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A new method for the detection of genomic RNA combines RNA cleavage by the 10-23 DNAzyme and use of the cleavage fragments as primers to initiate rolling circle amplification (RCA). 230 different 10-23 DNAzyme variants were screened to identify those that target accessible RNA sites within the highly structured RNA transcripts of SARS-CoV-2. A total of 28 DNAzymes were identified with >20 % cleavage, 5 with >40 % cleavage and one with >60 % in 10 min. The cleavage fragments from these reactions were then screened for coupling to an RCA reaction, leading to the identification of several cleavage fragments that could efficiently initiate RCA. Using a newly developed quasi-exponential RCA method with a detection limit of 500 aM of RNA, 14 RT-PCR positive and 15 RT-PCR negative patient saliva samples were evaluated for SARS-CoV-2 genomic RNA, achieving a clinical sensitivity of 86 % and specificity of 100 % for detection of the virus in <2.5 h., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Invited for the cover of this issue are John Brennan, Yingfu Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078., (© 2022 Wiley-VCH GmbH.)

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed K d values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with K d values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5'-end protects it from 3'-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay., (© 2021 Wiley-VCH GmbH.)

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There is growing interest in developing printable paper sensors to enable rapid testing of analytes for environmental, food safety, and clinical applications. A major challenge is to find suitable bioinks that are amenable to high-speed printing and remain functional after printing. We report on a simple and effective approach wherein an aqueous ink composed of megadalton-sized tandem repeating structure-switching DNA aptamers (concatemeric aptamers) is used to rapidly create patterned paper sensors on filter paper by inkjet printing. These concatemeric aptamer reporters remain immobilized at the point of printing through strong adsorption but retain sufficient segmental mobility to undergo structure switching and fluorescence signaling to provide both qualitative and quantitative detection of small molecules and protein targets. The convenience of inkjet printing allows for the patterning of internally referenced sensors with multiplexed detection, and provides a generic platform for on-demand printing of sensors even in remote locations., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)