Single-molecule analysis of nucleic acid biomarkers – A review

Gilboa, Tal; Garden, Padric M.; Cohen, Limor

doi:10.1016/j.aca.2020.03.001

Cited by 41 publications

(30 citation statements)

References 260 publications

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“…The detection time window potentially can be further shortened with fast cameras, high laser intensities, stable fluorophores, and sensors that can withstand higher formamide concentrations and/or temperatures. iSiMREPS showed a linear dynamic range of ~3-4 orders of magnitude, which is comparable to or better than existing single-molecule techniques for nucleic acid detection 36 . While iSiMREPS has somewhat lower sensitivity than existing technologies for detecting nucleic acids like droplet digital PCR (ddPCR 10,37 ) and NGS 38 , it exhibits superior specificity, comparable or better dynamic range and faster detection of nucleic acids than ddPCR 39 , NGS 38,40 , or Simoa 13 .…”

Section: Discussionmentioning

confidence: 67%

Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET-based Dynamic Nanosensor

Khanna

Mandal

Blanchard

et al. 2021

Preprint

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Biofluids contain cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) that have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that increase the risk of sample loss and cross-contamination, respectively. To achieve the direct, rapid detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein describe an accelerated amplification-free single-molecule kinetic fingerprinting. This approach, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS), exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures individual target molecules onto the surface, while the other transiently interrogates them to generate kinetic fingerprints by intramolecular sin-gle-molecule Forster resonance energy transfer (smFRET). Formamide is used to further accelerate the kinetics of probe-target interactions and fingerprinting, while background signals are reduced by removing non-target-bound probes from the surface using toehold-mediated strand displacement. We show that iSiMREPS can detect in as little as 10 seconds two distinct, promising cancer biomarkers, miR-141 and a common EGFR exon 19 deletion, reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.

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Section: Discussionmentioning

confidence: 67%

Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET-based Dynamic Nanosensor

Khanna

Mandal

Blanchard

et al. 2021

Preprint

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“…The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ones [16,17]. This study applies several modular DNA sequences containing the restriction sites of interest and uses CFPS as a readout means to interrogate the restriction digest efficiency of a wide variety of REs and other DNA-degrading enzymes.…”

Section: Introductionmentioning

confidence: 99%

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

et al. 2020

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Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases’ behaviors and functions in daily genetic engineering experiments.

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“…Malignant gene mutations usually result in cancer, and there are usually marker RNA transcriptions after cells became cancerous. The detection of those marker RNAs has high confidence in the early detection of cancers [ 50 , 51 ]. For instance, miRNA−21 is one of the early markers of glioma cancer.…”

Section: Paper-based Sensor For Biomolecule Sensingmentioning

confidence: 99%

Hospitals and Laboratories on Paper-Based Sensors: A Mini Review

Zhang

Xia

Feng

et al. 2021

Sensors

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With characters of low cost, portability, easy disposal, and high accuracy, as well as bulky reduced laboratory equipment, paper-based sensors are getting increasing attention for reliable indoor/outdoor onsite detection with nonexpert operation. They have become powerful analysis tools in trace detection with ultra-low detection limits and extremely high accuracy, resulting in their great popularity in medical detection, environmental inspection, and other applications. Herein, we summarize and generalize the recently reported paper-based sensors based on their application for mechanics, biomolecules, food safety, and environmental inspection. Based on the biological, physical, and chemical analytes-sensitive electrical or optical signals, extensive detections of a large number of factors such as humidity, pressure, nucleic acid, protein, sugar, biomarkers, metal ions, and organic/inorganic chemical substances have been reported via paper-based sensors. Challenges faced by the current paper-based sensors from the fundamental problems and practical applications are subsequently analyzed; thus, the future directions of paper-based sensors are specified for their rapid handheld testing.

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Single-molecule analysis of nucleic acid biomarkers – A review

Cited by 41 publications

References 260 publications

Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET-based Dynamic Nanosensor

Rapid Kinetic Fingerprinting of Single Nucleic Acid Molecules by a FRET-based Dynamic Nanosensor

A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

Hospitals and Laboratories on Paper-Based Sensors: A Mini Review

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