Toward Complete Sequence Flexibility of Nucleic Acid Base Analogue FRET
Artikel i vetenskaplig tidskrift, 2017
Forster resonance energy transfer (FRET) using fluorescent base analogues is a powerful means of obtaining high-resolution nucleic acid structure and dynamics information that favorably complements techniques such as NMR and X-ray crystallography. Here, we expand the base base FRET repertoire with an adenine analogue FRET-pair. Phosphoramidite-protected quadracyclic 2'-deoxyadenosine analogues qAN1 (donor) and qA(nitro) (acceptor) were synthesized and incorporated into DNA by a genetic, reliable, and high-yielding route, and both constitute excellent adenine analogues. The donor, qAN1, has quantum yields reaching 21% and 11% in single- and double-strands, respectively. To the best of our knowledge, this results in the highest average brightness of an adenine analogue inside DNA. Its potent emissive features overlap well with the absorption of qA(nitro), and thus enable accurate FRET-measurements over more than one turn of B-DNA. As we have shown previously for our cytosine analogue FRET-pair, FRET between qAN1 and qA(nitro) positioned at different base separations inside DNA results in efficiencies that are highly dependent on both distance and orientation. This facilitates significantly enhanced resolution in FRET structure determinations, demonstrated here in a study of conformational changes of DNA upon binding of the minor groove binder netropsin. Finally, we note that the donor and acceptor, of our cytosine FRET-pair, tC degrees and tC(nitro), can be conveniently combined with the acceptor and donor of our current adenine pair, respectively. Consequently, our base analogues can now measure base base FRET between 3 of the 10 possible base combinations and, through base-complementarity, between all sequence positions in a duplex.