DNA Photocleavage by DNA and DNA−LNA Amino Acid−Dye Conjugates

Abstract

DNA photocleavage by triplex forming oligonucleotides (TFO) has potential implications in both biotechnology and medicine. We have synthesized a series of homopurine DNA and DNA/LNA 14-mers to which an amino acid (glycine or l-tryptophan) and a cyanine dye are covalently linked. Two cyanine dyes were examined that include a quinolinium ring linked to a benzothiazolium ring through a monomethine (TO1) or trimethine (TO2) linker. The 14-mer sequence was chosen to target mdm2, a ubiquitin ligase (E3) that regulates p53 by promoting its ubiquitylation and proteosomal degradation. Such inhibition has been previously proposed as a therapeutic approach to target wild-type p53-expressing cancers. To examine whether our TFO conjugates photocleave the mdm2 target, we incubated the various conjugates with the mdm2 plasmid and irradiated the samples with visible light. We show that only the TFO with the complementary sequence and with an intervening l-tryptophan leads to the linearization of the plasmid after a short irradiation time (10 min) exciting the dye (lambda(max)(TO1) = 500 nm and lambda(max)(TO2) = 630 nm) with visible light. Furthermore, the photoreactivity is more pronounced for the LNA/DNA conjugate, an observation that is consistent with improved hybridization to the DNA target. Sequence specificity of the photoreaction is further corroborated on a synthetic 44-mer duplex containing the TFO site. Evidence for a ROS-dependent mechanism is also given and discussed.

Molecular Engineering of Guanine-Rich Sequences: Z-DNA, DNA Triplexes, and G-Quadruplexes

“…An inhibition of firefly luciferase expression, which was dependent on the TFO concentration, was observed with IC 50 values around 1.5 μM, which decreased even further (0.5 μM) when Ps was attached to the oligonucleotide. In a recent study 294 the photocleavage of plasmid DNA was achieved using an antiparallel GA 14-mer DNA/LNA-TFO possessing six evenly distributed LNA monomers, which also had an Ltryptophan and a cyanine dye TO1 attached at the 5′-end (Figure 15C). The photoreactivity, derived from the production of reactive oxygen species upon irradiation with visible light, was more pronounced for LNA-modified TFO than for DNA-TFOs; this was attributed to the increased thermal stability of the triplex formed.…”

Stability of Oligonucleotide–Small Molecule Conjugates to DNA-Deprotection Conditions

“…DNA conjugation has been a common practice since the development of automated DNA synthesis and numerous examples of small peptides, , fluorophores, , small molecules, , and bio- or synthetic macromolecules have been conjugated to DNA. In addition, there are many unnatural nucleosides that are commercially available or custom-made in research laboratories .…”

Site-Specific DNA Photocleavage and Photomodulation by Oligonucleotide Conjugates

“…The product of this reaction, a stable peroxide, was then potent to cause frank strand breaks in DNA without the need of piperidine treatment. Based on these studies, we have recently studied 2 cyanine dye systems where a TO (l max ¼ 500 nm) and a red-shifted analog of TO (TO-2, l max ¼ 630 nm) were chosen as chromophores (Biton et al, 2010). DNA-LNA TFOs were conjugated to L-Trp-dye at the 5 0 end of the sequence.…”