ISOALLOXAZINE DERIVATIVES PROMOTE PHOTOCLEAVAGE OF NATURAL RNAS AT G-CENTER-DOT-U BASE-PAIRS EMBEDDED WITHIN HELICES

We have recently shown that isoalloxazine derivatives are able to phot ocleave RNA specifically at G.U base pairs embedded within a helical s tack, The reaction involves the selective molecular recognition of G.U base pairs by the isoalloxazine ring and the removal of one nucleosid e downstream of the uracil residue, Divalent metal ions are absolutely required for cleavage, Here we extend our studies to complex natural RNA molecules with known secondary and tertiary structures, such as tR NAs and a group I intron (td), G.U pairs were cleaved in accordance wi th the phylogenetically and experimentally derived secondary and terti ary structures, Tandem G.U pairs or certain G.U pairs located at a hel ix extremity were not affected, These new cleavage data, together with the RNA crystal structure, allowed us to perform molecular dynamics s imulations to provide a structural basis for the observed specificity, We present a stable structural model for the ternary complex of the G .U-containing helical stack, the isoalloxazine molecule and a metal io n, This model provides significant new insight into several aspects of the cleavage phenomenon, mechanism and specificity for G.U pairs, Our study shows that in large natural RNAs a secondary structure motif ma de of an unusual base pair can be recognized and cleaved with high spe cificity by a low molecular weight molecule, This photocleavage reacti on thus opens up the possibility of probing the accessibility of G.U b ase pairs, which are endowed with specific structural and functional r oles in numerous structured and catalytic RNAs and interactions of RNA with proteins, in folded RNAs.