Ravi Vinayak

AN EFFICIENT METHOD FOR THE ISOLATION AND PURIFICATION OF OLIGORIBONUCLEOTIDES

Abstract Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.

Fast oligonucleotide deprotection phosphoramidite chemistry for DNA synthesis

Abstract A new set of base protecting groups for cyanoethylphosphoramidite nucleosides and supports has been developed which decreases the post-synthesis time requirements. The traditional purine amide protecting groups, which require 8-16 hours at 55°C for deprotection in ammonia, have been replaced with the dimethylformamidine group. Oligonucleotides made with the new reagents require only 1 hour at 55°C or 8 hours at room temperature for complete deprotection. Dialkylformamidine phosphoramidites exhibit enhanced resistance to depurination compared to the traditional, or even the phenoxyacetyl, phosphoramidites.

Solvent, not palladium oxidation state, is the primary determinant for successful coupling of terminal alkynes with iodo-nucleosides☆

Abstract Coupling of iodo-nucleosides with terminal alkynes such as 3-(acylamino)propynes, whose initial products readily undergo secondary cyclization reactions, can be effected smoothly by the standard catalysis with (Ph 3 P) 2 PdCl 2 /CuI/Et 3 N in dimethylformamide.

A convenient automated solid-phase synthesis of PNA-(5′)-DNA-(3′)-PNA chimera

An automated online solid-phase synthesis of Ac-cac ct T∗GG TC t∗ac ct-Gly-OH1 using standard DNA and appropriately protected PNA building blocks (2–5) is described. This chimera forms stable duplexes with complementary DNA and RNA.

Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA.

The Hin recombinase of Salmonella catalyzes a site‐specific recombination event which leads to flagellar phase variation. Starting with a fully symmetrical recombination site, hixC, a set of 40 recombination sites which vary by pairs of single base substitutions was constructed. This set was incorporated into the Salmonella‐specific bacteriophage P22 based challenge phage selection and used to define the DNA sequence determinants for the binding of Hin to DNA in vivo. The critical sequence‐specific contacts between a Hin monomer and a 13 bp hix half‐site are at two T:A base pairs in the major groove of the DNA which are separated by one base pair, and two consecutive A:T contacts in the minor groove. The base substitutions in the major groove recognition portion which were defective in binding Hin still retained residual binding capability in vivo, while the base pair substitutions affecting the minor groove recognition region lost all in vivo binding. Using in vitro binding assays, Hin was found to bind to hix symmetrical sites with A:T base pairs or I:C base pairs in the minor groove recognition sequences, but not to G:C base pairs. In separate in vitro binding assays, Hin was equally defective in binding to either a G:C or a I:C contact in a major groove recognition sequence. Results from in vitro binding assays to hix sites in which 3‐deazaadenine was substituted for adenine are consistent with Hin making a specific contact to either the N3 of adenine or O2 of thymine in the minor groove within the hix recombination site on each symmetric half‐site. These results taken with the results of previous studies on the DNA binding domain of Hin suggest a sequence‐specific minor groove DNA binding motif.

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology.

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.

Large Scale Synthesis of Oligoribonucleotides on High-Loaded Polystyrene (HLP) Support

Abstract Large quantities of oligoribonucleotides (up to 200 μmole) were synthesized on the high-loaded polystyrene (HLP) support with phosphoramidite nucleosides and 5-ethylthio-1H-tetrazole as activator. The HLP support significantly reduces solvent and reagent consumption. RNA synthesized on HLP support at large scale was shown to have full biological activity by a comparative ribozyme-substrate assay.

Automated Chemical Synthesis of PNA and PNA-DNA Chimera on a Nucleic Acid Synthesizer

Abstract Automated chemical synthesis of PNA and PNA-DNA chimera on a DNA synthesizer using the monomethoxytrityl/acyl protecting group strategy is described.

A CONVENIENT, SOLID-PHASE COUPLING OF RHODAMINE DYE ACIDS TO 5' AMINO-OLIGONUCLEOTIDES

Abstract A convenient method has been developed for directly labeling the 5′ amino group of oligonucleotides on solid-support with rhodamine dyes to give rhodamine-labeled oligonucleotides.

Thermal melting studies with PNA and PNA-DNA chimera

Abstract We have performed thermal melting studies toward developing an empirical formula for PNA-DNA duplexes. Our results indicate the identity and sequence of the junction between the phosphodiester and 2-aminoethylglycine backbones are critical to the overall stability of the duplex and an important design consideration. Thermal melting studies toward developing an empirical formula for PNA-DNA duplexes were performed. The results indicate the identity and sequence of the junction between the phosphodiester and 2-aminoethylglycine backbones is critical to the overall stability of the duplex.