Nucleic acids through condensation of nucleosides and phosphorous acid in the presence of sulfur

Short phosphorothioate oligonucleotides have been prepared by refluxing an equimolar mixture of thymidine and triethylammonium phosphite in toluene in the presence of elemental sulfur. Desulfurization and subsequent digestion of the products by P1 nuclease revealed that nearly 80% of the internucleosidic linkages thus formed were of the canonical 3',5'-type

Understanding Catalysis of Phosphate-Transfer Reactions by the Large Ribozymes

Large ribozymes are unique among catalytic RNA molecules in that their reactions involve intermolecular nucleophilic attack on an RNA phosphodiester linkage. Crystal structures of near-atomic resolution are now available for the group I and group II self-splicing introns and the RNA subunit of RNase P. The structural data agrees well with the earlier models proposed on the basis of biochemical studies and the evidence at hand suggests that all of the large ribozymes utilize a mechanism in which coordination of MgII ions reduces the negative charge on the scissile phosphodiester linkage, as well as assists both the nucleophilic attack and the departure of the leaving group

Impact of steric constraints on the product distribution of phosphate-branched oligonucleotide models of the large ribozymes

To assess the extent to which steric constraints may influence the product distribution of the reactions of the large ribozymes, phosphate-branched oligonucleotides of varying length and sequence have been synthesized and their alkaline hydrolysis studied over a wide temperature range. At low temperatures, the branching trinucleoside-3 ',3 ',5 '-monophosphate moiety is hydrolyzed almost exclusively by P-O3 ' fission. At higher temperatures, P-O5 ' fission competes, accounting at most for 22% of the overall reaction. The results suggest that steric constraints imposed by the secondary structure of the reaction site may significantly contribute to the observed regioselectivity of the transesterification reactions catalyzed by the large ribozymes

Intracomplex general acid/base catalyzed cleavage of RNA phosphodiester bonds: the leaving group effect

The general acid/base catalyzed cleavage of a number of alkyl esters of uridine-3'-(and -5'-) phosphate has been studied by utilizing a cleaving agent, in which the catalytic moiety (a substituted 1,3,5-triazine) is tethered to an anchoring Zn-II: cyclen moiety. Around pH 7, formation of a strong ternary complex between uracil, Zn-II and cyclen brings the general acid/base catalyst close to the scissile phosphodiester linkage, resulting in rate acceleration of 1-2 orders of magnitude with the uridine-3'-phosphodiesters. Curiously, no acceleration was observed with their 5'-counterparts. A beta(lg) value of -0.7 has been determined for the general acid/base catalyzed cleavage, consistent with a proton transfer to the leaving group in the rate-limiting step

Oligonucleotides Incorporating Palladacyclic Nucleobase Surrogates

An oligonucleotide incorporating a palladacyclic nucleobase has been prepared by ligand-directed metalation of a phenylpyridine moiety. This oligonucleotide hybridized with natural counterparts placing any of the canonical nucleobases opposite to the palladacyclic residue. The palladated duplexes had B-type conformation and melting temperatures comparable to those of respective unmodified duplexes with a single mismatch. In the duplexes placing C, G or T (but not A) opposite to the palladacyclic residue, greatly increased absorptivity suggested formation of a Pd-II-mediated base pair. Absorptivity and ellipticity of these duplexes persisted even at the highest temperatures applicable in T-m and CD experiments (90 degrees C). Evidently the Pd-II-mediated base pairs do not dissociate under the experimental conditions

Mimics of small ribozymes utilizing a supramolecular scaffold

For elucidating the mechanism of the general acid/base catalysis of the hydrolysis of RNA phosphodiester bonds, a number of cleaving agents having two cyclen moieties tethered to a 1,3,5-triazine core have been prepared and their ability to bind and cleave uridylyl-3', 5'-uridine (UpU) studied over a wide pH range. Around neutral pH, the cleaving agents form a highly stable ternary complex with UpU and Zn-II through coordination of the uracil N3 and the cyclen nitrogen atoms to the Zn-II ions. Under conditions where the triazine core exists in the deprotonated neutral form, hydrolysis of UpU, but not of adenylyl-3',5'-adenosine (ApA), is accelerated by approximately two orders of magnitude in the presence of the cleaving agents, suggesting general base rather than metal ion catalysis. The probable mechanism of the observed catalysis and implications to understanding the general acid/base-catalyzed phosphodiester hydrolysis by ribozymes are discussed.</p

Oligonucleotide-Palladacycle Conjugates as Splice-Correcting Agents

2'-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5'-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 degrees C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents

Oxidation of an Oligonucleotide-Bound Ce-III/Multiphosphonate Complex for Site-Selective DNA Scission

Oligodeoxyribonucleotide conjugates of ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid) (EDTP) have been used to place a Ce-III/EDTP complex in close proximity to predetermined phosphodiester linkages of a complementary target oligonucleotide. In the presence of atmospheric oxygen, the Ce-III is oxidized into Ce-IV which, in turn, efficiently cleaves the target phosphodiester linkage. No cleavage occurs at the other single-stranded regions, which suggests that the catalytic Ce species is strictly localized next to the target phosphodiester linkage. No decrease in the reaction rate is observed upon introduction of scavengers for hydroxyl radicals (such as DMSO or MeOH) or singlet oxygen (such as NaN3) to the system; this indicates that the reaction proceeds via a hydrolytic pathway. Any significant contribution by an oxidative pathway is further ruled out by the observation that nucleosides remain intact after incubation with Ce-IV/EDTP complex for extended periods

Stabilization of ribozyme-like cis-noncoding rRNAs induces apoptotic and nonapoptotic death in lung cells

Bidirectional non-protein-coding RNAs are ubiquitously transcribed from the genome. Convergent sense and antisense transcripts may regulate each other. Here, we examined the convergent cis-noncoding rRNAs (nc-rRNAs) in A5 and E9 lung cancer models. Sense nc-rRNAs extending from rDNA intergenic region to internal transcribed spacer of around 10 kb in length were identified. nc-rRNAs in sense direction exhibited in vitro characteristics of ribozymes, namely, degradation upon incubation with MgCl2 and stabilization by complementary oligonucleotides. Detection of endogenous cleavage-ligation products carrying internal deletion of hundreds to thousands nucleotides by massively parallel sequencing confirmed the catalytic properties. Transfection of oligonucleotides pairing with antisense nc-rRNAs stabilized both target and complementary transcripts, perturbed rRNA biogenesis, and induced massive cell death via apoptotic and/or nonapoptotic mechanisms depending on cell type and treatment. Oligonucleotides targeting cellular sense transcripts are less responsive. Spontaneously detached cells, though rare, also showed accumulation of nc-rRNAs and perturbation of rRNA biogenesis. Direct participation of nc-rRNAs in apoptotic and nonapoptotic death was demonstrated by transfection of synthetic nc-rRNAs encompassing the rDNA promoter. In sum, convergent cis-nc-rRNAs follow a feed-forward mechanism to regulate each other and rRNA biogenesis. This opens an opportunity to disrupt rRNA biogenesis, commonly upregulated in cancers, via inhibition of ribozyme-like activities in nc-rRNAs