Multi-Amplified Sensing of MicroRNA by a Small DNA Fragment-Driven Enzymatic Cascade Reaction
File(s)
Accepted version
Author(s)
Kim, E
Howes, PD
Crowder, SW
Stevens, MM
Type
Journal Article
Abstract
Combining technological developments such as
nanomaterials, DNA nanotechnology, and functional enzymes
has great potential to facilitate next generation high performance
molecular diagnostic systems. In this work, we describe a
microRNA (miRNA) detection assay that combines target
recycling and isothermal amplification in an elegantly designed
enzyme-mediated cascade reaction. Target recycling is driven
by the action of duplex-specific nuclease (DSN), resulting in
highly amplified translation of input miRNA to short output
DNA fragments. These fragments act as highly specific
initiators of rolling circle amplification (RCA), an isothermal
reaction that outputs a large volume of polymeric DNAzymes
per initiator, and finally a fluorogenic output signal. Based on
careful electrophoretic analysis we observed that the DSN produces ca. 10 nt DNA fragments from DNA/miRNA duplexes,
regardless of the length of DNA strands. Target recycling yielded ca. 5 orders of magnitude amplification through the DSNassisted
recycling system on magnetic particles, and the RCA yielded a further 2 orders of magnitude. The final assay exhibited a
limit of detection of 1.8 fM of miRNA spiked into 20% human serum, and showed excellent selectivity for miR-21 versus single
base-mismatched sequences and other cancer-related miRNAs. The developed assay was further employed to determine accurate
amounts of miR-21 in total RNA samples extracted from human cancer cell lines and normal cells, confirming the applicability of
the assay for direct and absolute quantification of mature specific miRNA in real biological samples.
nanomaterials, DNA nanotechnology, and functional enzymes
has great potential to facilitate next generation high performance
molecular diagnostic systems. In this work, we describe a
microRNA (miRNA) detection assay that combines target
recycling and isothermal amplification in an elegantly designed
enzyme-mediated cascade reaction. Target recycling is driven
by the action of duplex-specific nuclease (DSN), resulting in
highly amplified translation of input miRNA to short output
DNA fragments. These fragments act as highly specific
initiators of rolling circle amplification (RCA), an isothermal
reaction that outputs a large volume of polymeric DNAzymes
per initiator, and finally a fluorogenic output signal. Based on
careful electrophoretic analysis we observed that the DSN produces ca. 10 nt DNA fragments from DNA/miRNA duplexes,
regardless of the length of DNA strands. Target recycling yielded ca. 5 orders of magnitude amplification through the DSNassisted
recycling system on magnetic particles, and the RCA yielded a further 2 orders of magnitude. The final assay exhibited a
limit of detection of 1.8 fM of miRNA spiked into 20% human serum, and showed excellent selectivity for miR-21 versus single
base-mismatched sequences and other cancer-related miRNAs. The developed assay was further employed to determine accurate
amounts of miR-21 in total RNA samples extracted from human cancer cell lines and normal cells, confirming the applicability of
the assay for direct and absolute quantification of mature specific miRNA in real biological samples.
Date Issued
2016-10-10
Date Acceptance
2016-10-10
Citation
ACS Sensors, 2016, 2 (1), pp.111-118
ISSN
2379-3694
Publisher
American Chemical Society
Start Page
111
End Page
118
Journal / Book Title
ACS Sensors
Volume
2
Issue
1
Copyright Statement
© 2016 American Chemical Society. This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Sensors, after peer review and technical editing by the publisher. To access the final edited and published work see https://dx.doi.org/10.1021/acssensors.6b00601
Sponsor
Engineering & Physical Science Research Council (E
Engineering & Physical Science Research Council (EPSRC)
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000393088300015&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Grant Number
EP/K031953/1
EP/K020641/1
Subjects
Science & Technology
Physical Sciences
Chemistry, Multidisciplinary
Nanoscience & Nanotechnology
Chemistry
Science & Technology - Other Topics
biosensing
miRNA
DNA fragments
target recycling
isothermal amplification
DUPLEX-SPECIFIC NUCLEASE
SIGNAL AMPLIFICATION
NONCODING RNAS
LABEL-FREE
ONE-STEP
STRATEGY
QUANTIFICATION
HEPATOPANCREAS
DNAZYMES
DISEASES
Publication Status
Published