How to Perform miRacles: A Step‐by‐Step microRNA Detection Protocol Using DNA Nanoswitches

Abstract

MicroRNAs are short non‐coding RNAs involved in post‐transcriptional gene regulation, and are increasingly considered to be biomarkers for numerous biological processes and human diseases. Current techniques used for microRNA detection can be expensive and labor‐intensive, and typically require amplification, labeling, or radioactive probes. In this protocol, we describe a DNA nanoswitch–based microRNA detection assay termed “miRacles”: microRNA‐activated conditional looping of engineered switches. This method uses conformationally responsive DNA nanoswitches that detect the presence of specific microRNAs with a simple and unambiguous gel‐shift assay that can be performed on the benchtop. The assay is low cost, minimalistic, and capable of direct detection of specific microRNAs in unprocessed total RNA samples, with no enzymatic amplification, labeling, or special equipment. The protocol for detection of microRNAs in total RNA can be completed in as little as a few hours, making this assay a compelling alternative to qPCR and Northern blotting. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of DNA nanoswitches Basic Protocol 2: Detection of microRNAs from total RNA samples Support Protocol 1: Optional nanoswitch purification by PEG precipitation Support Protocol 2: Optional nanoswitch purification by liquid chromatography

DNA Nanotechnology in the Undergraduate Laboratory: Analysis of Molecular Topology Using DNA Nanoswitches

“…These results suggest that the DNA nanoswitch system is a feasible platform for adaptation to undergraduate laboratories, not just in a topological context but also in potential biosensing and biophysical applications . Instructors can adapt the nanoswitch for other nucleic acid targets or to explain different concepts using guidelines in our earlier work. , …”

“…29 Instructors can adapt the nanoswitch for other nucleic acid targets or to explain different concepts using guidelines in our earlier work. 33,35 From the outcome of these experiments, we list here a few considerations for adaptation to the undergraduate laboratory, as well as steps that might not be successful:…”

High-throughput single-molecule quantification of individual base stacking energies in nucleic acids

“…DNA constructs were prepared by hybridizing 124 oligonucleotides (Integrated DNA Technologies) to 7249 nt single-stranded M13mp18 DNA (New England Biolabs). The construction method largely follows our approach for DNA nanoswitch construction [48]. Briefly, the M13 DNA is enzymatically linearized and then incubated with a 10 fold molar excess of backbone oligos 1-122, 150 fold overhang oligo and 500 fold stacking end oligo (Table S2) with an annealing temperature ramp from 90 ˚C to 20 ˚C.…”

Using ultraviolet absorption spectroscopy to study nanoswitches based on non-canonical DNA structures

“…DNA nanoswitches can be used to selectively open and close drug delivery capsules [ [11] , [12] , [13] ] and they can also play a role in molecular computing systems [ 14 ], which offer scope for low-power information processing in a biocompatible framework [ 15 ]. In addition, DNA nanoswitches find applications in a range of biosensors [ 16 , 17 ].…”