Ribonuclease-Responsive DNA Nanoswitches

Abstract

SUMMARY DNA has been used in the construction of dynamic DNA devices that can reconfigure in the presence of external stimuli. These nanodevices have found uses in fields ranging from biomedical to materials science applications. Here, we report a DNA nanoswitch that can be reconfigured using ribonucleases (RNases) and explore two applications: biosensing and molecular computing. For biosensing, we show the detection of RNase H and other RNases in relevant biological fluids and temperatures, as well as inhibition by the known enzyme inhibitor kanamycin. For molecular computing, we show that RNases can be used to enable erasing, write protection, and erase-rewrite functionality for information-encoding DNA nanoswitches. The simplistic mix-and-read nature of the ribonuclease-activated DNA nanoswitches could facilitate their use in assays for identifying RNase contamination in biological samples or for the screening and characterization of RNase inhibitors.

DNA-based ribonuclease detection assays

“…2c). 19 In catalytic hairpin assembly, a single stranded DNA triggers the self-assembly 34 Fluorescence 0.09 pg ml À1 1 h None DNA nanoswitch 35 Gel electrophoresis 0.02 U in 10 ml reaction 1 h Kanamycin of two metastable hairpin DNA probes via toehold-mediated strand displacement, thus producing a large number of hybridized duplexes. 37 In this CHA-based method, RNase H degrades the RNA in a DNA:RNA hybrid duplex substrate, releasing the DNA strand that binds to hairpin 1 via toehold based strand displacement.…”

“…22 enzyme presence using gel electrophoresis. 35 The group used DNA nanoswitches locked by an RNA strand into a looped conformation (Fig. 4c).…”

“…The interaction of ellipticine with RNase H effectively inhibits enzymatic activity either by blocking enzyme binding or by interaction with the cleavage substrate. 44 In the literature discussed here, several assays were used to screen RNase H inhibitors such as ellipticine, 19,28,32 ethidium bromide, 28,29 gentamycin, 22,29,30 oxoglaucine, 19 streptomycin sulfate, 22 kanamycin 22,35 and ampicillin. 22 Further analysis of the efficiency of RNase H inhibition could lead to promising drug development routes for AIDS treatment.…”

DNA Nanoswitch Barcodes for Multiplexed Biomarker Profiling

“…The signal comes from the intercalation of thousands of dye molecules from regularly used DNA gel stains (GelRed in this case). We previously used similar DNA nanoswitches for single molecule experiments, [21] detection of microRNAs, [22] viral RNAs, [23] antigens [24] and enzymes, [25] as well as in molecular memory. [16,26] Here, we expand the use of nanoswitches to a multiplexed DNA barcode system that can be used to detect any combination of up to 6 different biomarkers.…”

Detection of Alzheimer’s associated microRNAs using a DNA-based smart reagent

“…17 We previously used the DNA nanoswitches for detecting nucleic acids (DNA, 18 microRNAs, 17 viral RNAs 19 ) and other biomarkers such as proteins 20 and enzymes. 21 In this work, we use the nanoswitches to detect AD-related microRNAs with femtomolar sensitivity, specificity to discriminate microRNAs from the same family and demonstrate multiplexed detection of four microRNAs in a single assay. We further screened for AD-related microRNAs in brain tissue total RNA from healthy and AD samples, and quantified dysregulation of specific microRNA expression levels in these samples.…”