No abstract

A cytidine-free ribozyme with RNA ligase activity was obtained by in vitro evolution, starting from a pool of randomsequence RNAs that contained only guanosine, adenosine, and uridine. This ribozyme contains 74 nt and catalyzes formation of a 39,59-phosphodiester linkage with a catalytic rate of 0.016 min -1 . The RNA adopts a simple secondary structure based on a three-way junction motif, with ligation occurring at the end of a stem region located several nucleotides away from the junction. Cytidine was introduced to the cytidine-free ribozyme in a combinatorial fashion and additional rounds of in vitro evolution were carried out to allow the molecule to adapt to this added component. The resulting cytidine-containing ribozyme formed a 39,59 linkage with a catalytic rate of 0.32 min -1 . The improved rate of the cytidine-containing ribozyme was the result of 12 mutations, including seven added cytidines, that remodeled the internal bulge loops located adjacent to the three-way junction and stabilized the peripheral stem regions.

Failures in trials for Alzheimer's disease (AD) may be attributable to inadequate dosing, population selection, drug inefficacy, or insufficient design optimization. The Coalition Against Major Diseases (CAMD) was formed in 2008 to develop drug development tools (DDT) to expedite drug development for AD and Parkinson's disease. CAMD led a process that successfully advanced a clinical trial simulation (CTS) tool for AD through the formal regulatory review process at the US Food and Drug Administration (FDA) and European Medicines Agency (EMA).

The RNA-world hypothesis proposes that, before the advent of DNA and protein, life was based on RNA, with RNA serving as both the repository of genetic information and the chief agent of catalytic function. An argument against an RNA world is that the components of RNA lack the chemical diversity necessary to sustain life. Unlike proteins, which contain 20 different amino-acid subunits, nucleic acids are composed of only four subunits which have very similar chemical properties. Yet RNA is capable of a broad range of catalytic functions. Here we show that even three nucleic-acid subunits are sufficient to provide a substantial increase in the catalytic rate. Starting from a molecule that contained roughly equal proportions of all four nucleosides, we used in vitro evolution to obtain an RNA ligase ribozyme that lacks cytidine. This ribozyme folds into a defined structure and has a catalytic rate that is about 10(5)-fold faster than the uncatalysed rate of template-directed RNA ligation.