Solid‐Phase Synthesis of Branched Oligonucleotides

Branched nucleic acids (bNAs) have been of particular interest since the discovery of RNA forks and lariats as intermediates of nuclear mRNA splicing, as well as multicopy, single‐stranded DNA (msDNA). Such molecules contain the inherent trait of vicinal 2′,5′‐ and 3′,5′‐phosphodiester linkages. bNAs have many potential applications in nucleic acid biochemistry, particularly as tools for studying the substrate specificity of lariat debranching enzymes, and as biological probes for the investigation of branch recognition during pre‐mRNA splicing. The protocols described herein allow for the facile solid‐phase synthesis of branched DNA and/or RNA oligonucleotides of varying chain length, containing symmetrical or asymmetrical sequences immediate to an RNA branch point. The synthetic methodology utilizes widely adopted phosphoramidite chemistry. Methods for efficient purification of bNAs via anion‐exchange HPLC and PAGE are also illustrated.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143634/1/cpnc0414.pd

A Brief History, Status, and Perspective of Modified Oligonucleotides for Chemotherapeutic Applications

The advent of rapid and efficient methods of oligonucleotide synthesis has allowed the design of modified oligonucleotides that are complementary to specific nucleotide sequences in mRNA targets. Such modified oligonucleotides can be used to disrupt the flow of genetic information from transcribed mRNAs to proteins. This antisense strategy has been used to develop therapeutic oligonucleotides against cancer and various infectious diseases in humans. This overview reports recent advances in the application of oligonucleotides as drug candidates, describes the relationship between oligonucleotide modifications and their therapeutic profiles, and provides general guidelines for enhancing oligonucleotide drug properties.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143788/1/cpnc0401.pd

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme

Chemische Synthese von katalytischen Oligonucleotiden und ihre Erprobung an Tumormodellsystemen Abschlussbericht

SIGLEAvailable from TIB Hannover: F97B2258+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman