scholarly journals Highly specific enrichment of rare nucleic acids using Thermus thermophilus Argonaute

2018 ◽  
Author(s):  
Jinzhao Song ◽  
Jorrit W. Hegge ◽  
Michael G. Mauk ◽  
Neha Bhagwat ◽  
Jacob E. Till ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Thermus Thermophilus ◽  
Wild Type ◽  
Single Nucleotide ◽  
Blood Samples ◽  
Complementary Dna ◽  
Enzymatic Amplification ◽  
Dna And Rna ◽  
Rare Alleles

ABSTRACTCharacterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early detection, genotyping, and personalized therapy; but is challenged by alleles of interest differing by single nucleotide from and residing among large abundance of wild-type alleles. We describe a new multiplexed enrichment assay, dubbed NAVIGATER, that utilizes short nucleic acid-guided endonucleases Argonaute (Ago), derived from the bacterium Thermus thermophilus (TtAgo), to specifically cleave complementary DNA and RNA while sparing alleles having single nucleotide mismatches with the guides. NAVIGATER greatly increases the fractions of rare alleles of interest in samples and enhances sensitivity of downstream procedures such ddPCR, sequencing, and clamped enzymatic amplification. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and detection of KRAS, EGFR, and BRAF mutants with XNA-PCR at 0.01% fraction.

Highly sensitive detection of rare mutant alleles by combining argonaute-based enrichment and XNA-PCR.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14605-e14605
Author(s):  
Jinzhao Song ◽  
Michael Joseph Powell ◽  
Wei Liu ◽  
Junman Chen ◽  
Haim Bau
Keyword(s):  
Nucleic Acids ◽  
High Efficiency ◽  
Elevated Temperatures ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Detection Methods ◽  
Single Nucleotide ◽  
Complementary Dna ◽  
Dna And Rna ◽  
Rna Targets

e14605 Background: Characterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early disease detection, genotyping, and personalized therapy. Detection of alleles of clinical interest is often challenged by their low concentration and sequence homology with the much more abundant wildtype nucleic acids. Methods: Argonuate (Ago) from the thermophilic bacterium Thermus thermophilus ( TtAgo) utilizes short DNA guides to specifically cleave complementary DNA and RNA targets. We found that under optimized conditions, TtAgo cleaves DNA and RNA complementary to the guide DNA with high efficiency, but spares nucleic acids with a single nucleotide mismatch at and around its catalytic site with high sensitivity. Based on these findings, we designed a new multiplexed enrichment assay, dubbed NAVIGATER (Nucleic Acid enrichment Via DNA Guided Argonaute from Thermus thermophilus), that utilizes TtAgo, to specifically cleave perfectly complementary DNA and RNA while sparing alleles of interest. Results: NAVIGATER greatly increases the fractions of rare mutant alleles with single nucleotide precision enhancing the sensitivity of downstream detection methods such as XNA-PCR. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and over ten-fold improved sensitivity of XNA-PCR, enabling multiplex detection of KRAS and EGFR mutants at 0.01% fractions. Conclusions: NAVIGATER has important advantages over other mutant allele enrichment assays such as the ones based on CRISPR-Cas. It does not require the target to contain a protospacer-adjacent motif; is a true (turnover) catalyst; can cleave both DNA and associated exosomal RNA targets, improving sensitivity; and can operate at elevated temperatures for higher selectivity and compatibility with detection schemes.

scholarly journals Polarization spectroscopy methods in the determination of interactions of small molecules with nucleic acids – tutorial

2018 ◽  
Vol 14 ◽  
pp. 84-105 ◽  
Author(s):  
Tamara Šmidlehner ◽  
Ivo Piantanida ◽  
Gennaro Pescitelli
Keyword(s):  
Nucleic Acids ◽  
Small Molecules ◽  
Helical Structure ◽  
Linear Dichroism ◽  
Research Field ◽  
X Ray Diffraction ◽  
Polarization Spectroscopy ◽  
Dna And Rna

The structural characterization of non-covalent complexes between nucleic acids and small molecules (ligands) is of a paramount significance to bioorganic research. Highly informative methods about nucleic acid/ligand complexes such as single crystal X-ray diffraction or NMR spectroscopy cannot be performed under biologically compatible conditions and are extensively time consuming. Therefore, in search for faster methods which can be applied to conditions that are at least similar to the naturally occurring ones, a set of polarization spectroscopy methods has shown highly promising results. Electronic circular dichroism (ECD) is the most commonly used method for the characterization of the helical structure of DNA and RNA and their complexes with ligands. Less common but complementary to ECD, is flow-oriented linear dichroism (LD). Other methods such as vibrational CD (VCD) and emission-based methods (FDCD, CPL), can also be used for suitable samples. Despite the popularity of polarization spectroscopy in biophysics, aside several highly focused reviews on the application of these methods to DNA/RNA research, there is no systematic tutorial covering all mentioned methods as a tool for the characterization of adducts between nucleic acids and small ligands. This tutorial aims to help researchers entering the research field to organize experiments accurately and to interpret the obtained data reliably.

Histidine-rich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway

2016 ◽  
Vol 115 (01) ◽  
pp. 89-98 ◽  
Author(s):  
Trang T. Vu ◽  
Beverly A. Leslie ◽  
Alan R. Stafford ◽  
Ji Zhou ◽  
James C. Fredenburgh ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Wild Type ◽  
Contact Activation ◽  
Clotting Time ◽  
Intrinsic Pathway ◽  
Dna And Rna ◽  
Multiple Levels ◽  
Coagulation Pathway ◽  
Control Plasma ◽  
Deficient Mice

SummaryWhen triggered by factor (F) XII and nucleic acids, we showed that thrombosis in HRG-deficient mice is accelerated compared with that in wild-type mice. In this study, we set out to identify the mechanisms by which nucleic acids promote contact activation, and to determine whether HRG attenuates their effects. DNA or RNA addition to human plasma enhances thrombin generation via the intrinsic pathway and shortens the clotting time. Their effect on the clotting time is seven- to 14-fold greater in HRG-deficient plasma than in control plasma. Investigations into the mechanisms of activation reveal that nucleic acids a) promote FXII activation in the presence of prekallikrein- and high molecular weight kininogen (HK), and b) enhance thrombin-mediated FXI activation by 10– to 12-fold. Surface plasmon resonance studies show that DNA and RNA bind FXII, FXIIa, HK, FXI, FXIa and thrombin with high affinity. HRG attenuates DNA- and RNA-mediated FXII activation, and FXI activation by FXIIa or by thrombin, suggesting that HRG down regulates the capacity of DNA and RNA to activate the intrinsic pathway. Therefore, HRG attenuates the procoagulant activity of nucleic acids at multiple levels.

scholarly journals Highly specific enrichment of rare nucleic acid fractions using Thermus thermophilus argonaute with applications in cancer diagnostics

2019 ◽  
Vol 48 (4) ◽  
pp. e19-e19 ◽  
Author(s):  
Jinzhao Song ◽  
Jorrit W Hegge ◽  
Michael G Mauk ◽  
Junman Chen ◽  
Jacob E Till ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Elevated Temperatures ◽  
Thermus Thermophilus ◽  
Low Frequency ◽  
Cancer Diagnostics ◽  
Detection Methods ◽  
Dna And Rna ◽  
Novel Approach ◽  
Clinically Significant

Abstract Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

Thermodynamic and Kinetic Characterization of Duplex Formation between 2′-O, 4′-C-Methylene-modified Oligoribonucleotides, DNA and RNA

2007 ◽  
Vol 27 (6) ◽  
pp. 327-333 ◽  
Author(s):  
Ulla Christensen
Keyword(s):  
Nucleic Acid ◽  
Full Length ◽  
Locked Nucleic Acid ◽  
Complementary Dna ◽  
High Affinity ◽  
Thermodynamics And Kinetics ◽  
Dna And Rna ◽  
Reaction Thermodynamics ◽  
Duplex Formation

2′-O,4′-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA's with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg2+- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA's thus may be a better strategy, than using full length LNA's to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.

DNA and RNA: NMR studies of conformations and dynamics in solution

1987 ◽  
Vol 20 (1-2) ◽  
pp. 35-112 ◽  
Author(s):  
Dinshaw J. Patel ◽  
Lawrence Shapiro ◽  
Dennis Hare
Keyword(s):  
Aqueous Solution ◽  
Data Analysis ◽  
Nucleic Acids ◽  
Nmr Studies ◽  
Magnet Design ◽  
Dna And Rna ◽  
Qualitative Nature ◽  
Abundant Supply

The early NMR research on nucleic acids was of a qualitative nature and was restricted to partial characterization of short oligonucleotides in aqueous solution. Major advances in magnet design, spectrometer electronics, pulse techniques, data analysis and computational capabilities coupled with the availability of pure and abundant supply of long oligonucleotides have extended these studies towards the determination of the 3-D structure of nucleic acids in solution.

scholarly journals Characterization of a Mannose-6-Phosphate Isomerase fromThermus thermophilusand Increased l-Ribose Production by Its R142N Mutant

2010 ◽  
Vol 77 (3) ◽  
pp. 762-767 ◽  
Author(s):  
Soo-Jin Yeom ◽  
Eun-Sun Seo ◽  
Bi-Na Kim ◽  
Yeong-Su Kim ◽  
Deok-Kun Oh
Keyword(s):  
Specific Activity ◽  
Thermus Thermophilus ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Wild Type ◽  
Active Site Residues ◽  
Geobacillus Thermodenitrificans ◽  
Wild Type Enzyme ◽  
Mannose 6 Phosphate

ABSTRACTAn uncharacterized gene fromThermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed inEscherichia coli. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. Thekcat/Kmof the R142N mutant was 3.8-fold higher than that ofGeobacillus thermodenitrificansmannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reportedkcat/Km. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter−1h−1, which was 1.5-fold higher than that of the wild-type enzyme.

scholarly journals Characterization of Spontaneously Arising Chlorhexidine-Tolerant Variants inStreptococcus mutans

2019 ◽  
Author(s):  
Justin R. Kaspar ◽  
Matthew J. Godwin ◽  
Irina M. Velsko ◽  
Vincent P. Richards ◽  
Robert A. Burne
Keyword(s):  
Antimicrobial Agents ◽  
Acid Tolerance ◽  
Oral Streptococci ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Oral Biofilms ◽  
Glycolipid Synthesis ◽  
Increased Sensitivity

ABSTRACTBroad spectrum antimicrobials, both in dental products and within the clinic, have been used in the suppression of cariogenic bacteria such asStreptococcus mutansfor over 40 years. One such antimicrobial is chlorhexidine (CHX), and serves as a standard in dental research against which other antimicrobial therapies are compared against for their efficacy. However, very little is known about the mode of action for CHX against Streptococci and whether tolerance can be developed from repeated exposures. Here, we begin to answer such questions by passagingS. mutanswith increasing concentrations of CHX and isolating spontaneously-arising tolerant variants (CTVs) from separate lineages. We find that these CTVs display a higher minimal inhibitory concentration (MIC) against CHX than the wild-type strain and have altered virulence properties such as acid tolerance and biofilm formation. We record higher MICs for the variants against both daptomycin and bacitracin, but find increased sensitivity to triclosan and sodium fluoride. Measurements of antagonistic capabilities against other health-associated oral streptococci show decreased bacteriocin production compared to wild-type and increased sensitivity to hydrogen peroxide. Finally whole genome sequencing of the CTVs show common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, altering LTA accumulation and potentially lipid profile of the cell wall. Together, these findings confirm that streptococci may develop tolerance to antimicrobial agents such as CHX but in the case ofS. mutans,increased tolerance may come at a fitness cost for survival within oral biofilms that keeps variants suppressed within the population.

Sign in / Sign up

Export Citation Format

Share Document