Melting and Premelting Transitions of an Oligomer Measured by DNA Base Fluorescence and Absorption

Xu, Daguang; Evans, Kervin O.; Nordlund, Thomas M.

doi:10.1021/bi00198a027

Cited by 132 publications

(153 citation statements)

References 33 publications

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“…The melting profiles from these two sets of measurements overlap for each of the four 2AP-substituted analogs, indicating an all-or-none melting process. The melting profiles obtained from changes in the fluorescence of 2AP are, however, shifted to lower temperatures, suggesting a premelted state in which the bases are mobile while maintaining a time-averaged helical structure (21).…”

Section: Resultsmentioning

confidence: 94%

“…1). The fluorescence of 2AP decreases by about a factor of 2 when the base is hydrogenbonded and stacked compared with the single-stranded state and provides both a local and a global probe for hairpin melting (20)(21)(22). Equilibrium melting profiles that monitor the average fraction of broken bonds (absorbance at 266 nm) as well as the fraction of broken bonds at the 2AP substituted site (absorbance at 330 nm) were monitored (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins

Ansari

Kuznetsov

Shen

2001

Proc. Natl. Acad. Sci. U.S.A.

175

259

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Elucidating the mechanism of folding of polynucleotides depends on accurate estimates of free energy surfaces and a quantitative description of the kinetics of structure formation. Here, the kinetics of hairpin formation in single-stranded DNA are measured after a laser temperature jump. The kinetics are modeled as configurational diffusion on a free energy surface obtained from a statistical mechanical description of equilibrium melting profiles. The effective diffusion coefficient is found to be strongly temperaturedependent in the nucleation step as a result of formation of misfolded loops that do not lead to subsequent zipping. This simple system exhibits many of the features predicted from theoretical studies of protein folding, including a funnel-like energy surface with many folding pathways, trapping in misfolded conformations, and non-Arrhenius folding rates.H airpin loops are ubiquitous in single-stranded DNA and RNA. Knowing the time scales and mechanism of formation of these loops is an essential first step toward understanding the folding problem. Although the stability of hairpin loops and the kinetics of hairpin formation have been a subject of intense investigation for over 30 years (1-5), our understanding of the kinetics is limited. In particular, there is no simple physical model that describes in a consistent way both the thermodynamics and kinetics of hairpin formation. With the exception of some early work on the helix-to-coil transition, in which the kinetics were described in terms of a statistical mechanical kinetic ''zipper'' model (6, 7), the kinetics of hairpin-to-coil transition have been described more recently in terms of a two-state system with Arrhenius temperature dependence for the rates of hairpin formation and unwinding (5, 8). The equilibrium dynamics of hairpins, obtained from fluctuation correlation spectroscopy measurements of hairpins labeled with fluorescent donor and acceptor pairs (8, 9), have revealed a number of kinetic features that are not easily explained within the framework of a simple two-state analysis. First, the data of Libchaber and coworkers show that the rate coefficient corresponding to the closing of hairpins has a non-Arrhenius temperature dependence (8). Second, they report a puzzling result in which the apparent activation energy for forming hairpins with poly(dA) loops increases as the loop size increases. Third, Klenerman and coworkers report stretched exponential kinetics at temperatures well below the melting temperature (9). These observations suggest a failure of the simplest two-state analysis and require a modification of even the more rigorous kinetic ''zipper'' model in the form in which it was applied to helix-coil kinetics (6).Here we present a model for the dynamics of hairpins that is consistent with many of the apparently anomalous kinetic observations. The dynamics are described as configurational diffusion along a free energy profile that we calculate from a statistical mechanical ''zipper'' model that describes the equilibrium melt...

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins

Ansari

Kuznetsov

Shen

2001

Proc. Natl. Acad. Sci. U.S.A.

175

259

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“…Investigations of the structure and stability of Ap-containing DNA duplexes by NMR, fluorescence, and calorimetry have shown that Ap undergoes normal Watson-Crick pairing with T and is well stacked within the DNA helix in a manner that is similar to adenine (37)(38)(39)(40). The guanine analog, I, is base-paired with C. DNA duplexes substituted with I are not identical to their G-containing counterparts as the exchange of G for I results in the loss of the exocyclic amino group in the minor groove and the loss of one H bond in the base pair with C. However, the structural consequences of a single I substitution are relatively minor, based on crystal structures (41,42) and theoretical investigations (43).…”

Section: Resultsmentioning

confidence: 99%

“…Many such investigations rely on the notion that base stacking is solely responsible for quenching Ap* fluorescence. Yet, various mechanisms and͞or conditions, including stacking (39,48), hydrogen bonding (34,38), base-base collisions (48), and CT reactions (6,7,32) have been proposed to explain the quenching of Ap* fluorescence in DNA. Furthermore, the dynamic exchange of Ap between conformational states in DNA modulates stacking, H bonding, and other base-base interactions and reactions on the fluorescence time scale, and thereby regulates all quenching reactions.…”

Section: Discussionmentioning

confidence: 99%

Effects of strand and directional asymmetry on base–base coupling and charge transfer in double-helical DNA

O’Neill

Barton²

2002

Proc. Natl. Acad. Sci. U.S.A.

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Mechanistic models of charge transfer (CT) in macromolecules often focus on CT energetics and distance as the chief parameters governing CT rates and efficiencies. However, in DNA, features unique to the DNA molecule, in particular, the structure and dynamics of the DNA base stack, also have a dramatic impact on CT. Here we probe the influence of subtle structural variations on base-base CT within a DNA duplex by examining photoinduced quenching of 2-aminopurine (Ap) as a result of hole transfer (HT) to guanine (G). Photoexcited Ap is used as a dual reporter of variations in base stacking and CT efficiency. Significantly, the unique features of DNA, including the strandedness and directional asymmetry of the double helix, play a defining role in CT efficiency. For an (AT)n bridge, the orientation of the base pairs is critical; the yield of intrastrand HT is markedly higher through (A)n compared with (T)n bridges, whereas HT via intrastrand pathways is more efficient than through interstrand pathways. Remarkably, for reactions through the same DNA bridge, over the same distance, and with the same driving force, HT from photoexcited Ap to G in the 5 to 3 direction is more efficient and less dependent on distance than HT from 3 to 5 . We attribute these differences in HT efficiency to variations in base-base coupling within the DNA assemblies. Thus base-base coupling is a critical parameter in DNA CT and strongly depends on subtle structural nuances of duplex DNA.T he transport of electronic charge through double-helical DNA continues to fascinate and surprise us (1-5). A wealth of experimental evidence has established, undeniably, that DNA can act as a conduit for rapid and long-range charge transfer (CT), not only in DNA assemblies designed in the laboratory (6-24), but also in biologically significant environments (25,26). DNA, remarkable for its role in the molecular basis of life, may also play a role as a mediator of CT in diverse areas of chemistry and biology. Indeed, the ability of DNA to mediate CT and the exquisite sensitivity of this chemistry to DNA structure has spawned the development of a completely new family of diagnostic tools (27,28). Yet, while we continue to probe and exploit the distinctive ability of DNA to transport charge, fundamental questions concerning the mechanisms and features of DNA CT remain.We investigate DNA CT by using a combination of spectroscopic, biochemical, and electrochemical tools to interrogate well-characterized assemblies that incorporate redox-active molecules from a large repertoire including metallointercalators, organic intercalators, and modified DNA bases (6)(7)(8)(9)(10)(11)(12)(25)(26)(27)(28). Consistently, we have observed that DNA CT is remarkably sensitive to the structure and dynamics of the DNA bases. Experiments in which the DNA base stack is altered by, for instance, sequence variation (10), mismatches (9, 28), structural perturbations (11), or protein binding (12,27) emphasize the importance of the integrity of the -stack to DNA CT. In addition, time-...

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“…Upon phosphodiester cleavage, the base stacking, solvation, and dynamics of 2AP are likely to change substantially. 22 Since the photophysical properties of 2AP are known to change in response to changes in its local environment, 23À26 it is expected that continuous fluorescence measurements can be used to monitor the progress of ribozyme-mediated cleavage in real time. 27 …”

Section: Designmentioning

confidence: 99%

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

Kirk

2001

Bioorganic &Amp; Medicinal Chemistry

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Abstract-The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidinoneomycin B, and [Á-(Eilatin)Ru(bpy) 2 ] 2+ , prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [Á-(Eilatin)Ru(bpy) 2 ] 2+ , and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500 mM. #

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Melting and Premelting Transitions of an Oligomer Measured by DNA Base Fluorescence and Absorption

Cited by 132 publications

References 33 publications

Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins

Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins

Effects of strand and directional asymmetry on base–base coupling and charge transfer in double-helical DNA

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

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