases and can therefore replace them inside nucleic acids without significantly perturbing the properties of the nucleic acid. The design of new FBAs is often challenging due to the limitations imposed on their structure by the overall structure of nucleic acids.
This thesis starts by describing the development and characterization of a large set of new potential adenine analogues, all based on the structure of the FBA qA, and how TDDFT calculations were utilized to aid the design. Among these, three fluorescent (qAN1, qAN4 and pA) and one non-fluorescent (qAnitro) analogue have been incorporated and characterized inside DNA as well. They are all good adenine analogues, i.e. they do not perturb the structure or stability of DNA duplexes significantly. The three fluorescent analogues are all significantly brighter than the parent compound qA, and importantly, pA is the brightest adenine analogue inside DNA reported to date. The thesis also describes the development and characterization of a good thymine analogue, bT, which might serve as the starting point for development of brighter thymine analogues, much like qA did for the adenine analogues mentioned above.
The second half of the thesis focuses on interbase FRET (Förster resonance energy transfer) using the new adenine analogues and the previously reported FRET-pair tCO-tCnitro. FRET is confirmed and characterized inside DNA using the three adenine donors (qAN1, qAN4 and pA) with the acceptor qAnitro. These FRET-pairs can monitor energy transfer up to 1.5 turns of DNA and are hence suitable for monitoring structural changes in short DNA. This is exemplified by a study of the effect on DNA structure by binding of netropsin, showing that the interbase FRET is sensitive to small changes in DNA structure. The previously reported tCO-tCnitro are here both incorporated into RNA and interbase FRET in RNA is measured for the first time. This is an important step since RNA, among other things, has proved to be a key player in cell regulation and hence of high interest and importance. Lastly the change in interbase FRET upon inducing a change from A- to Z-form RNA is shown to be significant, again highlighting the potential of interbase FRET in nucleic acid structure investigations.
Fluorescent base analogue