ABSTRACT
Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.
Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , DNA, Single-Stranded/genetics , Electrophoresis, Agar Gel/methods , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Sequence Analysis, RNA/methods , Base Sequence , COVID-19 , Cell Line, Tumor , Coronavirus Infections/virology , Dengue/diagnosis , Dengue/virology , Dengue Virus/genetics , Electrophoresis, Agar Gel/economics , Humans , Limit of Detection , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Saliva/virology , Sequence Analysis, RNA/economics , Zika Virus/genetics , Zika Virus Infection/diagnosis , Zika Virus Infection/virologyABSTRACT
MicroRNAs are short noncoding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive and often requires amplification, labeling, or radioactive probes. Here, we report a single-step, nonenzymatic microRNA detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA-activated conditional looping of engineered switches), our assay has subattomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells and multiplex our detection for several microRNAs from one biological sample. We demonstrate 1-hour detection without expensive equipment or reagents, making this assay a compelling alternative to quantitative polymerase chain reaction and Northern blotting.
Subject(s)
DNA, Single-Stranded/metabolism , Electrophoresis, Agar Gel/methods , Genetic Engineering/methods , Inverted Repeat Sequences , MicroRNAs/analysis , Animals , Base Pairing , Cell Differentiation , Cell Line , DNA, Single-Stranded/genetics , Electrophoresis, Agar Gel/standards , Fluorescent Dyes/chemistry , Humans , Intercalating Agents/chemistry , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Myoblasts/cytology , Myoblasts/metabolism , Sensitivity and SpecificityABSTRACT
DNA serves as nature's information storage molecule, and has been the primary focus of engineered systems for biological computing and data storage. Here we combine recent efforts in DNA self-assembly and toehold-mediated strand displacement to develop a rewritable multi-bit DNA memory system. The system operates by encoding information in distinct and reversible conformations of a DNA nanoswitch and decoding by gel electrophoresis. We demonstrate a 5-bit system capable of writing, erasing, and rewriting binary representations of alphanumeric symbols, as well as compatibility with 'OR' and 'AND' logic operations. Our strategy is simple to implement, requiring only a single mixing step at room temperature for each operation and standard gel electrophoresis to read the data. We envision such systems could find use in covert product labeling and barcoding, as well as secure messaging and authentication when combined with previously developed encryption strategies. Ultimately, this type of memory has exciting potential in biomedical sciences as data storage can be coupled to sensing of biological molecules.
Subject(s)
Computers, Molecular , DNA, Viral/chemistry , Information Storage and Retrieval/methods , Nanostructures/chemistry , Bacteriophage M13/genetics , DNA, Viral/genetics , Electrophoresis, Agar Gel , Reproducibility of ResultsABSTRACT
DNA has emerged as a versatile building block for programmable self-assembly. DNA-based nanostructures have been widely applied in biosensing, bioimaging, drug delivery, molecular computation and macromolecular scaffolding. A variety of strategies have been developed to functionalize these nanostructures. In this study, we report a facile click-based strategy to incorporate a metal chelating ligand and a fluorescent tag into a three-point-star DNA tile containing 2'-O-propargyl groups. Such a strategy opens up the possibility of functionalizing pre-assembled DNA strands to construct platforms for metal or drug delivery.
