Anthraquinone (AQ) has been extensively used as a photosensitizer to study charge transfer in DNA. Near-UV photolysis of AQ induces electron abstraction in oligonucleotides leading to AQ radical anions and base radical cations. In general, this reaction is followed by the transport of base radical cations to sites of low oxidation potential, that is, GG, and conversion of G radical cations to DNA breaks. Here, we show that AQ also produces interstrand cross-links in DNA duplexes. About half of the cross-links collapse to single strands in hot piperidine treatment. The structure of stable interstrand cross-links was deduced by MS, NMR, and sequence substitution. The cross-links consist of a covalent link between the methyl group of T on one strand with either C6 or C7 of AQ on the other strand. The formation of interstrand cross-links decreased in O2 compared to deoxygenated solutions. In the presence of O2, the yield of breaks at GG doublets was 10-fold greater than that of cross-links for end tethered AQ, while cross-links exceeded breaks for centrally located AQ. The formation of stable cross-links can be explained by initial charge transfer from T to excited AQ, deprotonation of T radical cations, and condensation of the latter species with AQ radicals. These studies reveal a novel pathway of damage in the photolysis of AQ-DNA duplexes.

Fabricated an inexpensive bi-electrode electrochemical sensor for nucleic acid amplification detection with a user-friendly interface in a short detection time.Electronic conduction of the metal oxide sensing layer was successfully optimised by manipulating the concentration of oxygen vacancies.The sensing layer was annealed at various temperatures and optimised by testing their electrical, optical, and chemical properties.The fabricated device was able to detect dengue virus sequence DNA and Staphylococcus aureus genomic DNA with clinically relevant limit of detection.

Rolling circle amplification (RCA) has been utilized for detecting a diverse range of analytes, molecular pathways, and in cellular imaging. However, non-specific amplification (NSA, amplification in absence of a target analyte) in RCA assays, especially those involving pre-synthesized circular DNA (cDNA), affects its sensitivity and specificity. NSA could originate from inefficient ligation or the succeeding cDNA purification steps. To quantify the NSA in RCA here, cDNA substrates were prepared using either self-annealing, splint-padlock, or cohesive end ligations. The cDNAs were then each subjected to nine different exonuclease digestion steps and quantified for NSA under both linear and hyperbranched RCA conditions. We investigated buffer composition, divalent ion concentration, single or dual enzyme digestion, overhang length in cohesive end ligation, and splint length in splint-padlock ligation. When applied in tandem, the conditions successfully mitigated the NSA end-fluorescence 30 – 100 fold while reducing the relative NSA (with respect to primer assisted RCA) to ~5%. Besides understanding the mechanistic origin of NSA, novel aspects of enzyme-substrate selectivity, buffer composition, and the role of divalent ions in enzyme activity were discovered. With increasing analytical applications, this study will help standardize NSA-free RCA assays involving pre-synthesized cDNA.

DNA amplification detection is typically a lengthy and sophisticated procedure involving high-end instrumentation. Electrochemical detection has recently opened a relatively low budget, a straightforward and speedy method for detection with the aid of an electrochemically active redox probe. However, electrochemical sensors of nucleic acid amplification characteristically employ a tri-electrode geometry involving standard reference electrode usually made up of a noble metal that adds up the cost of detection. This work proposes a sensitive and rapid approach for detecting the endpoint of nucleic acid amplification test using a novel bi-electrode sensing geometry. The sensing layer of the electrode was comprised of a transition metal oxide to tune its electronic state for regulating its electrochemical response. The fabricated device was then used to detect dengue virus sequence DNA (using rolling circle amplification) and Staphylococcus aureus genomic DNA (using polymerase chain reaction) with the limit of detection in the order of 103 target DNA copies.

Rolling circle amplification (RCA) has been utilized for detecting a diverse range of analytes, molecular pathways, and in cellular imaging. However, non-specific amplification (NSA, amplification in absence of a target analyte) in RCA assays, especially those involving pre-synthesized circular DNA (cDNA), affects its sensitivity and specificity. NSA could originate from inefficient ligation or the succeeding cDNA purification steps. To quantify the NSA in RCA here, cDNA substrates were prepared using either self-annealing, splint-padlock, or cohesive end ligations. The cDNAs were then each subjected to nine different exonuclease digestion steps and quantified for NSA under both linear and hyperbranched RCA conditions. We investigated buffer composition, divalent ion concentration, single or dual enzyme digestion, overhang length in cohesive end ligation, and splint length in splint-padlock ligation. When applied in tandem, the conditions successfully mitigated the NSA end-fluorescence 30 – 100 fold while reducing the relative NSA (with respect to primer assisted RCA) to ~5%. Besides understanding the mechanistic origin of NSA, novel aspects of enzyme-substrate selectivity, buffer composition, and the role of divalent ions in enzyme activity were discovered. With increasing analytical applications, this study will help standardize NSA-free RCA assays involving pre-synthesized cDNA.