During the past several years, significant advances have been made in the development of oligonucleotides as therapeutic agents. However, effective implementation of DNA- and RNA-based therapeutics requires efficient delivery of these molecules in vivo. It is widely appreciated that controlling nucleic acid condensation represents a key step for the improvement of non-viral nucleic acid delivery systems (1). Unlike gene-length DNA polymers, short oligonucleotides do not readily condense into well-defined nanometer-scale particles. Most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. In contrast, our laboratory is investigating how the modulation of nucleic acid structure can be used to control condensate size and morphology (2, 3). We have developed a novel strategy for improving the packaging of oligonucleotides that is based on the self-assembly of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb) (4). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. Using transmission electron microscopy and dynamic light-scattering, we have studied the condensation behavior of nicked- and gapped-DNA duplexes by several cationic condensing agents, including the trivalent cation hexammine cobalt(III), an arginine-rich peptide and polymeric condensing agents. DNA oligonucleotides self-assembled into long duplexes were found to condense more readily than short oligonucleotide duplexes (i.e. 21 bp) and a 3 kb plasmid DNA. Self-assembled nicked- and gapped-DNA duplexes generally condense into smaller and more homogenous particles than short oligonucleotides duplexes. We have also observed an appreciable difference in the average size of nicked- and gapped-DNA condensates with respect to those formed by continuous DNA duplexes, which implies that the enhanced local flexibility of nicked and gapped sites provides both a kinetic and a thermodynamic advantage for DNA condensation. Based on our results, we propose that nucleic acids with nicks or gaps at regular intervals in the backbone represent a new class of nucleic acid structures that should prove useful for non-viral nucleic acid delivery systems. In addition, the results to be presented illustrate the fundamental role that DNA flexibility plays in condensate formation.

References and Footnotes

1. Luo, D. and Saltzman, W. Nat. Biotechnol. 18, 33-37 (2000).
2. Shen, M. R., Downing, K. H., Balhorn, R., and Hud, N. V. J. Am. Chem. Soc. 122, 4833?4834 (2000).
3. Conwell, C. C., Vilfan, I. D., Hud, N. V. Proc. Natl Acad. Sci. USA 100, 9296?9301 (2003).
4. Sarkar, T., Conwell, C. C., Harvey, L. C., Santai, C. T. and Hud, N. V. Nucleic Acids Res. 33, 143-151 (2005).