Condensation of oligonucleotides assembled into nicked and gapped duplexes: potential structures for oligonucleotide delivery

Abstract

The condensation of nucleic acids into well-defined particles is an integral part of several approaches to artificial cellular delivery. Improvements in the efficiency of nucleic acid delivery in vivo are important for the development of DNA- and RNA-based therapeutics. Presently, most efforts to improve the condensation and delivery of nucleic acids have focused on the synthesis of novel condensing agents. However, short oligonucleotides are not as easy to condense into well-defined particles as gene-length DNA polymers and present particular challenges for discrete particle formation. We describe a novel strategy for improving the condensation and packaging of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. 2 kb). These non-covalent assemblies possess single-stranded nicks or single-stranded gaps at regular intervals along the duplex backbones. The condensation behavior of nicked- and gapped-DNA duplexes was investigated using several cationic condensing agents. Transmission electron microscopy and light-scattering studies reveal that these DNA duplexes condense much more readily than short duplex oligonucleotides (i.e. 21 bp), and more easily than a 3 kb plasmid DNA. The polymeric condensing agents, poly-l-lysine and polyethylenimine, form condensates with nicked- and gapped-DNA that are significantly smaller than condensates formed by the 3 kb plasmid DNA. These results demonstrate the ability for DNA structure and topology to alter nucleic acid condensation and suggest the potential for the use of this form of DNA in the design of vectors for oligonucleotide and gene delivery. The results presented here also provide new insights into the role of DNA flexibility in condensate formation.

DNA-Based Soft Phases

“…The study of the N* and COL phase boundary thus provides a new direct route to evaluate the strength and the temperature dependence of the stacking and pairing forces [50]. Sticky ends were also recently proposed as an effective driving mechanism to aggregate short DNA strands into longer polymers that are more easily condensed into DNA particles, as a valuable method of oligonucleotide delivery in gene therapy [51].…”

Modified Concatemeric Oligonucleotide Complexes: New System for Efficient Oligonucleotide Transfer into Mammalian Cells

“…Gel-shift analysis of these concatemers revealed that in the presence of cholesterol-modified oligonucleotides there is some amount of supramolecular structures with low electrophoretic mobility. Therefore it may well be that formation of this supramolecular structures or complexes, arising from some additional organization of concatemer-forming oligonucleotides in the presence of covalently linked cholesterol, oligonucleotides or conventional long dsDNA (Sarkar et al, 2005). We observed that oligonucleotide distribution in cells treated with single-stranded oligonucleotides or concatemeric complexes in the presence of cholesterol-conjugated PEI is indeed different.…”

“…In contrast, long ds-DNA commonly does not display the same activity in nonimmune cells (Suzuki et al, 2002;Martin and Elkon, 2006). Concatemeric complexes were shown to have some structural differences from conventional DNA molecules (Sarkar et al, 2005), besides that little is known about the intracellular activity of such structures. Therefore we checked the influence of the cell treatment with concatemers on induction of the IFN response.…”

“…Formation of the nicked and gapped concatemeric complexes is known to promote condensation of the oligonucleotides by various DNA-condensing agents used for the delivery of nucleic acids into cells (Sarkar et al, 2005); one such agent is polyethylenimine (PEI). The effect of various chemical modifications on the efficiency of polyethylenimines as synthetic vectors for the delivery of nucleic acids has been systematically investigated (Nguyen et al, 2000;Han et al, 2001;Kircheis et al, 2001;Merlin et al, 2001;Ogris et al, 2001;Dolivet et al, 2002;Sochanik et al, 2004;Tseng et al, 2004).…”

Liquid Crystal Ordering of Four-Base-Long DNA Oligomers with Both G–C and A–T Pairing

“…Taking advantage of the selective Watson and Crick pairing of DNA nucleobases-adenine-thymine (A-T), guanine-cytosine (G-C)-the fine design of different DNA sequences has been used to obtain 2D and 3D self-assembled structures with nanometer dimensions, such as DNA origami [2] or DNA nanostructures [3], with many potential applications ranging from drug delivery [4] to the production of systems for basic condensed matter studies [5].…”