Identification of Structural Motif of RNAs Interacting with the G-rich Sequence RNA

Abstract

Guanine-rich tracts are observed in the terminal segments of eukaryotic genomes.1,2 These guanine-rich sequences have the potential to form the non-carnonical four-stranded topology called G-quadruplexes. The G-quadruplexes are built from the stacking of successive GGGG tetrads and stabilized by bound monovalent Na + and K + cations.3 The Gquadruplexes play a biological role in DNA telomere ends, the purine-rich DNA strands of the oncogenic promoter elements such as c-myc and c-kit, and RNA 5'-untranslated region (UTR) in close to translational start sites. Telomeres located at the ends of eukaryotic chromosomes are composed of the tandem DNA repeats of guanine-rich sequences and essential for chromosome stability. They appear to play a critical role in cellular aging and cancer.4-8 Telomeric DNA ends decrease 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 expressed in most cancer cells.10 So telomeres and telomerases correlated with cancer progression and have been used for the targets of anti-cancer agents.Attention has been paid to DNA quadruplexes and their potential role in biology. On the other hand, RNA quadruplexes have got less attention, despite the implication of their involvement at the site of translational control. The guanine-rich sequences, putative G-quadruplex-forming elements in the 5'-UTRs of the human genome have recently identified.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. This sequence is located 14-nucleotides downstream of the 5'-cap and 222-nucleotides upstream of the translation start site. The measured t m of this sequence was 63 o C in 1 mM K + cation and the stabilization decreased in the order KAccording to a cell-free 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 Gquadruplex formation can be the candidates for therapeutic agents, thereby inhibiting the translation of oncogenes.The expression of genetic information in RNA can be regulated by designing DNA or RNA oligonucleotide complementary to the target sequence. For example, antisense oligonucleotides were developed to specifically repress the complementary target genes.12,13 But the down regulation of specific mRNA with antisense oligonucleotide needs to be studied further because mRNAs have diverse conformers. To overcome this structural problem, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was applied to isolate RNA aptamers which specifically bind to the N-ras G-quadruplex RNA in this study. 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 ampl...

SELEX with Affinity Chromatography on RNA-RNA Interaction

“…After the 11th round of SELEX, some of the selected RNA aptamers had the consensus sequence GGGAUCCG-CAUGCAAGCUUA. 10,11 From these results, we knew that the identical RNA sequences could be selected irrespective of numbers of random nucleotides in RNA pool. The selected RNA aptamers binding to the stem-loop RNA for the synthesis of the gag-pol fusion protein of HIV-1 and the guanine-rich sequence in the 5'-UTR RNA of N-ras oncogene were thought to be the candidates of anti-viral or anti-cancer agents at genetic level.…”

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

“…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 between an RNA aptamer and a ligand RNA used for selection.…”

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

“…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.…”