Abstract
The fluorescent adenine analogue qAN4 was recently shown to possess promising photophysical properties, including a high brightness as a monomer. Here we report the synthesis of the phosphoramidite of qAN4 and its successful incorporation into DNA oligonucleotides using standard solid-phase synthesis. Circular dichroism and thermal melting studies indicate that the qAN4-modification has a stabilizing effect on the B-form of DNA. Moreover, qAN4 base-pairs selectively with thymine with mismatch penalties similar to those of mismatches of adenine. The low energy absorption band of qAN4 inside DNA has its peak around 358 nm and the emission in duplex DNA is partly quenched and blue-shifted (ca. 410 nm), compared to the monomeric form. The spectral properties of the fluorophore also show sensitivity to pH; a property that may find biological applications. Quantum yields in single-stranded DNA range from 1-29 % and in duplex DNA from 1-7 %. In combination with the absorptive properties, this gives an average brightness inside duplex DNA of 275 M-1 cm-1 , more than five times higher than the most used environment-sensitive fluorescent base analogue, 2-aminopurine. Finally, we show that qAN4 can be used to advantage as a donor for interbase FRET applications in combination with adenine analogue qAnitro as an acceptor.
Highlights
Introduction structure and dynamics are essentialNMR,[1] X-ray crystallography[2] and lately Cryo-EM[3] are frequently usedNucleic acids are vital in the biology of cells
In an effort to improve the brightness of the excellent adenine analogue quadracyclic adenine (qA),[14] we recently investigated a series of N9-ethylated derivatives in this series of compounds, and found that monomeric qAN4 had the highest quantum yield (32 %) and 40 brightness of the series, while retaining a large Stokes shift, making it a promising fluorescent adenine
Calculations, the orientation of the transition dipole moments of We present here a fluorescent adenine analogue, qAN4, which qAN4 and qAnitro has been predicted, and the associated phase is more than one order of magnitude brighter than the parent angle is 25 degrees
Summary
In combination Circular dichroism and thermal melting studies indicate that the with the absorptive properties, this gives an average brightness qAN4-modification has a stabilizing effect on the B-form of inside duplex DNA of 275 MÀ 1 cmÀ 1, more than five times higher DNA. We show that qAN4 can be The low energy absorption band of qAN4 inside DNA has its used to advantage as a donor for interbase FRET applications in peak around 358 nm and the emission in duplex DNA is partly combination with adenine analogue qAnitro as an acceptor. Drawbacks of these their interaction with other molecules, investigation of their techniques are, for example, that NMR requires high sample concentrations while X-ray crystallography requires crystals,.
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