Development of a selection assay for small guide RNAs that drive efficient site-directed RNA editing.

A major challenge confronting the clinical application of site-directed RNA editing (SDRE) is the design of small guide RNAs (gRNAs) that can drive efficient editing. Although many gRNA designs have effectively recruited endogenous Adenosine Deaminases that Act on RNA (ADARs), most of them exceed the size of currently FDA-approved antisense oligos. We developed an unbiased in vitro selection assay to identify short gRNAs that promote superior RNA editing of a premature termination codon.

Cationic oligonucleotide-peptide conjugates with aggregating properties enter efficiently into cells while maintaining hybridization properties and enzymatic recognition.

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems.

A novel, modification-dependent ATP-binding aptamer selected from an RNA library incorporating a cationic functionality.

An analogue of uridine triphosphate containing a cationic functional group was incorporated into a degenerate RNA library by enzymatic polymerization. In vitro selection experiments using this library yielded a novel receptor that binds ATP under physiological pH and salt conditions in a manner completely dependent on the presence of the cationic functionality. The consensus sequence and a secondary structure model for the ATP binding site were obtained by the analysis of functional sequences selected from a partially randomized pool based on the minimal parental sequence.

An HIV reverse transcriptase-selective nucleoside chain terminator.

The synthesis of a 2',3'-dideoxynucleoside cytidine analogue, but one that lacks the O2-carbonyl, is described from 2-aminopyridine in an overall yield of 60%. The synthesis of the 2-pyridone C-nucleoside relies upon the use of a Heck-type coupling between an appropriately protected sugar glycal and the 5-iodo derivative of 2-aminopyridone. Upon conversion of the dideoxynucleoside to the corresponding 5'-triphosphate, the analogue ddNTP is observed to be a reasonable substrate with HIV reverse transcriptase (for a template dG residue), but is not a substrate for calf thymus DNA polymerase alpha or for human DNA polymerase beta.