Table of Contents

Volume 3, Issue 1

  January/February/March 2012

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COMMENTARIES

Open Access Article

“Artifactual” arsenate DNA

Peter E. Nielsen
http://dx.doi.org/10.4161/adna.19672
Abstract | Full Text | PDF

The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA arose much scepticism, primarily due to the very limited chemical stability of arsenate esters. A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present evidence that the identification of arsenate DNA was artifactual.

Commentary to: Wolfe-Simon F, Switzer Blum J, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, et al. A bacterium that can grow by using arsenic instead of phosphorus. Science 2011; 332:1163-6; PMID:21127214; http://dx.doi.org/10.1126/science.1197258; and Reaves ML, Sinha S, Rabinowitz JD, Kruglyak L, Redfield RJ. Absence of arsenate in DNA from arsenate-grown GFAJ-1 cells. Science 2012; In press.

Open Access Article

A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery

Thomas Bentin
http://dx.doi.org/10.4161/adna.19843
Abstract | Full Text | PDF

A DNA capsule fitted with aptamer controlled target sensing has been “woven” using a 7308-base single-stranded DNA “thread” and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.




RESEARCH PAPERS

Open Access Article

(α,α-dimethyl)glycyl (dmg) PNAs: Achiral PNA analogs that form stronger hybrids with cDNA relative to isosequential RNA

Aland Gourishankar and Krishna N. Ganesh
http://dx.doi.org/10.4161/adna.19185
Abstract | PDF

The design and facile synthesis of sterically constrained new analogs of PNA having gem-dimethyl substitutions on glycine (dmg-PNA-T) is presented. The PNA oligomers [aminoethyl dimethylglycyl (aedmg) and aminopropyl dimethylglycyl (apdmg)] synthesized from the monomers 6 and 12) effected remarkable stabilization of homothyminePNA₂:homoadenine DNA/RNA triplexes and mixed base sequence duplexes with target cDNA or RNA. They show a higher binding to DNA relative to that with isosequential RNA. This may be a structural consequence of the sterically rigid gem-dimethyl group, imposing a pre-organized conformation favorable for complex formation with cDNA. The results complement our previous work that had demonstrated that cyclohexanyl-PNAs favor binding with cRNA compared with cDNA and imply that the biophysical and structural properties of PNAs can be directed by introduction of the right rigidity in PNA backbone devoid of chirality. This approach of tweaking selectivity in binding of PNA constructs by installing gem-dimethyl substitution in PNA backbone can be extended to further fine-tuning by similar substitution in the aminoethyl segment as well either individually or in conjunction with present substitution.

Open Access Article

Enzymatic synthesis of DNA strands containing α-L-LNA (α-L-configured locked nucleic acid) thymine nucleotides

Torben Højland, Rakesh N. Veedu, Birte Vester and Jesper Wengel
http://dx.doi.org/10.4161/adna.19272
Abstract | PDF

We describe the first enzymatic incorporation of an α-L-LNA nucleotide into an oligonucleotide. It was found that the 5′-triphosphate of α-L-LNA is a substrate for the DNA polymerases KOD, 9°N_m, Phusion and HIV RT. Three dispersed α-L-LNA thymine nucleotides can be incorporated into DNA strands by all four polymerases, but they were unable to perform consecutive incorporations of α-L-LNA nucleotides. In addition it was found that primer extension can be achieved using templates containing one α-L-LNA nucleotide.

Open Access Article

Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

Laia Llovera, Peter Berthold, Peter E. Nielsen and Takehiko Shiraishi
http://dx.doi.org/10.4161/adna.19906
Abstract | Full Text | PDF

Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.