Interrogating Aptamer Chemical Space Through Modified Nucleotide Substitution Facilitated by Enzymatic DNA Synthesis.

Chemical modification of aptamers is an important step to improve their performance and stability in biological media. This can be performed either during their identification (mod-SELEX) or after the in vitro selection process (post-SELEX). In order to reduce the complexity and workload of the post-SELEX modification of aptamers, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden often associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity. In addition, while bioisosteres often improve the potency of small molecule drugs, this does not extend to chemically modified aptamers. Overall, this versatile method can be applied for the post-SELEX modification of other aptamers and functional nucleic acids.

Multiplexed isothermal nucleic acid amplification.

Multiplexed isothermal amplification and detection of nucleic acid sequences and biomarkers is of increasing importance in diverse areas including advanced diagnostics, food quality control and environmental monitoring. Whilst there are several very elegant isothermal amplification approaches, multiplexed amplification remains a challenge, requiring careful experimental design and optimisation, from judicious primer design in order to avoid the formation of primer dimers and non-specific amplification, applied temperature as well as the ratio and concentration of primers. In this review, we describe the various approaches that have been reported to date for multiplexed isothermal amplification, for both "one-pot" multiplexing as well as parallelised multiplexing using loop-mediated isothermal amplification, strand-displacement amplification, helicase-dependent amplification, rolling circle amplification, nucleic acid sequence-based amplification, with a particular focus on recombinase polymerase amplification.

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10-15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Isothermal solid-phase amplification system for detection of Yersinia pestis.

DNA amplification is required for most molecular diagnostic applications, but conventional polymerase chain reaction (PCR) has disadvantages for field testing. Isothermal amplification techniques are being developed to respond to this problem. One of them is the recombinase polymerase amplification (RPA) that operates at isothermal conditions without sacrificing specificity and sensitivity in easy-to-use formats. In this work, RPA was used for the optical detection of solid-phase amplification of the potential biowarfare agent Yersinia pestis. Thiolated forward primers were immobilized on the surface of maleimide-activated microtitre plates for the quantitative detection of synthetic and genomic DNA, with elongation occurring only in the presence of the specific template DNA and solution phase reverse primers. Quantitative detection was achieved via the use of biotinylated reverse primers and post-amplification addition of streptavidin-HRP conjugate. The overall time of amplification and detection was less than 1 h at a constant temperature of 37 °C. Single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) sequences were detected, achieving detection limits of 4.04*10(-13) and 3.14*10(-16) M, respectively. The system demonstrated high specificity with negligible responses to non-specific targets.