Identification of a Conserved Sequence Motif of an RNA Aptamer Binding to a G-rich Sequence RNA with Structural Probes

Abstract

Guanine-rich sequences observed in terminal segments of eukaryotic genomes 1,2 have the potential to form the noncanonical four-stranded topology called G-quadruplexes, which are built from the stacking of successive GGGG tetrads and stabilized by bound monovalent Na + and K + cations.3 They appear to play a critical role in cellular aging and cancer.4-8 The end of telomeric DNA decreases in length after each round of cell division in somatic cells.9 But the telomere length can be maintained by the enzyme telomerase, a ribonucleoprotein complex with reverse transcriptase activity in most cancer cells.10 The guanine-rich sequences, putative G-quadruplex-forming elements in the 5'-UTRs of the human genome have been indentified.11 One of these sequences, an 18-mer containing four guanine-tracts, 5'-GGGAGGGGCGGGUCUGGG-3' is associated with the 5'-UTR of the oncogenic N-ras sequence. According to a cellfree translation system coupled to a reporter gene assay, the N-ras G-quadruplex can inhibit gene expression at the translational level.11 This result indicates that molecules stabilizing 5'-UTR RNA G-quadruplex formation can be the candidates for therapeutic agents, thereby inhibiting the translation of the oncogene.SELEX is a technique for isolating nucleic acid molecules (aptamers) with affinities for a target molecule from a random pool with a large number of sequences by the iterative rounds of affinity selection and amplification. Target molecules include proteins, amino acids, nucleotides, antibiotics and RNA.12-24 We isolated RNA molecules binding to the guanine-rich RNA (Fig. 1) in the 5'-UTR RNA of N-ras oncogene from an RNA pool.25 After the 11th round of in vitro selection, the sequences of the selected RNA aptamers were closely related and had the consensus sequence GGGAUCCGCAUGCAAGCUUA.26 This sequence is thought to be important to the interaction between the selected RNA aptamer and the ligand G-rich sequence RNA. 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. So it is important to determine the structure of the selected RNA aptamers and to get the information for the interaction between an RNA aptamer and a ligand RNA used for selection. In this study, the secondary structure of a selected RNA aptamer was determined with RNA structural probes such as RNase T1 and RNase V1 and the conserved sequence motif for the interaction between an RNA aptamer and a ligand G-rich sequence RNA was identified.The secondary structure of 11-30-24 (Fig. 1), one of RNA aptamers selected from an RNA pool including the constant primer sequences, is predicted and shown in Figure 2. This structure was found by the CLC RNA workbench ver. 4.2 program accessed on the internet (www.clcbio.com). The biochemical experiments to investigate the predicted secondary structure of the RNA aptamer were performed by using RNase T1 that has the specificity for a guanine in singlestranded region and RNase V1 that is double-...