A Unified Sensor Architecture for Isothermal Detection of Double‐Stranded DNA, Oligonucleotides, and Small Molecules

Abstract

Pathogen detection is an important problem in many areas of medicine and agriculture, which may involve genomic or transcriptomic signatures, or small molecule metabolites. We report a unified, DNA-based sensor architecture capable of isothermal detection of double-stranded DNA targets, single-stranded oligonucleotides, and small molecules. Each sensor contains independent target detection and reporter modules, enabling rapid design. We detected gene variants on plasmids via a straightforward isothermal denaturation protocol. The sensors were highly specific, even with a randomized DNA background. We achieved a limit of detection of ~15 pM for single-stranded targets and ~5 nM for targets on denatured plasmids. By incorporating a blocked aptamer sequence, we also detected small molecules using the same sensor architecture. This work provides a starting point for multiplexed detection of multi-strain pathogens, and disease states caused by genetic variants (e.g., sickle cell anemia).

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“…1,2 DNA-based biomolecular computation has been used for bioassays targeting pathogen signatures and to integrate detection of multiple pathogen signatures into a single reaction. 6–13 However, non-specific interactions, arising from background environmental nucleic acids and from molecular crosstalk due to lack of component separation in solution, can severely limit assay performance. 6 …”

“…6–13 However, non-specific interactions, arising from background environmental nucleic acids and from molecular crosstalk due to lack of component separation in solution, can severely limit assay performance. 6 …”

“…14,15 Of particular interest are DNAzymes that cleave partially complementary chimeric DNA substrate molecules at a single RNA base, such as the 8–17 DNAzyme, chosen for this work due to its small size and high catalytic activity. 1,6,10,16–18 In DNAzyme-driven molecular computation, input signals activate DNAzymes, each of which can cleave multiple substrate molecules, enabling signal amplification via catalysis.…”

“…The field of DNA nanotechnology uses biomolecules to construct computing components analogous to those in electronic computers − and to build synthetic nanostructures to study molecular interactions. , DNA is attractive for these applications as Watson–Crick base pairing enables straightforward programming of molecular interactions. , DNA-based biomolecular computation has been used for bioassays targeting pathogen signatures and to integrate detection of multiple pathogen signatures into a single reaction. − However, nonspecific interactions, arising from background environmental nucleic acids and from molecular crosstalk due to lack of component separation in solution, can severely limit assay performance …”

“…DNA-based molecular computation is typically performed in solution using toehold-mediated strand displacement (TMSD), DNAzymes, or a combination of the two approaches. , TMSD molecular computing systems are an enzyme-free mechanism wherein target strands interact with a gate complex composed of a template strand partially hybridized to an incumbent strand . The gate has a double-stranded DNA (dsDNA) region with a short single-stranded DNA (ssDNA) overhang on the template strand, referred to as a toehold , which serves as a nucleation site for target strand binding.…”