Cleavage of RNA bulge loops by artificial RNases

Abstract

Sensitivity of phosphodiester bonds in RNA bulge loops to cleavage by short cationic peptides and compounds based on 1,4 diazabicyclo[2.2.2]octane and its conjugates with imid azole was studied. Bulge loops containing from one to seven nucleotides were formed in RNA upon its hybridization with partially complementary oligodeoxyribonucleotides. The efficiency of RNA cleavage depends on the length of a bulge loop, the position of the cleaved phosphodiester bond in the loop, and the nature of the RNA binding fragment of chemical ribonuclease (1,4 diazabicyclo[2.2.2]octane or a cationic peptide). In the absence of Mg 2+ ions, the phosphodiester bond in the CA motif located in the apical position in 4 , 6 , or 7 membered loops is cleaved with the highest efficiency. In the presence of magnesium ions, the selectivity of RNA cleavage within bulge loops is substantially enhanced. In the case of 1,4 diaza bicyclo[2.2.2]octane based compounds, RNA is subjected to cleavage predominantly at the bonds in 4 , 6 , and 7 membered loops, whereas cleavage of other bonds is greatly suppressed.In recent years, numerous low molecular weight com pounds capable of cleaving RNA under physiological con ditions have been designed. 1-4 The RNA cleavage by these compounds occurs predominantly at the regions that are not involved in double stranded structures, such as various loops, single stranded regions, and junctions. The influence of the RNA sequence and secondary struc ture on the efficiency and specificity of cleavage was ex amined only for divalent metal ions. 5-9 The cleavage of phosphodiester bonds located in hairpin loops was stud ied in detail 5-7 since a hairpin is the main structural element of natural RNA and because of important bio logical functions of hairpin loops. 10-12 Among various RNA loops, bulge loops, i.e., RNA regions containing nucleotides that have no complementary bases in the op posite RNA strand are of special interest. These loops arrange the RNA tertiary structure, 13,14 serve as specific RNA protein interaction sites, 15 and form ribozyme ac tive sites. 16 RNA bulge loops are sensitive to cleavage by a wide range of compounds and are potential targets for antisense oligonucleotide based conjugates exhibiting site selective cleavage of RNA. Phosphodiester bonds within RNA bulge loops display a high sensitivity to cleavage by metal ions. 5,8,9 Bonds in most of RNA bulge loops or RNA:DNA complexes are cleaved by divalent metal ions, the bonds located at the 3´ side (in some cases, at the 5´ side) of each nucleotide in the loop being predominantly cleaved. 5In addition, bonds that are adjacent to the bulge loop but are involved in an RNA:DNA duplex were subject to cleavage. 5Earlier, 17 we have studied the sensitivity of phospho diester bonds in one to seven membered RNA bulge loops to cleavage by the 1,4 diazabicyclo[2.2.2]oc tane-histidine conjugate (compound ABL4C3), which cleaves RNA selectively at bonds in 4 and 7 membered loops in the presence of magnesium ions. In the present study, we carried out a comp...

Strict conformational demands of RNA cleavage in bulge-loops created by peptidyl-oligonucleotide conjugates

“…This is due to the orientation of the 2′- hydroxyl in an RNA duplex being unsuitable for intramolecular transesterification, and a conformational change would require unfavorable duplex disruption ( 29 ). Cleavage within RNA bulge-loops is widely regarded as a method to encourage catalytic turnover of chemical nucleases ( 30 , 31 ). In order to induce bulge-loops in a target RNA, the sequence of the conjugate recognition motif must be designed so that a short region (2–5nt) in the middle of the target RNA sequence remains non-complementary, thus forcing the RNA to adopt a bulge-loop structure upon hybridization.…”

“…The size of the bulge-loop can be fine-tuned by altering the size of the non-complementary regions. Previously, 3- and 4-member bulge-loops have shown the greatest potential for cleavage ( 30 , 31 ) by different catalysts, presumably due to their ability to provide the optimum flexibility for the ‘in-line’ attack of the 2′-oxyanion onto the bridging phosphodiester group, the rate-limiting step of the phosphodiester cleavage mechanism ( 30 , 31 ). Recently developed bulge-loop inducing peptide nucleic acid-based conjugates, PNAzymes, carrying toxic Cu 2+ -2,9-dimethylphenanthroline or tris(2-aminobenzimidazole) cleaving units demonstrated multiple substrate turnover against a model 15-nt RNA ( 23 , 32 , 33 ).…”