Abstract
Förster 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 qAnitro (acceptor) were synthesized and incorporated into DNA by a generic, 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 qAnitro 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 qAnitro 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, tCO and tCnitro, 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.
Highlights
Fluorescence is a versatile and sensitive tool for studying a wide range of chemical, physical, and biological processes
Exact knowledge of the position and orientation of the donor and acceptor is essential in detailed Förster resonance energy transfer (FRET) structure and dynamics investigations, except in a few cases where single molecule measurements are combined with molecular dynamics (MD) simulations, in an ingenious but elaborative way, to give detailed 3D-structures of DNA.[10]
In an effort to improve the brightness of the promising adenine analogue qA,[23] we recently investigated a series of N9-ethylated model compounds, qAN1−4, where the outer benzene ring is replaced with a pyridine ring.[22]
Summary
Fluorescence is a versatile and sensitive tool for studying a wide range of chemical, physical, and biological processes. To avoid the uncertainties in position and orientation mentioned above, we have put significant effort into developing rotationally restricted internal fluorophores This allows us to obtain high-resolution FRET by taking full advantage of the orientation dependence.[5,6,14,15] We have previously reported on the first FBA FRET-pair that uses the tricyclic cytosine analogues tCO and tCnitro as the donor and acceptor, respectively.[16] While the vast majority of fluorescent base analogues are useful due to their high sensitivity to the surrounding microenvironment,[11−13,17,18] the tricyclic cytosines tCO and tC are two rare examples of FBAs that are both bright (Φf = 22% and 19%, respectively) and virtually insensitive to the microenvironment inside duplex DNA.[19,20] These unique characteristics are, in combination with the restricted motion of the tricyclic cytosines within duplexes, critical for FRETdistance measurements, and for the control and accuracy of R0. (C−C, A−A, and C−A/A−C) of the 10 possible nucleobase combinations
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