Single-Stranded Helices and Base-Stacking in RNA
Contact : Arnaud BUHOT

A schematic of a single stranded helix stabilized by base-stacking interactions (grey boxes). b is the rise per nucleotide in the helix. The random coil domains are modeled as freely-jointed chains in which the base associated with each chain segment is oriented randomly with respect to its neighbors. Each Kuhn segment of the chain contains 2l/a bases, with a the interphosphate distance per nucleotide in the random coil, and l the persistence length.

We have found strong supporting evidence for the helical structures of single-stranded nucleic acids by stretching individual molecules of polyadenylic acid [poly(A)] and polycytidylic acid [poly(C)]. Analyzing the force versus extension data using a two-state elastic model in which random-coil domains alternate with rigid helical domains allows one to extract the thermodynamic and structural properties. In addition, it also yields moderate to low cooperativity of the helix-coil transition for poly(A) and poly(C), respectively.

Single stranded (ss) nucleic acids (NA) can form rigid helical domains. These are thought to arise because of stacking interactions favoring parallel orientation of adjacent aromatic rings of the bases leading to weakly cooperative helix-coil transition. The evidence for stacking of NA in solution is mostly based on calorimetric measurements and spectroscopy. The thermodynamic parameters extracted from these measurements are not consistent and vary over a wide range. Furthermore, there is little direct evidence for ss helices at ambient temperatures. Attempts to use scattering techniques to probe the chain configurations yield inconclusive data because rod-like configurations are only attained at low temperatures. At room temperature the chains behave as living rod-coil multi-block copolymers with short helical domains whose hydrodynamic radius is similar to that of a flexible coil. In the following paper we present direct evidence for the occurrence of ss helices in ribonucleic acid homopolymers (RNA) in solution, at room temperature, and at neutral pH, as obtained from single-chain extension experiments using optical tweezers. The resulting force vs. extension curves exhibit plateaus indicative of the presence of ss helical domains due to base stacking interactions.

 

Two examples of force-extension curves for poly(A) (circles) and for poly(C) (squares) with their respective fits. For comparison a force extension curve for poly(U) is also shown (triangles), offset by +100 nm along the horizontal axis for clarity.

Renewed interest in stacking effects has arisen because of a controversy concerning the interpretation of ”molecular beacon” experiments. These involve short ssDNA chains capable of forming stem-loop structures. Experiments by the Libchaber group revealed differences in cyclization behavior of polydeoxythymidylate[poly(dT)] and polydeoxyadenylate [poly(dA)] loops that were attributed to stacking and its effects on the rigidity of the chains. In particular, it was argued that the elasticity of the two species is different because stacking is important in poly(dA) but negligible in poly(dT). This interpretation was disputed by Ansari et al. who ascribed the effects to transient trapping of misfolded loops while arguing that both poly(dT) and poly(dA) behave as flexible polymers and their elastic properties are indistinguishable. Although our results concern much longer chains of poly(A) and poly(C), we present the clear evidence of deviations from simple random coil elasticity models; the deviations indicative of helical structure formation. The interest in ss helices is however of wider origins. The occurrence of ss helices is expected to affect the secondary structure and elastic properties of RNA, and the thermodynamics of hairpin/loop formation can be expected to depend upon the fraction of ss helices. This also affects the elastic penalty incurred upon bending and/or stretching. From a biological point of view it is important to note the prevalence of homopolymers of RNA where stacking interactions are of importance. For example, the majority of eukaryotic messenger RNA is modified to include a 3’ poly(A) tail which may reach a final length of up to 250 nucleotides, with longer tails yielding increased mRNA stability and enhanced translation. In Escherichia coli, on the other hand, the poly(A) tail decreases mRNA stability, presumably because of the absence of poly(A) binding proteins which may protect the tail from exonucleases. Homopolymeric tracts and tails of poly(C) and polyuridylic acid [poly(U)] are thought to play similar roles. Such interactions between regulatory protein complexes and RNA can be expected to depend upon the local sequence, but also the tails at play.

Stretching of Homopolymeric RNA Reveals Single-Stranded Helices and Base-Stacking Yeonee Seol, Gary M. Skinner, Koen Visscher, Arnaud Buhot and Avraham Halperin, Phys. Rev. Lett. 98, 158103 (2007)

 

Maj : 13/10/2011 (456)

 

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