DNA oxidation causes a variety of diseases including cancer. The oxidized DNA nucleobases are excised by cellular repair enzymes and released into extracellular fluids. Specifically, the excised DNA oxidation product, such as 8-oxoGua, has been suggested as a biomarker for early cancer diagnosis. We previously developed an artificial receptor for the free base of 8-oxoGua on a triplex DNA backbone. The receptor contained a pre-organized cavity, which bounded 8-oxoGua with strong affinity and excellent selectivity over other nucleobases. However, accurate detection of 8-oxoGua in urine samples was affected by the presence of a large excess of guanine. Herein, we report a strategy to convert our receptor to a colorimetric biosensor by conjugating DNA strands to gold nanoparticles (GNP), specifically for 8-oxoGua. By simply incubating our sensor with a urine sample, 8-oxoGua can be detected at submicromolar concentrations with UV–vis spectrometer or even by naked eye.The gold nanoparticle/triplex DNA conjugates contained a pre-organized cavity that can bind 8-oxoGua with strong affinity and excellent selectivity over other nucleobases. Nanoparticle assemblies interconnected with DNA triple helices bound with 8-oxoGua show much sharper and significantly higher melting temperatures than the analogous free DNA triplex. By simply incubating our sensor with a urine sample, 8-oxoGua can be detected colorimetrically at submicromolar concentrations with a UV–vis spectrometer or even by naked eyes.

Effects of hydrogen bonding clamps on the selectivity of triplex DNA receptors were studied. Incorporation of a 5-methyl-2-thiocytosine base to the parallel homopyrimidine region of a triplex receptor enabled selective molecular recognition of an inosine ligand.

Triplex DNA receptors are known for their strong and specific binding of a variety of purine ligands. However, not many studies have been reported on their binding of pyrimidine ligands. Here we used fluorophore-labeled DNA receptors to study these binding interactions. The results suggest that in general, cytosine binds more strongly than thymine and uracil, although the selectivity between two pyrimidine ligands can be tuned by changing the surrounding nucleobases of the DNA receptor. In contrast, binding of pyrimidine nucleosides is much weaker, possibly due to lack of the N1 hydrogen bonding donor.

8-Oxo-7,8-dihydroguanine (8-oxoG, or OG) as a free base has been widely considered as a biomarker for DNA oxidative damage. Currently no fluorescence sensor has been developed to directly detect 8-oxoG less than 100 nM. In this study, two triple-stranded DNAs were selected as the scaffolds to rationally design DNA aptamer sensors for 8-oxoG. The cavity was created by deleting the 8-oxodG nucleoside in a triplex containing an A·OG-C triad or a C·OG-A triad. The results showed that the fluorescence of both sensors were completely quenched by 8-oxoG. The detection ranges of the two sensors were different, while the combined range was comparable to the detection range of an antibody-based method. This result is expected to enable a fast, low-cost, and reusable method to measure 8-oxoG concentration.

N7-methyl-9-deaza-dG was synthesized and incorporated into oligonucleotides. Thermal melting studies showed that replacement of dG by N7-methyl-9-deaza-dG only slightly decreased DNA duplex stability. Replication of DNA templates containing N7-methyl-9-deaza-dG and the related 7-methyl-7-deaza-dG and 7-deaza-dG by the Klenow fragment of Escherichia coli DNA polymerase I was examined. The dNTP misinsertion frequencies on all three templates were comparably low, although the 7-methyl group significantly slowed down the turnover rates of the polymerase when dCTP was incorporated. The stabilities of N7-methyl-9-deaza-dG and 7-methyl-7-deaza-dG against the actions of formamidopyrimidine DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) were also examined. N7-methyl-9-deaza-dG was stable in the presence of both enzymes. In contrast, 7-methyl-7-deaza-dG was cleaved by Fpg, and possibly by hAAG but at an extremely slow rate. This study suggests that N7-alkyl-9-deaza-dG is a better analogue than 7-alkyl-7-deaza-dG for cellular studies.

Abasic site-containing DNA duplex is a versatile structural motif that can be used for the design of purine aptamers and sensors. In this study, several modifications were introduced to the abasic site to explore possible specific binding of free 8-oxoG, a product of DNA base excision repair. The nucleoside opposite the abasic site was replaced by pyrrolo-dC as a reporter group. Binding of 8-oxoG quenched pyrrolo-dC fluorescence by as much as 70%. In contrast, adenine, guanine, thymine, and cytosine showed only minimal fluorescence quenching effect. The best aptamer binds 8-oxoG with a dissociation constant of 5.5 ± 0.8 μM. This sensor can be used to accurately measure 8-oxoG concentrations in the presence of guanine.

Effects of hydrogen bonding clamps on the selectivity of triplex DNA receptors were studied. Incorporation of a 5-methyl-2-thiocytosine base to the parallel homopyrimidine region of a triplex receptor enabled selective molecular recognition of an inosine ligand.