An in vitro evolution procedure was used to obtain RNA enzymes with a particular catalytic function. A population of 10(13) variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce "progeny" ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

The catalytic specificity of T7 RNA polymerase (RNAP) for ribonucleoside triphosphates vs deoxynucleoside triphosphates {(kcat/Km)rNTP/(kcat/Km)dNTP} during transcript elongation is approximately 80. Mutation of tyrosine 639 to phenylalanine reduces specificity by a factor of approximately 20 and largely eliminates the Km difference between rNTPs and dNTPs. The remaining specificity factor of approximately 4 is kcat-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the Km difference between rNTPs and dNTPs. Mn2+ substitution also enhances the activity of poorly active mutant enzymes carrying nonconservative substitutions in the active site, and its effects are generally consistent with the Mn2+-catalyzed reaction being less restrictive in its requirements for alignment of the reactive groups. In addition to discrimination occurring at the level of nucleoside monophosphate (NMP) incorporation, it is also found that transcripts containing deoxynucleoside monophosphates (dNMPs) are more poorly extended than transcripts of canonical structure, though a severe barrier to transcript extension is seen only when the 3' region of the transcript is heavily substituted with dNMPs. The barrier to extension of transcripts heavily substituted with dNMPs is reduced for sequences known to be amenable to forming A-like helices and is larger for sequences that resist transformation from B-form DNA.DNA structures. The barrier to extension of dNMP-substituted transcripts is also reduced by solution conditions known to destabilize B-form DNA and to stabilize A-form structures. These observations imply a requirement for a non-B-form, possibly A-like, conformation in the transcript.template hybrid that is disrupted when the transcript is of predominantly deoxyribose structure.

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA (siRNA), QPI-1007, following intravitreal administration. QPI-1007 is a chemically modified siRNA designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the caspase 2 protein and is being developed as a neuroprotectant for the treatment of nonarteritic anterior ischemic optic neuropathy and other optic neuropathies such as glaucoma that result in the death of retinal ganglion cells. The half-life of QPI-1007 in the vitreous and retina/choroid in the Dutch Belted rabbit was about 2 days, and there was no sign of accumulation after repeated administrations at either 2- or 4-week dosing intervals in the rabbit. QPI-1007 was well tolerated in Dutch Belted rabbits following single or repeated intravitreal administrations of up to 11 doses over 9 months. Test-article-related effects were limited to the eyes, with minimal to mild vitreal cellular infiltration being the major finding, which was reversible. In repeated-dose studies, a modest reduction in B-wave amplitude obtained by electroretinography was observed in animals treated with the highest dose level tested (3 mg, which is equivalent to a 12 mg/eye human dose) that was not considered to be clinically meaningful. Administration in the rat of either a single bolus intravenous (i.v.) injection of 100 mg/kg or daily bolus i.v. injections of 75 mg/kg/day for 28 days failed to elicit any macroscopic or microscopic changes, suggesting a low risk for systemic toxicity. QPI-1007 was negative in three genetic toxicity studies. Overall, the nonclinical studies support the further development of QPI-1007.

We have completed a comprehensive deletion analysis of the Tetrahymena ribozyme in order to define the minimum secondary structure requirements for phosphoester transfer activity of a self-splicing group I intron. A total of 299 nucleotides were removed in a piecewise fashion, leaving a catalytic core of 114 nucleotides that form 7 base-paired structural elements. Among the various deletion mutants are a 300-nucleotide single-deletion mutant and a 281-nucleotide double-deletion mutant whose activity exceeds that of the wild type when tested under physiologic conditions. Consideration of those structural elements that are essential for catalytic activity leads to a simplified secondary structure model of the catalytic core of a group I intron.

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (␤), single-stranded DNA-binding protein, a complex containing the polymerase (␣) and exonuclease (⑀) subunits, and the essential components of the DnaX complex ( 3 ␦␦). Efficient primer elongation requires the presence of ␣⑀, ␤, and 3 ␦␦. Pseudomonas aeruginosa ␣⑀ can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas ␤ and 3 ␦␦ exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli subunit was not apparent in sequence similarity searches, addition of purified E. coli and (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas 3 ␦␦ complex ϳ10-fold and enables the reconstituted enzyme to function better under physiological salt conditions.