The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5′ single-stranded overhang, indicating a 5′o 3′ polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT₂₄ binding over 10 times more strongly than dA₂₄ as observed by the apparent K_m .

Highly specific and tight-binding nucleic acid aptamers have been selected against a variety of molecular targets for over 20 years. A significant proportion of these oligonucleotides display G-quadruplex structures, particularly for DNA aptamers, that enable molecular recognition of their ligands. G-quadruplex structures couple a common scaffold to varying loop motifs that act in target recognition. Here, we review DNA G-quadruplex aptamers and their ligands from a structural and functional perspective. We compare the diversity of DNA G-quadruplex aptamers selected against multiple ligand targets, and consider structure with a particular focus on dissecting the thrombin binding aptamer – thrombin interaction. Therapeutic and analytical applications of DNA G-quadruplex aptamers are also discussed. Understanding DNA G-quadruplex aptamers carries implications not only for therapeutics and diagnostics, but also in the natural biochemistry of guanine-rich nucleic acids.

Aptamers are short nucleic acid sequences capable of specific, high-affinity molecular binding. They are isolated via SELEX (Systematic Evolution of Ligands by Exponential Enrichment), an evolutionary process that involves iterative rounds of selection and amplification before sequencing and aptamer characterization. As aptamers are genetic in nature, bioinformatic approaches have been used to improve both aptamers and their selection. This review will discuss the advancements made in several enclaves of aptamer bioinformatics, including simulation of aptamer selection, fragment-based aptamer design, patterning of libraries, identification of lead aptamers from high-throughput sequencing (HTS) data and in silico aptamer optimization.

Development of plasmonic biosensors combining reliability and ease of use is still a challenge. Gold nanoparticle arrays made by block copolymer micelle nanolithography (BCMN) stand out for their scalability, cost-effectiveness and tunable plasmonic properties, making them ideal substrates for fluorescence enhancement. Here, we describe a plasmon-enhanced fluorescence immunosensor for the specific and ultrasensitive detection of Plasmodium falciparum lactate dehydrogenase (PfLDH)—a malaria marker—in whole blood. Analyte recognition is realized by oriented antibodies immobilized in a close-packed configuration via the photochemical immobilization technique (PIT), with a top bioreceptor of nucleic acid aptamers recognizing a different surface of PfLDH in a sandwich conformation. The combination of BCMN and PIT enabled maximum control over the nanoparticle size and lattice constant as well as the distance of the fluorophore from the sensing surface. The device achieved a limit of detection smaller than 1 pg/mL (<30 fM) with very high specificity without any sample pretreatment. This limit of detection is several orders of magnitude lower than that found in malaria rapid diagnostic tests or even commercial ELISA kits. Thanks to its overall dimensions, ease of use and high-throughput analysis, the device can be used as a substrate in automated multi-well plate readers and improve the efficiency of conventional fluorescence immunoassays.