Thermodynamic parameters for internal loops of unpaired adenosines in oligoribonucleotides have been measured by optical melting studies. Comparisons are made between helices containing symmetric and asymmetric loops. Asymmetric loops destabilize a helix more than symmetric loops. The differences in free energy between symmetric and asymmetric loops are roughly half the magnitude suggested from a study of parameters required to give accurate predictions of RNA secondary structure [Papanicolaou, C., Gouy, M., & Ninio, J. (1984) Nucleic Acids Res. 12, 31-44]. Circular dichroism spectra indicate no major structural difference between helices containing symmetric and asymmetric loops. The measured sequence dependence of internal loop stability is not consistent with approximations used in current algorithms for predicting RNA secondary structure.

Glycol nucleic acid (GNA) has an acyclic backbone of propylene glycol nucleosides that are connected by phosphodiester bonds. This paper characterizes the duplex-formation properties of this simplified nucleic acid. Although single and multiple GNA nucleotides are highly destabilizing if incorporated into DNA duplexes, the two enantiomeric oligomers (S)-GNA and (R)-GNA form antiparallel homoduplexes that are thermally and thermodynamically significantly more stable than analogous duplexes of DNA and RNA. The salt-dependence and Watson-Crick-pairing fidelity of GNA duplexes are similar to those of DNA duplexes, but, apparently, the 2'-deoxyribonucleotide and the propylene glycol backbones are not compatible with each other. This conclusion is further supported by cross-pairing experiments. Accordingly, both (S)- and (R)-GNA strands do not generally pair with DNA. However, (S)-GNA, but not (R)-GNA, forms stable heteroduplexes with RNA in sequences that are low in G:C content. Altogether, the high stability and fidelity of GNA duplex formation in combination with the economical accessibility of propylene glycol building blocks for oligonucleotide synthesis render GNA an attractive candidate for the design of self-assembling materials. They further suggest that GNA could be considered as a potential candidate for a predecessor of RNA during the evolution of life on Earth.

The variability in gene conversion frequency by an RNAapproximately 0.1%, determined by bacterial transform-DNA oligonucleotide (RDO) prompted us to develop a sysation. Using the in vitro reaction, frequency of gene convertem as a means of measuring the conversion frequency sion in different cell types was measured. The embryonic rapidly and reproducibly. A shuttle vector was constructed fibroblasts from p53−/− mouse showed higher gene correcto measure the frequency of targeted gene correction by tion than that of the isogenic p53+/+ cells. Nuclear extracts RDO of the E. coli ␤-galactosidase gene containing a sinfrom DT40 cells, which have a higher homologous recomgle point mutation (G → A), that resulted in inactivation of bination rate than any other mammalian cells exhibited enzymatic activity. An RDO corrected the point mutation 0.1-0.6% of gene correction. These results indicated that and restored the enzymatic activity, approximately 1%, recombination may be rate-limiting in gene conversion by determined by a histochemical staining in mammalian cells RDO in cells with competent mismatch repair activities. Utiand by a color selection (blue or white) of bacteria transforlizing transfection and in vitro reaction, we demonstrated med with Hirt DNA. In addition, we established an in vitro that such a shuttle system might be useful in comparing system capable of gene correction using nuclear extracts. the frequency of targeting among different cell types and CHO-K1 nuclear extracts corrected the point mutation to investigate the mechanism of gene conversion by RDO.

A unique hybrid oligonucleotide composed of both RNA and DNA has been shown to correct a point mutation in a site-specific and inheritable manner in extrachromosomal and chromosomal targets. In order to develop new gene therapeutics for skin, we tested two oligonucleotides that were shown to create a point mutation in alkaline phosphatase and beta-globin genes in several epithelial cell types. Highly transformed epithelial cells (HeLa) exhibited a conversion frequency of 5% by both RNA-DNA oligonucleotides. In comparison, other immortalized epithelial cells (HaCaT) or human primary keratinocytes did not show any detectable level of gene conversion by the restriction fragment length polymorphism analysis, indicating less than 1% conversion frequency. The concentration of the oligonucleotide in the nuclei of HeLa cells was similar to that of HaCaT or human primary keratinocytes measured by a radiolabeled or a fluorescein-conjugated oligonucleotide. Moreover, the RNA-DNA oligonucleotide exhibited a prolonged stability in the nucleus. Thus, neither uptake nor nuclear stability of the oligonucleotide appears to be a limiting factor in gene targeting events under our experimental conditions. These results indicate that the frequency of gene targeting varies among different cells, suggesting that cellular recombination and DNA repair activities may be important.