Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1.

In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mug of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mug of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential.

Suppression of eukaryotic translation termination by selected RNAs.

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis.

Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine.

In vitro selection of a novel nuclease-resistant RNA phosphodiesterase.

BACKGROUND: Ribonucleotide-based enzymes (ribozymes) that cleave pathological RNAs are being developed as therapeutic agents. Chemical modification of the hammerhead ribozyme has produced nuclease-resistant catalysts that cleave targeted mRNAs in cell culture and exhibit antitumor activity in animals. Unfortunately, stabilizing modifications usually reduce the catalytic rate in vitro. An alternative to rationally designed chemical modifications of existing ribozymes is to identify novel motifs through in vitro selection of nuclease-stable sequence space. This approach is desirable because the catalysts can be optimized to function under simulated physiological conditions. RESULTS: Utilizing in vitro selection, we have identified a nuclease-stable phosphodiesterase that demonstrated optimal activity at simulated physiological conditions. The initial library of 10(14) unique molecules contained 40 randomized nucleotides with all pyrimidines in a nuclease-stabilized 2'-deoxy-2'-amino format. The selection required trans-cleaving activity and base-pairing specificity towards a resin-bound RNA substrate. Initial selective pressure was permissive, with a 30 min reaction time and 25 mM Mg(2+). Stringency of selection pressure was gradually increased until final conditions of 1 mM Mg(2+) and less than 1 min reaction times were achieved. The resulting 61-mer catalyst required the 2'-amino substitutions at selected pyrimidine positions and was stable in human serum (half-life of 16 h). CONCLUSIONS: We demonstrated that it is possible to identify completely novel, nuclease-resistant ribozymes capable of trans-cleaving target RNAs at physiologically relevant Mg(2+) concentrations. The new ribozyme motif has minimal substrate requirements, allowing for a wide range of potential RNA targets.

An RNA pocket for the planar aromatic side chains of phenylalanine and tryptophane.

This is a preliminary report of RNA's with affinity for the aromatic side chains of the amino acids phenylalanine and tryptophane that have been isolated utilizing in vitro selection to phenylalanine-affinity matrix. Cloning and sequencing of a binding pool identified a frequently occurring 6-9 base motif (-CUCGUGU-) common to most of the RNA's; other potential binding motifs were revealed but occurred less frequently. Four of five clones analyzed bind free phenylalanine and tryptophane with dissociation constants in the low millimolar range, the fifth binds resin only. Isolate F7, the most common sequence in the binding pool, demonstrated moderate specificity for aromatic rings, no stereoselectivity was observed. Preliminary Pb cutting structural analysis agrees with the most stable predicted secondary structure. A striking similarity between the sequence and predicted structure of F7 and the bridged-biphenylisomerase RNA (J.R. Prudent, T. Uno, and P.G. Schultz, Science 264,1924 (1994)) suggests a 19 base loop contains the important aromatic binding elements.

Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase.

Pre-steady-state methods were used to study the fidelity of human immunodeficiency virus reverse transcriptase. Fidelity of DNA-directed DNA synthesis can be attributed to a 1-2 order of magnitude reduction in affinity for noncomplementary dNTPs, and a 1-4 order of magnitude reduction in the rate of the conformational change that limits the rate of nucleotide addition. Affinities of reverse transcriptase for paired or mispaired primer termini are similar. Discrimination against a mispaired primer is due to reduction in affinity for the next dNTP and reduction in rate of extension. Extension of mispaired termini proceeds 20-700-fold faster than the rate of dissociation of reverse transcriptase from the primer-template and is 2-3 orders of magnitude more frequent than nucleotide misincorporation. The rate-limiting step for extension of a mispaired terminus occurs at the conformational change or chemical step, depending on the nature of the mispair. Presence of a mismatch at the 3' penultimate position reduces pyrophosphorolysis of the primer by a factor of 10(3), indicating that mispairs 5' to the site of chemistry can also affect catalysis.

Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase.

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.