Sharon T. Cload

SELEX with modified nucleotides

Aptamers, a promising new class of therapeutics, are single-stranded oligonucleotides generated via an in vitro selection process that bind to and inhibit the activity of target proteins in a manner similar to therapeutic antibodies. In order to enhance the drug-like character of aptamers, oligonucleotide libraries containing modified nucleotides are increasingly being used for selection. Principally, the choice of modifications aims to increase aptamer potency by enhancing nuclease-resistance, or increasing target affinity by providing more target recognition functionality or generating more stable aptamer structures.

Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids

BACKGROUND Chemically aminoacylated suppressor tRNAs have previously been used in vitro to generate mutant proteins in which unnatural amino acids are incorporated site-specifically. Although the existing methodology often provides adequate quantities of mutant proteins, the suppression efficiencies of some unnatural amino acids are not high enough to yield useful amounts of protein. In an effort to make this useful mutagenesis strategy more general, we report here the results of a search to find alternative tRNAs as a way of increasing suppression efficiencies. RESULTS Three suppressor tRNAs have been generated by runoff transcription and their ability to deliver unnatural amino acids site-specifically into proteins has been assessed in an E. coli-derived in vitro transcription/translation system. Analysis of their ability to insert both polar and nonpolar residues in response to an amber codon in two proteins suggests that an E. coli tRNAAsn-derived suppressor offers a significant improvement in suppression efficiency over other previously used tRNAs. CONCLUSIONS Use of an E. coli tRNAAsn-derived suppressor may provide substantially higher yields of proteins containing unnatural amino acids, in addition to offering a broader tolerance for polar amino acids. A comparison of suppressor tRNAs derived from tRNAAsn, tRNAGln or tRNAAsp with that derived from tRNAPhe supports emerging evidence that the identity of an amino acid may be important in message recognition.

Novel Therapeutic Modalities to Address Nondrugable Protein Interaction Targets

Small molecule drugs are relatively effective in working on ‘drugable’ targets such as GPCRs, ion channels, kinases, proteases, etc but ineffective at blocking protein–protein interactions that represent an emerging class of ‘nondrugable’ central nervous system (CNS) targets. This article provides an overview of novel therapeutic modalities such as biologics (in particular antibodies) and emerging oligonucleotide therapeutics such as antisense, small-interfering RNA, and aptamers. Their key properties, overall strengths and limitations, and their utility as tools for target validation are presented. In addition, issues with regard to CNS targets as it relates to the blood–brain barrier penetration are discussed. Finally, examples of their application as therapeutics for the treatment of pain and some neurological disorders such as Alzheimers disease, multiple sclerosis, Huntingtons disease, and Parkinsons disease are provided.