A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots.

H D Gliddon,P D Howes,M Levin,M Kaforou,M M Stevens

doi:10.1039/c6nr00484a

Abstract

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.

Highlights

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research
A population of quantum dots (QDs) were functionalized with ssDNA surface probes that are complementary to the QProbes (Scheme 1b)
Specific hybridization of the QProbe and CProbe brings the dye into close proximity of the QD and allows Förster resonance energy transfer (FRET) to occur from the QD to the dye

Summary

Introduction

The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. The ‘-omics’ areas of biomedical research (transcriptomics, genomics, proteomics, metabolomics and lipidomics) are revealing ever more complex and subtle relationships between biomolecular signatures and health status Such studies are identifying biomarkers with the potential to be developed into diagnostic tests. DNA nanotechnology, where nucleic acids are used to build structural and/or mechanistic systems, has emerged as an area that could prove transformative in the way we detect genomic biomarkers, both in vitro and in cell studies.[1,2,3,4] Of specific interest here is a process called ‘toehold-mediated strand displacement’, which has become an important mechanism upon which to base dynamic DNA systems.[5] Here, an incoming invading strand binds to an overhanging toehold region of a DNA duplex, proceeds to displace the incumbent DNA strand by branch migration. The switching event can either lead directly to a signal change itself,[12] or it can initiate a downstream event that leads to a signal change.[11]

Methods

Results

Conclusion