•nDNA can be formed by changing the negative-charge DNA phosphodiester to neutral methyl phosphotriester.•nDNA reduces DNA duplex formation and stability by causing high entropy penalty and steric ...hindrance.•Na+ cations help forming DNA duplexes by reducing entropy costs but not the strand charge repulsion.•By destabilizing duplexes, nDNA is a novel oligonucleotide for discriminating single nucleotide polymerphisms.
To exploit the formation of DNA duplexes, the charge repulsion between complementary DNA strands can be reduced by adding salts or by replacing the negatively charged natural phosphodiester linkage(s) with charge-neutral methyl phosphotriester(s) (MPTE) linkage(s) of the synthetic nDNA oligonucleotides. Recently, we reported prominent improvements of detecting target nucleic acids with nDNA probes on liquid-phase PCR and solid/liquid-interface biosensing. To understand the observed improvements from thermodynamics perspectives, we studied the formation and stability of the double-stranded DNA (dsDNA) containing nDNA and/or natural DNA. With the natural DNA oligonucleotides, increment of Na+ cations caused unexpected reductions of favorable enthaply (ΔH) of duplex formation and expected increases of favorable free energy (ΔG) and temperature of melting transition (Tm). With one nDNA in duplex formation, the increase of Na+ yielded extra faborable ΔH, yet unfavorable ΔG and Tm. Possibly, Na+ cations (> 50 mM) promote the formation of dsDNA through the endothermic release of DNA (and nDNA)-hydrating water molecules more than through the reduction of inter-strand repulsions. The methyl groups of MPTE of nDNA also destabilize the DNA duplexes by hindering the formation of standard double helices. These novel findings explain the duplex-destabilization property of nDNA which enables its discrimination of single-nucleotide polymorphisms.
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The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While ...CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs).
Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected.
Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.
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Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching ...multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01–25, 0.01–50, and 0.1–50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%–17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.
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•A sensitive immunoassay based on DNA/RNA hybridization and RNase H was developed for the detection of organophosphate pesticides.•Different fluorescent groups are labeled on the RNA chain to excite different fluorescent signals.•The use of RNase H enzyme can only specifically hydrolyze the RNA chain in the DNA/RNA hybridization chain.
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FFLJ, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, ODKLJ, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Here, we report a simple and sensitive colorimetric detection method for Hg
2+ ions with a tunable detection range based on DNA oligonucleotides and unmodified gold nanoparticles (DNA/AuNPs) sensing ...system. Complementary DNA strands with T–T mismatches could effectively protect AuNPs from salt-induced aggregation. While in the presence of Hg
2+ ions T–Hg
2+–T coordination chemistry leads to the formation of DNA duplexes, and AuNPs are less well protected thus aggregate at the same salt concentration, accompanying by color change from red to blue. By rationally varying the number of T–T mismatches in DNA oligonucleotides, the detection range could be tuned. Employing duplex oligonucleotides with 4 T–T mismatches in the sensing system, a sensitive linear range for Hg
2+ ions from 0 to 5
μM and a detection limit of 0.5
μM are obtained. Adding the number of T–T mismatches to 6 and 8, the assay region is enlarged and linear range is tuned. A low proportion of T–T mismatches makes the detection range narrow but the sensitivity high while a high proportion influences the detection limit but enlarges assay region. Besides, the sensor also shows a good selectivity for Hg
2+.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically ...modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1. Time-dependent inhibitory effects were validated in multiple loci of different human cells. Further studies demonstrated that PS-modified DNA oligonucleotides were able to serve as Cpf1 inhibitors in a sequence-independent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder target DNA binding and recognition by Cpf1. Consequently, these synthetic DNA molecules expand the sources of CRISPR inhibitors, providing a platform to inactivate Cpf1-mediated genome editing.
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•Phosphorothioate-modified DNA oligonucleotides inhibit Cpf1-mediated genome editing•Inhibition of Cpf1 activity is time and dose dependent and target sequence independent•psDNA oligonucleotides may block the complex formation of Cpf1-crRNA-DNA•psDNA oligonucleotides inhibit Cas9-mediated genome editing
Li et al. show that phosphorothioate-modified DNA (psDNA) oligonucleotides inhibit Cpf1-mediated genome-editing activity in a sequence-independent manner in human cells. These psDNA oligonucleotides interact with Cpf1 protein and block the formation of Cpf1-crRNA-target DNA complex. They also display inhibitory effects on the CRISPR-Cas9 system.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Towards an Analytical Biology Garzon, Max H; Colorado, Fredy A
Current genomics,
01/2024, Volume:
25, Issue:
2
Journal Article
Peer reviewed
This article draws a perspective on the increasingly unavoidable question of whether steps can be taken in genomics and biology at large to move them more rapidly towards more analytical and ...deductive biology, akin to similar developments that occurred in other natural sciences, such as physics and chemistry, centuries ago. It provides a summary of recent advances in other relevant sciences in the last 3 decades that are likely to pull it in that direction in the next decade or so, as well as what methods and tools will make it possible.
Graphite-epoxy composites (GECs) are alternative construction materials for electrochemical sensors. For these materials, the electron transfer rate constant of some redox reaction depends ...additionally on the stoichiometric relationship between the insulating and conducting phases of the composite. In this work, the influence of different ratios of araldite/hardener/graphite on the electrochemical properties of GEC electrodes is evaluated for the simultaneous determination of adenine and guanine in the single chain DNA, using the square wave voltammetry technique. Six GEC electrodes were prepared with different ratios of components, and electrochemically characterized by cyclic voltammetry in the presence of ferri/ferrocyanide redox couple as a redox probe. GEC electrodes that showed the best electrochemical responses of redox probe were characterized by thermogravimetric analysis (TGA) and used for the simultaneous determination of free adenine and guanine in a solution, and DNA oligonucleotides. The best results were obtained for GEC electrodes containing twice higher volume of araldite resin with respect to the hardener. TGA analysis revealed presence of 15-26 % of resin for these GEC electrodes. The obtained results revealed potential application of these GEC electrodes as DNA sensors based on the oxidation signal of guanine.
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•UV photoproducts are removed from DNA in the form of sedDNAs.•sedDNAs have been detected for the first time in UVB-irradiated human skin ex vivo.•sedDNA detection may allow for ...quantification of DNA repair activity in human skin.
UVB radiation results in the formation of potentially mutagenic photoproducts in the DNA of epidermal skin cells. In vitro approaches have demonstrated that the nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small (∼30-nt-long), excised, damage-containing DNA oligonucleotides (sedDNAs). Though this process presumably takes place in human skin exposed to UVB radiation, sedDNAs have not previously been detected in human skin. Using surgically discarded human skin, we have optimized the detection of the sedDNA products of NER from small amounts of human epidermal tissue ex vivo within minutes of UVB exposure and after UVB doses that normally lead to minimal erythema. Moreover, sedDNA generation was inhibited by treatment of skin explants with spironolactone, which depletes the epidermis of the essential NER protein XPB to mimic the skin of xeroderma pigmentosum patients. Time course experiments revealed that a partially degraded form of the sedDNAs could be readily detected even 12 hours following UVB exposure, which indicates that these repair products are relatively stable in human skin epidermis. Together, these data suggest that sedDNA detection may be a useful assay for determining how genetic, environmental, and other factors influence NER activity in human skin epidermis and whether abnormal sedDNA processing contributes to photosensitive skin disorders.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Terahertz absorption spectroscopy based on attenuated total reflection (ATR) from a microfluidic sample cell was designed and implemented to detect gene mutations leading to Huntington's disease ...(HD). The self‐developed compact ATR microfluidic system was employed to detect two groups of base‐repeated DNA molecules combined with a terahertz time‐domain spectrometer in a marker‐free manner. The first group featured different repetition patterns of oligonucleotide fragments, and the second group included the HD gene. For the oligonucleotides of different repetition patterns, there were significant differences among the three oligonucleotides with three repeats of the double bases, which could be unambiguously classified and identified; For the HD gene, it was found that the magnitude of the terahertz absorption coefficients of the four oligonucleotide solutions was, in ascending order, CAG‐4 < CAG‐16 < CAG‐32 < CAG‐40 (the numbers are the repeat times of the CAG base segment, with 40 repeats belonging to the HD gene), when the concentration of oligonucleotide was 1 mg/mL. Principal component analysis result indicated that the spectral differences of the four oligonucleotide solutions with different CAG repeat times were statistically significant and clearly distinguishable. These results demonstrate the potential of terahertz spectroscopy as a noninvasive, unmarked, fast and low‐cost assay for gene diagnosis and clinical disease detection.
The self‐developed compact THz‐ATR microfluidic system was employed to detect base‐repeated DNA molecules in a marker‐free manner. THz absorption coefficient can be used to identify trace Huntington's disease gene at the same mass or molar concentration. Oligonucleotides with different repetition patterns and times were distinguished by principal component analysis.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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