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Biophysical techniques, such as single molecule FRET, fluorescence microscopy, single ion-channel patch clamping, and optical tweezers often yield data that are noisy time series containing discrete steps. Here we present a method enabling objective identification of nonuniform steps present in such noisy data. Our method does not require the assumption of any underlying kinetic or state models and is thus particularly useful for analysis of novel and poorly understood systems. In contrast to other model-independent methods, no parameters or other information is taken from the user. We find that, at high noise levels, our method exceeds the performance of other model-independent methods in accurately locating steps in simulated noisy data.

The RNA homopolymer poly(A) forms single-stranded helices due to base-stacking, the mechanical properties of which may affect the biological function of naturally-occurring A-repeat sequences. Since RNA is a polyanion, screening and binding by cations enhances this helix formation by shielding charges along its backbone from each other.The poly(A) single helix may be disrupted by application of tension, as by optical tweezers, allowing the energetics of helix formation to be estimated in addition to the mechanical properties of the helical and randomly-coiled states.Using optical tweezers, we measured the force-extension behavior of poly(A) under a variety of buffer conditions, finding that the effect of sodium is consistent with Debye screening but that magnesium and other Group II cations are likely to be binding and forming inner-sphere (direct) contacts with poly(A), stabilizing the helix more than would be expected for screening alone. The mode of binding appears to be dependent on cation species, with distinct differences between binding by strontium and by magnesium and calcium.To obtain quantitative information from these force-extension measurements, we have developed a new model for the force-extension behavior of poly(A) and similar helicogenic polymers, which unlike previous models takes into account Debye screening effects and the semiflexible nature of the helical state. Fitting this model to our data, we obtain estimates of helix formation energies and find evidence that magnesium binding distorts the poly(A) single helix into a mechanically distinct state.

Using mechanical unfolding by optical tweezers (OT) and steered molecular dynamics (SMD) simulations, we have demonstrated the critical role of Mg(2+) ions for the resistance of the Beet Western Yellow Virus (BWYV) pseudoknot (PK) to unfolding. The two techniques were found to be complementary, providing information at different levels of molecular scale. Findings from the OT experiments indicated a critical role of stem 1 for unfolding of the PK, which was confirmed in the SMD simulations. The unfolding pathways of wild type and mutant appeared to depend upon pH and nucleotide sequence. SMD simulations support the notion that the stability of stem 1 is critical for -1 frameshifting. The all-atom scale nature of the SMD enabled clarification of the precise role of two Mg(2+) ions, Mg45 and Mg52, as identified in the BWYV X-ray crystallography structure, in -1 frameshifting. On the basis of simulations with "partially" and "fully" hydrated Mg(2+) ions, two possible mechanisms of stabilizing stem 1 are proposed. In both these cases Mg(2+) ions play a critical role in stabilizing stem 1, either by directly forming a salt bridge between the strands of stem 1 or by stabilizing parallel orientation of the strands in stem 1, respectively. These findings explain the unexpected drop in frameshifting efficiency to null levels of the C8U mutant in a manner consistent with experimental observations.