7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli.

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N6-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N6-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.

Phenoxazine pseudonucleotides in DNA i-motifs allow precise profiling of small molecule binders by fluorescence monitoring.

The lack of high throughput screening (HTS) techniques for small molecules that stabilize DNA iMs limits their development as perspective drug candidates. Here we showed that fluorescence monitoring for probing the effects of ligands on the iM stability using the FAM-BHQ1 pair provides incorrect results due to additional dye-related interactions. We developed an alternative system with fluorescent phenoxazine pseudonucleotides in loops that do not alter iM unfolding. At the same time, the fluorescence of phenoxazine residues is sensitive to iM unfolding that enables accurate evaluation of ligand-induced changes of iM stability. Our results provide the basis for new approaches for HTS of iM ligands.

Catalytically synthesized Prussian Blue nanozymes as labels for electrochemical DNA/RNA sensors.

We propose catalytically synthesized nanozymes based on Prussian Blue (PB) and azidomethyl-substituted poly (3,4-ethylenedioxythiophene) (azidomethyl-PEDOT) as novel electrocatalytic labels for DNA/RNA sensors. Catalytic approach allowed to synthesize highly redox and electrocatalytically active Prussian Blue nanoparticles functionalized with azide groups that enable 'click' conjugation with alkyne-modified oligonucleotides. Both competitive and sandwich-type schemes were realized. As the sensor response the direct (mediator-free) electrocatalytic current of H2O2 reduction can be measured, which is proportional to the concentration of the hybridized labeled sequences. The current of H2O2 electrocatalytic reduction is only 3-8 times increased in the presence of the freely diffusing mediator catechol, which indicates high efficiency of direct electrocatalysis with the elaborated labels. Electrocatalytic amplification of the signal allows robust detection of (63-70)-base target sequences with concentrations below 0.2 nM in blood serum within an hour. We believe, the use of advanced Prussian Blue based electrocatalytic labels sets new avenues for point-of-care DNA/RNA sensing.

Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support.

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.