MacIsaac, M. | Search bioRxiv

bioRxiv2020

DNA nanoswitch barcodes for multiplexed biomarker profiling

The detection of molecular biomarkers plays a key role in the clinic, aiding in diagnostics and prognostics, and in the research laboratory, contributing to our basic understanding of diseases. The ability to detect multiple and diverse molecular biomarkers within a single accessible assay would have great utility, providing a more comprehensive picture for clinical evaluation and research, but is a challenge with standard methods. One promising approach is the use of dynamic DNA nanostructures that can respond to molecular biomarkers, which have recently been used in a variety of biosensing strategies. In this work, we report the use of programmable DNA nanoswitches for the multiplexed detection of up to 6 biomarkers within a single pot through the use of a barcoded gel-based readout. We demonstrate the barcoding capability using gene fragments that correspond to 6 different diseases, with each fragment or combination of fragments producing a unique barcode signature. As a defining feature of our method, we show "mixed multiplexing" for simultaneous barcoded detection of different types of biomolecules - DNA, RNA, antibody and protein in a single assay. To demonstrate clinical potential, we show multiplexed detection of a prostate cancer biomarker panel in serum that includes two microRNA sequences and prostate specific antigen (PSA). This strategy holds promise in clinical diagnostics for profiling complex and diverse biomarker panels.

bioengineering↗

bioRxiv2018

Detection of cellular microRNAs with programmable DNA nanoswitches

MicroRNAs are short non-coding 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, non-enzymatic detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA activated conditional looping of engineered switches), our assay has sub-attomole 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 demonstrate multiplexed detection of several microRNAs from one biological sample. We demonstrate one-hour detection without expensive equipment or reagents, making this assay a compelling alternative to qPCR and Northern blotting.\n\nSignificance statementDetection of microRNAs play a key role in biological research and medical diagnostics, and current detection methods are expensive and require sophisticated processes. We present microRNA activated conditional looping of engineered switches (miRacles), a mix-and-read strategy that is based on conformational changes of DNA nanoswitches upon binding a target microRNA. MiRacles has a sensitivity of [~]4 copies/cell and specificity of a single nucleotide, and can be performed in one hour at a fraction of the cost of traditional microRNA detection techniques. Our method can also be multiplexed to detect multiple microRNAs from one biological sample. The minimalistic miRacles assay has immediate application in biomedical research and longer term potential as a clinical tool.

bioengineering↗