Optimization of hammerhead ribozymes for the cleavage of S100A4 (CAPL) mRNA.

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.

Tyrosine-PEG-derived poly(ether carbonate)s as new biomaterials. Part II: study of inverse temperature transitions.

Tyrosine-poly(alkylene oxide)-derived poly(ether carbonate)s represent a new group of degradable biomaterials that exhibit inverse temperature transitions. Poly(DTE co 70%PEG,1000 carbonate) was chosen as an example to study this special phase transition behavior of the polymers. The observed transition temperature varied slightly depending on the technique used, e.g. CD always gave a lower temperature than UV/Vis. CD and UV/Vis studies indicated that the transition temperature was both heating rate and concentration dependent. Thermodynamic parameters of the transition (enthalpy, entropy, and free energy) were determined by DSC. The molecularity of the transition was 2.6, as calculated from UV and DSC data. The transition temperature could be varied from 18 to 58 degrees C by changing the polymer structure. The new poly(ether carbonate)s may be used in medical applications such as injectable drug delivery formulations and bioresorbable barriers for the prevention of surgical adhesions.

Binding studies of a triple-helical peptide model of macrophage scavenger receptor to tetraplex nucleic acids.

The macrophage scavenger receptor (MSR), involved in the uptake of oxidized LDL, binds a variety of polyanionic ligands, and in particular shows selectivity for tetraplex forms of nucleic acids. The ligand binding region has been shown to lie in the triple-helical collagen-like domain of MSR. A model peptide-nucleic acid system is presented here to clarify how the triple-helical motif of MSR recognizes and binds tetraplex nucleic acids. The triple-helical peptide MSR-1, with the sequence (POG)3PKGQKGEKG(POG)4, contains a nine amino acid basic sequence implicated in MSR ligand binding, flanked by Pro-Hyp-Gly triplets to provide stability. The ability of this triple-helical MSR-1 peptide to bind to and perturb the conformation of nucleic acids in tetraplex, duplex, and single-stranded states was assessed by monitoring changes in the nucleic acid circular dichroism spectrum in the 240-300 nm region. Our results show that the triple-helical MSR-1 peptide binds to tetraplex poly(I) in a stoichiometric manner and is capable of reproducing the discrimination exhibited by the native MSR molecule for tetraplex over double-stranded or single-stranded nucleic acid states. The triple-helical reference peptide (POG)10 does not bind to tetraplex poly(I), suggesting that binding requires the highly basic 9-mer sequence from MSR that is included in MSR-1. The MSR-1 peptide did not perturb the CD spectra of a series of other tetraplex nucleic acids, indicating that it does not model the broader specificity that MSR shows under physiological conditions. Models of possible interactions between a triple-helical peptide and a tetraplex polynucleotide are proposed on the basis of the stoichiometry observed for the complex between triple-helical MSR-1 and tetraplex poly(I).