Secondary Structure Analysis of an RNA Interacting with Guanine-rich Sequence

Abstract

RNA structure has sometimes been analyzed by biophysical method such as NMR (nuclear magnetic resonance), which has not been popular because the RNA with small size only is available and a large amount of RNA is required for the structural analysis with NMR.1 So biochemical methods using structure specific enzymes and chemicals have widely been used for the analysis of RNA structure. 2-11Enzymes which have mainly been used for probing RNA structure in solution, are double-strand-specific RNase V1, single-strand-specific nuclease S1 and RNase T1 which has the specificity for a guanine in single strand region. Chemicals used for probing RNA structure, are DMS (dimethyl sulfate) which methylates position N-1 of adenines and, to a lower extent, N-3 of cytosines, CMCT (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate) which modifies position N-3 of uridine and, to a weaker degree, N-1 of guanines and kethoxal (3-ethoxy-1,1-dihydroxy-2-butanone) which modifies position N-1 and N-2 of guanine in the single strand.Hydroxyl radical (·OH) has also been used for the highorder structure analysis of RNA. Exposed nucleotides are damaged by hydroxyl radical while nucleotides involved in tertiary contacts are protected from damage, making it a favorable approach for establishing exterior/interior relations for RNA.12-18 Radicals are generated from hydrogen peroxide (H 2 O 2 ) with Fe(II)-EDTA. Ascorbate (or DTT) is added to reduce Fe(III) to Fe(II). Hydrogen abstraction from the ribose 4' carbon leads to strand scission.In-line probing is also an RNA-structure probing method developed by Breaker group.19-21 This method has been used to examine secondary structure of RNAs and whether RNAs undergo structural rearrangements under the different incubation conditions. In-line probing takes advantage of the fact that the spontaneous cleavage of RNA is dependent on the local structure at each inter-nucleotide linkage. RNA degrades through a nucleophilic attack by the 2' oxygen on the adjacent phosphorus. Cleavage occurs efficiently when the attacking 2' oxygen, the phosphorus and the departing 5' oxygen of the phosphodiester linkage are in a linear configuration. Linkages in double strand region of a folded RNA show resistance to cleavage because it is difficult for the atoms to be held in an in-line configuration. However, if folding does not restrict its structure, linkages occasionally take on in-line geometry through random motion and therefore are subject to a spontaneous cleavage.RNA aptamers which are capable of binding to the guaninerich sequence RNA (5'-GGGAGGGGCGGGUCUGGG-3') in the 5'-UTR (untranslated region) of N-ras oncogene, were selected from a random-sequence RNA library with RNA-RNA interaction. 22 The selected RNA aptamers can recognize the specific domain of RNA structure like a monoclonal antibody and be candidates of the anticancer agent at the genetic level. The determination of the structure of selected RNA aptamers is very important prior to getting the information for the interaction be...