Distinguishing “Looped-Out” and “Stacked-In” DNA Bulge Conformation Using Fluorescent 2-Aminopurine Replacing a Purine Base

Abstract

The conformation of a bulged DNA base, whether looped-out of the DNA helix or stacked-in between the flanking bases, can be distinguished using fluorescence spectroscopy of an inserted fluorescent base. If 2-aminopurine, a structural analog of adenine and guanine, is placed in duplex DNA as the bulged base replacing an adenine or guanine, it loops out of the DNA helix into solution. This is determined by the decrease or increase of 2-aminopurine fluorescence during DNA thermomelting: if the 2-aminopurine base stacks into the helix, its fluorescence increases or remains about the same during DNA duplex melting, but if the 2-aminopurine base loops out of the helix, its fluorescence decreases upon melting of the DNA duplex.

Site-selective probing of cTAR destabilization highlights the necessary plasticity of the HIV-1 nucleocapsid protein to chaperone the first strand transfer

“…The 2-Ap9 presented a similar transition pattern, suggesting that 2-Ap9 is likely base paired with C 48 . On the contrary, downward transitions were observed for 2-Ap53 and 2-Ap55 (Figure 3B), evidencing an extra-helical conformation of G 54 when the last two ds-segments are formed (60). Indeed, such an extra-helical conformation is thought to limit the dynamical quenching of G 54 with both 2-Ap53 and 2-Ap55, explaining a higher QY for the folded state than for the melted form.…”

Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine

“…Substitution of 2-AP for A has been shown previously to have a small influence on the stability of RNA (Zagórowska and Adamiak 1996;Jiao et al 2002;Liu et al 2008;Sarkar et al 2010). It was important to determine if the 2-AP/U and A/U base pair were similar in stability in the context of a hairpin stem.…”

“…The change in fluorescence with temperature has been used to examine the change in local structure as a molecule denatures. Typically, when a basepaired 2-AP base denatures, the fluorescence intensity of the base increases due to the decrease in stacking of the base with its neighbors; conversely, an unpaired 2-AP base decreases its fluorescence as it denatures due to increased interactions with its neighbors in the single-stranded form (Menger et al 2000;Jiao et al 2002;Hardman and Thompson 2006;Ballin et al 2007;Sarkar et al 2009Sarkar et al , 2010Rau et al 2012;). The major goal of this study was to use the change in 2-AP fluorescence to investigate the structural ambiguity of the group II single nucleotide bulge loops.…”

Low-lying excited states and nonradiative processes of 9-methyl-2-aminopurine

“…The threefold torsional barrier heights V 3 were obtained by fitting the torsional function V (τ ) = V 3 2 (1 − cos 3θ ) to the calculated energies, giving V 3 = 20 cm −1 for the S 0 and V 3 = 115 cm −1 for the 1 ππ * state. The corresponding calculated internal-rotation constants are F (S 0 ) = 5.3818 cm −1 and F (S 1 ) = 5.3724 cm −1 .…”

“…[1][2][3] In contrast to adenine (6-aminopurine) and its nucleotide, both 2AP and 2AP-nucleotide are highly fluorescent in aqueous solution. [4][5][6][7] Recent multireference ab initio and density-functional excited-state calculations have explained the nonfluorescence of 9H-adenine in terms of a highly efficient nonradiative process taking place along a barrierless reaction path from the initially populated lowest excited 1 ππ * state towards a low-lying conical intersection (CI) connected to the ground state.…”