No abstract

Most fluorescent nucleoside analogues are quenched when base stacked and some maintain their brightness, but there has been little progress toward developing nucleoside analogues that markedly increase their fluorescence upon duplex formation. Here, we report on the design and synthesis of a new tricyclic cytidine analogue, 8-diethylamino-tC (8-DEA-tC), that responds to DNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is the greatest of any reported nucleoside analogue that can participate in Watson–Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson–Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. These results, supported by DFT calculations, suggest a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of ag eneral method to predict their photophysics, ap roblem that is especially acute when base pairing and stacking changef luorescence. To better understand these effects, as eries of tricyclic cytidine (tC and tC O )a nalogues ranging from electron-rich to electron-deficient was designed and synthesized.T hey were then incorporated into oligonucleotides,a nd photophysical responses to base pairing and stackingw ere studied.W hen insertedi nto double-stranded DNA oligonucleotides, electron-rich analogues exhibit af luorescencet urn-on effect, in contrast with the electron-deficient compounds, whichs how diminished fluorescence. The magnitude of these fluores-cence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover,m atched base pairing enhances fluorescenceturn-on for the electron-rich compounds, and it causesaf luorescence decrease for the electron-deficientc ompounds. For the tC O compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering ap otentialn ew tool for studying nucleic acid structurea nd dynamics. These results, supported by DFT calculations,h elp to rationalize fluorescencec hangesi n the base stacka nd will be useful for selectingt he best fluorescent nucleoside analogues for ad esired application.

No abstract

DEAtC is a tricyclic 2’-deoxycytidine analogue that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with guanine base in a DNA–DNA duplex. The synthesis of DEAtC begins with 5-amino-2-methylbenzothiazole and provides the DEAtC nucleobase analogue over four synthetic steps. This nucleobase analogue is then silylated using BSA and conjugated to Hoffer’s chlorosugar to provide the protected DEAtC nucleoside in good yield. Following protective group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offered the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEAtC amidite and a short deprotection time are used to maximize efficiency. By following the protocol described in this unit, the DEAtC fluorescent probe can be synthesized and incorporated into any desired synthetic DNA oligonucleotide.