Toward Complete Sequence Flexibility of Nucleic Acid Base Analogue FRET

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 qA (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 qA 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 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 and tC, 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.

Lighting Up DNA with the Environment‐Sensitive Bright Adenine Analogue qAN4

“…To investigate the effect of the incorporation of qAN4 on duplex stability, we measured the melting temperature of all 16 qAN4‐modified duplexes and their unmodified counterparts (Table ). Introducing qAN4 results in an average increase in duplex melting temperature of 5.6 °C – a greater change than that observed for qA (3.0 °C) and qAN1 (2.9 °C) . Overall, the stability increase is lowest when qAN4 is surrounded by two purines (Δ T m =2.5 °C) and highest when qAN4 is surrounded by pyrimidines (Δ T m =8.7 °C).…”

“…We show that qAN4 works very well as an adenine analogue, and exhibits useful microenvironment‐ and pH‐sensitive emission properties. Furthermore, qAN4 forms an excellent base‐analogue FRET‐pair with the FRET acceptor qA nitro . Thus, we here also contribute to expanding the currently limited library of nucleoside analogues that are well suited for interbase FRET and fluorescence anisotropy investigations.…”

Approach to the Investigation of Nucleosome Structure by Using the Highly Emissive Nucleobase thdG–tC FRET Pair

“…Fluorescence decay profiles indicate that nucleosome with D 40th+A 27th have comparatively shorter average lifetimes ( τ =8.18 ns) than nucleosome with D 40th+A 41st ( τ =8.65 ns; Figure S7 and Table S1, Supporting Information). This shorter average lifetime supports that higher energy transfer occurred on nucleosome with D 40th+A 27th . On the lifetime analysis of these nucleosomes, we observed an unexpected shortest lifetime that can make the calculation of FRET efficiency inaccurate .…”

“…In the B‐form structure, the angle between the helical axis and the fluorescent nucleobase is almost 90° in the cylinder model of the DNA duplex because of the fixed parallel plan of the fluorescent nucleobase caused by hydrogen bonding with the counterbase and stacking interaction between neighboring bases, indicating that κ 2 can be assumed to between 0 and 1 . On the other hand, the base pair of the DNA duplex in the nucleosome undergoes geometrical changes such as roll, tilt, and twist unlike typical B‐form DNA .…”

New Size‐Expanded Fluorescent Thymine Analogue: Synthesis, Characterization, and Application

“…Interestingly,s equences in which diox T was flanked by two purine bases (PuPu), ap yrimidine and ap urine base (PyPu),apurine and ap yrimidine base (PuPy), and two pyrimidine bases (PyPy) all showeds imilart hermals tability (average 1.4-2.5 8C). [49] Despite this, the quantum yields for both single-and double-stranded DNA decreased significantly when Gw as located next to diox T. This quenching effect can be explainedbythe electron transfer from G. We calculated the order of the highest-occupied molecular orbital( HOMO) energyo fe ach nucleobase as G > diox T > A > C > T, which supports the concept that quenching was the result of electron transfer from G( Figure S11i nt he Supporting Information). Duplexes with AT as the neighboring nucleobases showedt he highest thermal stability,w hereas those with GG showed the lowest thermal stability.A sar esult, duplexesc ontaining diox T demonstrated better thermal stability than the previouslyr eported ring-fused fluorescent nucleobase BgQ.…”