Multitasking by multivalent circular DNA aptamers.

Nucleic acid aptamers are finding increasing applications in biology, especially as therapeutic candidates and diagnostic components. An important characteristic in meeting the needs of these applications is improved stability in physiological fluids, which is most often accomplished with chemical modification or unnatural nucleotides. In an alternative approach we have specified the design of a multivalent circular DNA aptamer topology that encompasses a number of properties relevant to nucleic acid therapeutic candidates, especially the ability to multitask by combining different activities together within a modular structure.

Plasminogen activator inhibitor-2 is highly tolerant to P8 residue substitution--implications for serpin mechanistic model and prediction of nsSNP activities.

The serine protease inhibitor (serpin) superfamily is involved in a wide range of cellular processes including fibrinolysis, angiogenesis, apoptosis, inflammation, metastasis and viral pathogenesis. Here, we investigate the unique mousetrap inhibition mechanism of serpins through saturation mutagenesis of the P8 residue for a typical family member, plasminogen activator inhibitor-2 (PAI-2). A number of studies have proposed an important role for the P8 residue in the efficient insertion and stabilisation of the cleaved reactive centre loop (RCL), which is a key event in the serpin inhibitory mechanism.

Proximity extension of circular DNA aptamers with real-time protein detection.

Multivalent circular aptamers or 'captamers' have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the 'proximity extension' assay.

Construction, stability, and activity of multivalent circular anticoagulant aptamers.

Here we describe the design and construction of multivalent circular DNA aptamers. Four aptameric binding motifs directed at blood-borne targets are used as a model set from which larger, multidomain aptamers are constructed in a straightforward manner. Intra- or intermolecular ligation of precursor oligonucleotides provides a stabilizing mechanism against degradation by the predominant exonuclease activity of blood products without the need for post-selection chemical modification.

Proofreading genotyping assays and electrochemical detection of SNPs.

The use of proofreading DNA polymerases in genotyping assays offers the prospect of improved performance. To this end, we have recently used compatible DNA polymerases, protected primers, and substrates to implement proofreading single base extension (P-SBE) and proofreading allele-specific extension (PRASE) assays. Key aspects of the P-SBE and related proofreading assay formats are described here.

Multipotential electrochemical detection of primer extension reactions on DNA self-assembled monolayers.

Electroactive nucleoside triphosphates ("electrotides") have been incorporated into primers by DNA polymerase and detected on oligonucleotide surface-assembled monolayers. Four electrotides bearing three different electroactive moieties-ferrocene, vinylferrocene, and anthraquinone-are detected in four alternative formats.

Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays.

The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3'-->5' exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance.