Dynamics of mismatched base pairs in DNA

Abstract

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.

Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site

“…The r(0) values for the biexponential fits range from 0.290 to 0.375, in good agreement with previously reported r(0) values for 2AP in both DNA and RNA, which range from 0.266 to 0.377. 4,5,8,10 The longer of the two rotational correlation times (φ 1 ) reflects the overall tumbling of the RNA duplex. 10 The shorter rotational correlation time (φ 2 ) reflects the internal motion of the 2AP base in the RNA, with the subnanosecond value of φ 2 (0.47 ± 0.09 ns) obtained in the absence of bound drug being characteristic of previously reported values for 2AP residues in short DNA and RNA duplex constructs at temperatures ≥ 20 °C.…”

“…By so doing, one is then in a position to use fluorescence-based approaches to characterize the structure, energetics, and dynamics of nucleic acids and their complexes with other biologically relevant molecules. [3][4][5][6][7][8][9][10] Here, we site specifically introduce 2AP into either of two different positions (1492 or 1493) of an RNA oligonucleotide (Ec) that models the E. coli 16 S rRNA A-site (see Figure 1). We determine the impact of each 2AP substitution on the structure and stability of the RNA, and use the intrinsic fluorescence properties of 2AP to characterize the energetics and dynamics associated with the interactions between the rRNA A-site model oligomer and the aminoglycoside antibiotics, paromomycin, geneticin (G418), neomycin, and ribostamycin (see drug structures in Figure 2).…”

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations

“…boring C6:G10 base pair, leading to quenching of its fluorescence (Nordland et al 1989;Guest et al 1991). By comparison, P8 stacking appears minimal.…”

“…This construct will lead to very rapid fluorescence decay ( ) through charge transfer to the neighboring base (Wan et al 2000;Jean and Hall 2001). Fluorescence emission decay components as short as 9-20 psec (Larsen et al 2001) and 30-70 psec (Guest et al 1991) have been measured for 2AP in a stacked conforma-tion. The short lifetime in the four-exponential fit is likely to result from stacking; this component is certainly present in the ∼ 300-psec component fit to the data.…”

“…Base dynamics in the IRE RNA loop www.rnajournal.org the fluorophore (Fleming 1986;Guest et al 1991;Rachofsky et al 2001b). To characterize the dynamic motions of 2AP in the IREs, the fluorescence emission anisotropy decay was measured as a function of temperature.…”