| Popis: |
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. |
