scholarly journals Detection of Target ssDNA Using a Microfabricated Hall Magnetometer with Correlated Optical Readout

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Steven M. Hira ◽  
Khaled Aledealat ◽  
Kan-Sheng Chen ◽  
Mark Field ◽  
Gerard J. Sullivan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Signal Amplification ◽  
Point Of Care ◽  
Target Sequence ◽  
Large Signal ◽  
Optical Readout ◽  
Hall Magnetometer ◽  
Target Dna ◽  
Dna Strands ◽  
Target Binding

Sensing biological agents at the genomic level, while enhancing the response time for biodetection over commonly used, optics-based techniques such as nucleic acid microarrays or enzyme-linked immunosorbent assays (ELISAs), is an important criterion for new biosensors. Here, we describe the successful detection of a 35-base, single-strand nucleic acid target by Hall-based magnetic transduction as a mimic for pathogenic DNA target detection. The detection platform has low background, large signal amplification following target binding and can discriminate a single, 350 nm superparamagnetic bead labeled with DNA. Detection of the target sequence was demonstrated at 364 pM (<2 target DNA strands per bead) target DNA in the presence of 36 μM nontarget (noncomplementary) DNA (<10 ppm target DNA) using optical microscopy detection on a GaAs Hall mimic. The use of Hall magnetometers as magnetic transduction biosensors holds promise for multiplexing applications that can greatly improve point-of-care (POC) diagnostics and subsequent medical care.

scholarly journals Nanomaterials for molecular signal amplification in electrochemical nucleic acid biosensing: recent advances and future prospects for point-of-care diagnostics

2020 ◽  
Vol 5 (1) ◽  
pp. 49-66 ◽  
Author(s):  
Léonard Bezinge ◽  
Akkapol Suea-Ngam ◽  
Andrew J. deMello ◽  
Chih-Jen Shih
Keyword(s):  
Nucleic Acid ◽  
Molecular Diagnostics ◽  
Signal Amplification ◽  
Point Of Care ◽  
Future Prospects ◽  
Electrochemical Biosensing ◽  
Recent Advances ◽  
Point Of Care Diagnostics ◽  
Molecular Signal

This account reviews the major amplification strategies utilizing nanomaterials in electrochemical biosensing for robust and sensitive molecular diagnostics.

scholarly journals Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

Micromachines ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1204
Author(s):  
Zackary A. Zimmers ◽  
Alexander D. Boyd ◽  
Hannah E. Stepp ◽  
Nicholas M. Adams ◽  
Frederick R. Haselton
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Magnetic Beads ◽  
Point Of Care ◽  
Dna Amplification ◽  
Magnetic Bead ◽  
Nucleic Acid Amplification ◽  
Diagnostic Strategies ◽  
Low Resource ◽  
Target Dna

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

scholarly journals Molecular mechanism of off-target effects in CRISPR-Cas9

2018 ◽  
Author(s):  
Clarisse G. Ricci ◽  
Janice S. Chen ◽  
Yinglong Miao ◽  
Martin Jinek ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Md Simulations ◽  
Target Sequence ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Mismatches ◽  
Open Issue ◽  
Conformational Freedom ◽  
Target Binding ◽  
Dna Strand

AbstractCRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

scholarly journals Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

2021 ◽  
Vol 7 (8) ◽  
pp. eabe5496
Author(s):  
Evan A. Boyle ◽  
Winston R. Becker ◽  
Hua B. Bai ◽  
Janice S. Chen ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Dna Cleavage ◽  
High Specificity ◽  
Biophysical Model ◽  
Target Sequence ◽  
Sequence Context ◽  
Guide Rnas ◽  
Target Dna ◽  
Target Binding ◽  
Model Sequence

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

scholarly journals Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

2020 ◽  
Author(s):  
Evan A Boyle ◽  
Winston R Becker ◽  
Hua B Bai ◽  
Janice S Chen ◽  
Jennifer A Doudna ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Dna Cleavage ◽  
High Specificity ◽  
Biophysical Model ◽  
Target Sequence ◽  
Sequence Context ◽  
Guide Rnas ◽  
Target Dna ◽  
Target Binding ◽  
Model Sequence

AbstractThe RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data has hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage, then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target significantly influences Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Finally, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

scholarly journals Type II and type V CRISPR effector nucleases from a structural biologist’s perspective

2016 ◽  
Vol 62 (3) ◽  
pp. 315-326
Author(s):  
Humberto Fernandes ◽  
Michal Pastor ◽  
Matthias Bochtler
Keyword(s):  
Target Sequence ◽  
Type Ii ◽  
Molecular Architecture ◽  
Genomic Engineering ◽  
Double Stranded Dna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strands ◽  
Catalytic Role ◽  
Type V

The type II and type V CRISPR effector nucleases Cas9 and Cpf1 are “universal” DNA endonucleases, which can be programmed by an appropriate crRNA or sgRNA strand to cleave almost any DNA duplex at a preselected position (constrained only by short, so-called PAMs). In this review, we briefly introduce CRISPR bacterial adaptive immunity as the biological context in which Cas9 and Cpf1 proteins operate, and then present the structural insights that have been obtained in the last two or three years that illustrate the mode of operation of these proteins. We describe the R-loop structures at the core of the Cas9 and Cpf1 complexes, and the structure of the 5’- or 3’-handles that help anchor the nucleic acid complexes to the proteins in a manner that is independent of the target sequence. Next, we describe the molecular architecture of the Cas9 and Cpf1 proteins. We illustrate how Cas9 and Cpf1 proteins scan double stranded DNA for so-called protospacer associated motifs (PAMs), we explain how the phosphate loop (PLL) and basic helix (BH) promote the separation of target and non-target DNA strands and the formation of hybrids between crRNA or sgRNA and the target strand of DNA. We also describe the current understanding of the catalytic mechanisms of RuvC and HNH domains, and a possible, but still very uncertain catalytic role of the Nuc domain. At the end of the review, we briefly summarize key developments that have initiated the field of genomic engineering using Cas9 or Cpf1 nucleases..

scholarly journals SPECIFICITY OF TRANSPOSON Tn5 INSERTION

Genetics ◽  
1983 ◽  
Vol 105 (4) ◽  
pp. 813-828
Author(s):  
Douglas E Berg ◽  
Margaret A Schmandt ◽  
John B Lowe
Keyword(s):  
Molecular Level ◽  
Target Sequence ◽  
Restriction Endonuclease Digestion ◽  
Plasmid Pbr322 ◽  
Base Pairs ◽  
Transposon Tn5 ◽  
Target Dna ◽  
Dna Strands ◽  
Endonuclease Digestion ◽  
Bacterial Transposon

ABSTRACT Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.

Gold-Aptamer-Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

2019 ◽  
Author(s):  
Veeren Chauhan ◽  
Mohamed M Elsutohy ◽  
C Patrick McClure ◽  
Will Irving ◽  
Neil Roddis ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
In Silico ◽  
Point Of Care ◽  
Lateral Flow ◽  
Nucleic Acid Sequence ◽  
In Silico Screening ◽  
Lateral Flow Assays ◽  
Life Threatening ◽  
Software And Hardware ◽  
Nucleic Acid Sequences

<p>Enteroviruses are a ubiquitous mammalian pathogen that can produce mild to life-threatening disease. Bearing this in mind, we have developed a rapid, accurate and economical point-of-care biosensor that can detect a nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and aptamers to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral RNA sequence (23 bases), which was identified through in silico screening. Aptamers were designed to demonstrate specific complementarity towards the target enteroviral RNA to produce aggregated gold-aptamer nanoconstructs. Conserved target enteroviral nucleic acid sequence (≥ 1x10<sup>-7</sup> M, ≥1.4×10<sup>-14</sup> g/mL), initiates gold-aptamer-nanoconstructs disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow-assays that utilise gold-aptamer-nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (< 60 s) and could be interpreted with a bespoke software and hardware electronic interface. We anticipate our methodology will translate in-silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave-the-way forward in the clinical evaluation of disease and complement existing strategies at overcoming antimicrobial resistance.</p>

scholarly journals A glucose meter interface for point-of-care gene circuit-based diagnostics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evan Amalfitano ◽  
Margot Karlikow ◽  
Masoud Norouzi ◽  
Katariina Jaenes ◽  
Seray Cicek ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Synthetic Biology ◽  
Molecular Diagnostics ◽  
Point Of Care ◽  
Low Cost ◽  
Gene Circuit ◽  
Reporter Systems ◽  
Glucose Meter ◽  
Pandemic Response ◽  
Glucose Output

AbstractRecent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

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