Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2′-deoxyribose, 2′-O-methyl-ribose, 2′-deoxy-2′-fluoro-ribose, arabinose and 2′-deoxy-2′-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2’ modifications gave a variety of effects. Arabinose and 2′-deoxy-2′-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2′-O-methyl and 2′-deoxy-2′-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability.

Oligonucleotides containing various adducts, including ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl groups, at the O atom of 5-fluoro-O -alkyl-2'-deoxyuridine were prepared by solid-phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5-fluoro-2'-deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B-form DNA structure. O -Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5-fluoro-O -benzyl-2'-deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O -ethyl and benzyl adducts of 5-fluoro-2-deoxyuridine. Computational assessment of N1-methyl analogues of the O -alkylated nucleobases revealed that the C5-fluorine modification had an influence on reducing the electron density of the O -C bond, relative to thymine (C5-methyl) and uracil (C5-hydrogen). These results reveal the positive influence of the C5-fluorine atom on the repair of larger O -alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT.

The chemical and self‐assembly properties of nucleic acids make them ideal for the construction of discrete structures and stimuli‐responsive devices for a diverse array of applications. Amongst the various three‐dimensional assemblies, DNA tetrahedra are of particular interest, as these structures have been shown to be readily taken up by the cell, by the process of caveolin‐mediated endocytosis, without the need for transfection agents. Moreover, these structures can be readily modified with a diverse range of pendant groups to confer greater functionality. This minireview highlights recent advances related to applications of this interesting DNA structure including the delivery of therapeutic agents ranging from small molecules to oligonucleotides in addition to its use for sensing and imaging various species within the cell.

A new fluorescent cytosine analog “tsC” containing a trans-stilbene moiety was synthesized and incorporated into hemiprotonated base pairs that comprise i-motif structures. Unlike previously reported fluorescent base analogs, tsC mimics the acid–base properties of cytosine (pK a ≈ 4.3) while exhibiting bright (ε × Φ ≈ 1000 cm–1 M–1) and red-shifted fluorescence (λem = 440 → 490 nm) upon its protonation in the water-excluded interface of tsC+:C base pairs. Ratiometric analyses of tsC emission wavelengths facilitate real-time tracking of reversible conversions between single-stranded, double-stranded, and i-motif structures derived from the human telomeric repeat sequence. Comparisons between local changes in tsC protonation with global structure changes according to circular dichroism suggest partial formation of hemiprotonated base pairs in the absence of global i-motif structures at pH = 6.0. In addition to providing a highly fluorescent and ionizable cytosine analog, these results suggest that hemiprotonated C+:C base pairs can form in partially folded single-stranded DNA in the absence of global i-motif structures.

Intrastrand crosslinked DNA has been shown to react with an AGT variant to produce well-defined protein–DNA conjugates.

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Tetrahedron DNA structures were formed by the assembly of three‐way junction (TWJ) oligonucleotides containing O6‐2′‐deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine (butylene and heptylene linked) intrastrand cross‐links (IaCLs) lacking a phosphodiester group between the 2′‐deoxyribose residues. The DNA tetrahedra containing TWJs were shown to undergo an unhooking reaction by the human DNA repair protein O6‐alkylguanine DNA alkyltransferase (hAGT) resulting in structure disassembly. The unhooking reaction of hAGT towards the DNA tetrahedra was observed to be moderate to virtually complete depending on the protein equivalents. DNA tetrahedron structures have been explored as drug delivery platforms that release their payload in response to triggers, such as light, chemical agents or hybridization of release strands. The dismantling of DNA tetrahedron structures by a DNA repair protein contributes to the armamentarium of approaches for drug release employing DNA nanostructures.