Synthetic DNA Oligonucleotides Research Articles

Development of a non-infectious control for viral hemorrhagic fever PCR assays.

Assay validation is an essential component of disease surveillance testing, but can be problematic in settings where access to positive control material is limited and a safety risk for handlers. Here we describe a single non-infectious synthetic control that can help develop and validate the PCR based detection of the viral causes of Crimean-Congo hemorrhagic fever, Ebola virus disease, Lassa fever, Marburg virus disease and Rift Valley fever. We designed non-infectious synthetic DNA oligonucleotide sequences incorporating primer binding sites suitable for five assays, and a T7 promotor site which was used to transcribe the sequence. Transcribed RNA was used as template in a dilution series, extracted and amplified with RT-PCR and RT-qPCR to demonstrate successful recovery and determine limits of detection in a range of laboratory settings. Our results show this approach is adaptable to any diagnostic assay requiring validation of nucleic acid extraction and/or amplification, particularly where sourcing reliable, safe material for positive controls is infeasible.

Improved melting temperature predictions for single LNA modifications in synthetic DNA oligonucleotides

Locked nucleic acids (LNA) is a modification to DNA that generally increases the melting temperature in double strands. The nearest-neighbour (NN) provides one of the best prediction accuracies and is attractive for its simplicity. Here, we revise the NN parametrization for single LNA modifications with a very large set of published melting temperatures and using a parameter optimization method especially aimed at accurate melting temperature predictions. We show that the new set of LNA-NN parameters achieves an improved prediction of 0.88 °C over older parameter sets that achieve 1.7 °C for this data. We also tested if the existing salt corrections for DNA could be used for LNA as well. Using a small set of low salt melting temperatures we show that by applying the usual DNA salt corrections one may obtain predictions within an average uncertainty of 3.0 °C.

Voltammetric oxidation behavior of single-stranded DNA on carbon screen printed electrodes: From short oligonucleotides to ultralong amplification products

To deeper understand the electrochemical behavior of nucleic acids, the synthetic DNA oligonucleotides of general formula (ACTG)n, where n = 1–12, as well as 16-mer DNA oligonucleotides varying in the number of guanine (dG; G-n, n = 0–7) or adenine (dA; A-n, n = 0–8) residues, and single-stranded DNA (ssDNA) amplification products were employed to systematically investigate their oxidation in the ‘dissolved’ and ‘adsorbed’ forms on carbon screen printed electrodes by square wave and cycling voltammetry in the potential range of 0.5–1.5 V (vs. Ag/AgCl). Two signals at 0.9–1.0 V and 1.1–1.2 V, attributed to the oxidation of dG and dA, respectively, were registered for the most of oligonucleotides. The oxidation process of ‘dissolved’ (ACTG)n with n = 1–2 via dG was found diffusion-controlled, while (ACTG)3 has demonstrated a mixed mechanism of oxidation reaction. At the same time, ‘adsorbed’ oligonucleotides demonstrated adsorption-limited responses, except for (ACTG)1 showing a mixed reaction mechanism. At a constant nucleotide or oligonucleotide molar concentration, the registered signals for (ACTG)n exponentially decreased with the molecule length. The signals of all studied oligonucleotides after pre-adsorption on electrode surface were up to 10-fold higher than those in the soluble form, except for (ACTG)1. The dG oxidation signal for G-n oligonucleotides, registered at about 0.9 V, was practically insensitive to the position of a single dG (at the end or in the middle of polynucleotide chain), while demonstrating a bell-shaped dependence on the dG amount with a maximum at n = 3–4. The dG oxidation signal for A-n oligonucleotides with a constant portion of dG also increased with the number of dA residues in the bell-shaped fashion with a maximum at n = 5. The folding of oligonucleotides into hairpins with a stem formed via dG/dC pairing resulted in a shift of the peak potential for dG oxidation to higher values. Finally, ultralong ssDNA products generated by rolling circle amplification (RCA) were found to produce strong non-Faradaic current masking the dG and dA oxidation signals. The electrochemical detection of RCA products via dG and dA oxidation was found possible if ultralong ssDNA is fragmented by carrying out RCA in the presence of an appropriate restrictase.

Identification of Ribosomal Footprints on the Electrophoretic Gel in Translatome Profiling: on the Use of DNA Size Standards

The commercial DNA ladder was tested as a substitute for RNA size standards to identify ribosomal footprints (RNA fragments of about 30 nucleotides long) on an electrophoretic polyacrylamide gel for the purposes of translatome profiling. It has been found that 25 and 35 nucleotides long synthetic RNA oligonucleotides do migrate slower than the synthetic DNA oligonucleotides of the matching length and sequences and their positions on the gel coincide with those of 30 and 40 nucleotides long DNA oligonucleotides, correspondingly, of the commercial IDT 20/100 DNA oligo length standards. By using this DNA ladder and RNA isolated from the preparation enriched in ribosomes (obtained by fractionating on MicroSpin S-400 columns the HepG2 cell lysate treated with RNase I), the position of a band of putative ribosomal footprints can be identified on a gel that has been verified by measuring in an RNA-seq experiment the length of RNA fragments extracted from the band.

Assay design for analysis of human uracil DNA glycosylase.

Human uracil DNA glycosylase (UNG2) is an enzyme whose primary function is to remove uracil bases from genomic DNA. UNG2 activity is critical when uracil bases are elevated in DNA during class switch recombination and somatic hypermutation, and additionally, UNG2 affects the efficacy of thymidylate synthase inhibitors that increase genomic uracil levels. Here, we summarize the enzymatic properties of UNG2 and its mitochondrial analog UNG1. To facilitate studies on the activity of these highly conserved proteins, we discuss three fluorescence-based enzyme assays that have informed much of our understanding on UNG2 function. The assays use synthetic DNA oligonucleotide substrates with uracil bases incorporated in the DNA, and the substrates can be single-stranded, double-stranded, or form other structures such as DNA hairpins or junctions. The fluorescence signal reporting uracil base excision by UNG2 is detected in different ways: (1) Excision of uracil from end-labeled oligonucleotides is measured by visualizing UNG2 reaction products with denaturing PAGE; (2) Uracil excision from dsDNA substrates is detected in solution by base pairing uracil with 2-aminopurine, whose intrinsic fluorescence is enhanced upon uracil excision; or (3) UNG2 excision of uracil from a hairpin molecular beacon substrate changes the structure of the substrate and turns on fluorescence by relieving a fluorescence quench. In addition to their utility in characterizing UNG2 properties, these assays are being adapted to discover inhibitors of the enzyme and to determine how protein-protein interactions affect UNG2 function.

Three-Way DNA Junction as an End Label for DNA in Atomic Force Microscopy Studies.

Atomic Force Microscopy (AFM) is widely used for topographic imaging of DNA and protein-DNA complexes in ambient conditions with nanometer resolution. In AFM studies of protein-DNA complexes, identifying the protein’s location on the DNA substrate is one of the major goals. Such studies require distinguishing between the DNA ends, which can be accomplished by end-specific labeling of the DNA substrate. We selected as labels three-way DNA junctions (3WJ) assembled from synthetic DNA oligonucleotides with two arms of 39–40 bp each. The third arm has a three-nucleotide overhang, GCT, which is paired with the sticky end of the DNA substrate generated by the SapI enzyme. Ligation of the 3WJ results in the formation of a Y-type structure at the end of the linear DNA mole cule, which is routinely identified in the AFM images. The yield of labeling is 69%. The relative orientation of arms in the Y-end varies, such dynamics were directly visualized with time-lapse AFM studies using high-speed AFM (HS-AFM). This labeling approach was applied to the characterization of the nucleosome arrays assembled on different DNA templates. HS-AFM experiments revealed a high dynamic of nucleosomes resulting in a spontaneous unraveling followed by disassembly of nucleosomes.

Abstract 496: Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities

Abstract Background: Translational cancer research is dependent on well annotated human samples, but scarcity of specimen DNA and RNA hinders research and novel diagnostic testing. We demonstrate a novel nucleic acid isolation and anchoring approach that facilitates uniform, consistent replication of the isolated and captured DNA and RNA. This approach allows for expanded investigations of precious samples and increases the value of biobanked material. Methods: Our simplSEQ replication methodology enzymatically adds homopolymeric tails to the 3’ end of DNA and RNA, with a terminal dideoxy biotin-modified NTP, to attach the nucleic acids to streptavidin beads. The 3’ immobilized nucleic acids are copied using complementary homopolymers. The simplSEQ advantage is that molecules of all sizes are attached and copied from their 3’ ends yielding cDNA copies that are full-length representations of the input. Various DNA samples were investigated to determine the characteristics of cDNA copies as well as stability of bound nucleic acids. Samples evaluated included Human 1,000 genome project, bacterial genomes, commercial DNA controls, and synthesized DNA molecules. The samples represent a range of DNA sizes, AT/GC content and Variant Allele Frequencies (VAF). NGS assays quantified the presence of full-length genomic sequences. Variant analysis determined accuracy of copies based on comparison with published data (NA12878). Synthetic DNA oligonucleotides representing regions in the human genome and constructed to enable molecular counting independent of adapter ligation efficiency. All sample types were replicated, sequenced, and analyzed for coverage and uniformity. This replication process was repeated multiple times. Results: Three replicates of each DNA type were attached to streptavidin beads and subjected to multiple rounds of the simplSEQ protocol. Analysis of overall coverage and variant calling for human NA12878 replicates compared to the platinum standard NA12878 genome show an average coverage ranging from 27x-32x with >99% correlation between replicates. Sensitivity ranging from 98.6%-99.5%, with a precision range from 96.0% to 98.0% for 6 replicates. Bacterial samples with coverage over 100x and >99% correlation. Synthetic DNA oligonucleotide pools with coverage over 10,000x and >99% correlation. Conclusion: Our novel enzymatic tailing methodology produces highly accurate copies of full-length nucleic acids. This technology obviates sample scarcity for genomic evaluations and allows for expanded hypothesis testing in translational research. This methodology also provides an option for extended storage of nucleic acids from which replicates can be repeatedly generated to allow for reinterrogation. Citation Format: John Christopher Spinosa, Brandon Young, Vincent Aguilar, Baiju Parikh. Novel sample isolation and immortalization method for DNA and RNA to extend translational research capabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 496.

Inhibition of Respiratory Syncytial Virus Infection by Small Non-Coding RNA Fragments

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infection in infants, immunocompromised individuals and the elderly. As the only current specific treatment options for RSV are monoclonal antibodies, there is a need for efficacious antiviral treatments against RSV to be developed. We have previously shown that a group of synthetic non-coding single-stranded DNA oligonucleotides with lengths of 25–40 nucleotides can inhibit RSV infection in vitro and in vivo. Based on this, herein, we investigate whether naturally occurring single-stranded small non-coding RNA (sncRNA) fragments present in the airways have antiviral effects against RSV infection. From publicly available sequencing data, we selected sncRNA fragments such as YRNAs, tRNAs and rRNAs present in human bronchoalveolar lavage fluid (BALF) from healthy individuals. We utilized a GFP-expressing RSV to show that pre-treatment with the selected sncRNA fragments inhibited RSV infection in A549 cells in vitro. Furthermore, by using a flow cytometry-based binding assay, we demonstrate that these naturally occurring sncRNAs fragments inhibit viral infection most likely by binding to the RSV entry receptor nucleolin and thereby preventing the virus from binding to host cells, either directly or via steric hindrance. This finding highlights a new function of sncRNAs and displays the possibility of using naturally occurring sncRNAs as treatments against RSV.

Nucleoside Analogues with a Seven-Membered Sugar Ring: Synthesis and Structural Compatibility in DNA-RNA Hybrids.

Herein we describe results on the pairing properties of synthetic DNA and RNA oligonucleotides that contain nucleotide analogues with a 7-membered sugar ring (oxepane nucleotides). Specifically, we describe the stereoselective synthesis of a set of three oxepane thymine nucleosides (OxT), their conversion to phosphoramidite derivatives, and their use in solid-phase synthesis to yield chimeric OxT-DNA and OxT-RNA strands. The different regioisomeric OxT phosphoramidites allowed for positional variations of the phosphate bridge and assessment of duplex stability when the oxepane nucleotides were incorporated in dsDNA, dsRNA, and DNA-RNA hybrids. Little to no destabilization was observed when two of the three regioisomeric OxT units were incorporated in the DNA strand of DNA-RNA hybrids, a remarkable result considering the dramatically different structure of oxepanes in comparison to 2'-deoxynucleosides. Extensive molecular modeling and dynamics studies further revealed the various structural features responsible for the tolerance of both OxT modifications in DNA-RNA duplexes, such as base-base stacking and sugar-phosphate H-bond interactions. These studies suggest that oxepane nucleotide analogues may find applications in synthetic biology, where synthetic oligonucleotides can be used to create new tools for biotechnology and medicine.

Recent developments in DNA-based mechanical nanodevices.

Cellular processes and functions can be regulated by mechanical forces. Nanodevices that can measure and manipulate these forces are critical tools in chemical and cellular biology. Synthetic DNA oligonucleotides have been used to develop a wide range of powerful nanodevices due to their programmable nature and precise and predictable self-assembly. In recent years, various types of DNA-based mechanical nanodevices have been engineered for studying molecular-level forces. With the help of these nanodevices, our understanding of cellular responses to physical forces has been significantly advanced. In this article, we have reviewed some recent developments in DNA-based mechanical sensors and regulators for application in the characterization of cellular biomechanics and the manipulation of cellular morphology, motion and other functions. The design principles discussed in this article can be further used to inspire other types of powerful DNA-based mechanical nanodevices.

Interactions between a dsDNA Oligonucleotide and Imidazolium Chloride Ionic Liquids: Effect of Alkyl Chain Length, Part I.

Ionic liquids (ILs) have become nearly ubiquitous solvents and their interactions with biomolecules has been a focus of study. Here, we used the fluorescence emission of DAPI, a groove binding fluorophore, coupled with molecular dynamics (MD) simulations to report on interactions between imidazolium chloride ([Imn,1]+) ionic liquids and a synthetic DNA oligonucleotide composed entirely of T/A bases (7(TA)) to elucidate the effects ILs on a model DNA duplex. Spectral shifts on the order of 500–1000 cm−1, spectral broadening (~1000 cm−1), and excitation and emission intensity ratio changes combine to give evidence of an increased DAPI environment heterogeneity on added IL. Fluorescence lifetimes for DAPI/IL solutions yielded two time constants 0.15 ns (~80% to 60% contribution) and 2.36–2.71 ns for IL up to 250 mM. With DNA, three time constants were required that varied with added IL (0.33–0.15 ns (1–58% contribution), ~1.7–1.0 ns (~5% contribution), and 3.8–3.6 ns (94–39% contribution)). MD radial distribution functions revealed that π-π stacking interactions between the imidazolium ring were dominant at lower IL concentration and that electrostatic and hydrophobic interactions become more prominent as IL concentration increased. Alkyl chain alignment with DNA and IL-IL interactions also varied with IL. Collectively, our data showed that, at low IL concentration, IL was primarily bound to the DNA minor groove and with increased IL concentration the phosphate regions and major groove binding sites were also important contributors to the complete set of IL-DNA duplex interactions.

A novel test strip assay based on gold nanoparticles-labeled nucleic acid probe for rapid detection of Vibrio splendidus

Vibrio splendidus is an opportunistic pathogen in aquaculture. It can infect a variety of aquaculture animals and has caused huge losses to the aquaculture industry. In this study, a novel and efficient method for detecting V. splendidus was developed by combining the exonuclease Ⅲ amplification strategy with a nucleic acid test strip developed based on gold nanoparticles-labeled DNA probe. The results could be directly visualized by naked eyes, and this system overcame the difficulty in preparation of the monoclonal antibody used in conventional immunostrip. Upon optimization of experimental conditions, the detection limit of the strip was 5 ng/mL for the synthetic oligonucleotide DNA fragment and 10 ng/mL for the actual genomic DNA sample of V. splendidus. This test strip was more sensitive compared with the PCR method and was specific for the detection of V. splendidus. The rapid preparation of nucleic acid strip and the efficient detection of V. splendidus open a new way for the prevention and control of aquatic diseases.

P13.19 Bi-modular G-quadruplex DNA-crypto-aptamers diminish viability of glioma primary cell cultures of patients

Abstract BACKGROUND G-quadruplex oligonucleotides (GQs) exhibit specific anti-survival activity in human cancer cell lines; they can selectively inhibit the viability/proliferation. The most studied, AS1411, had been in clinical trials. This anti-proliferative ability of GQs could be translated into glioma, which currently has poor prognosis and low-efficiency therapeutic treatments for glioblastoma multiform (GBM). Set of GQs have been designed, synthesized, and tested: they have different amount of Q-quartets, they have dimers of different GQ modules: either covalent dimers or non-covalent ones; all of them could be coined as ‘twins’. MATERIAL AND METHODS Folding of synthetic DNA oligonucleotides into GQs and thermal stability have been studied by circular dichroism, melting with unfolding-folding regimes, oligomerization was followed by original SE-HPLC. Conventional human cell lines U87 and fibroblasts from human embryo (HEF) were provided from the collection of the Centre of Neurosurgery (Moscow, Russia). GBM primary cell cultures N1, G11, Sus/fP2, G22, G23, and G01 were developed in Burdenko National Medical Research Centre of Neurosurgery (NMRCN) from the surgery samples of patients (PCC_SSP). All samples had WT IDH1. This study was approved by the Ethics Committee of Burdenko NMRCN, Russian Academy of Medical Sciences (№_12/2020, 15.12.2020). All subjects gave written informed consent in accordance with the guidelines of Declaration of Helsinki. Cell viability was tested by conventional MTT-test. RESULTS Novel bi-modular GQ, bi-(AID-1-T), twin of three-quartet AID-1-T, was designed by covalent conjugation of two AID-1-Ts via three thymidine link, TTT; and linking did not interfere with its GQ structure. Comparison of bi-(AID-1-T) with mono-modular AID-1-T, mono-modular two-quartet HD1, bi-modular bi-HD1, and two-quartet AS1411, was made. Among five GQs, bi-(AID-1-T) had the highest anti-survival activity for U87, while not affecting the control, HEF. GQs, for the first time, were tested on several PCC_SSP. Sensitivity of PCC_SSP toward GQs varied, with apparent IC50 of less than 1 μM for bi-(AID-1-T) toward the most sensitive G11 (glioma, Grade III). CONCLUSION GQs as anti-proliferative crypto-aptamers with moderate activity due to restricted functioning of apparent GQ-binding proteins could be applied toward real glioma PCC_SSP. Variety of effects reflects glioma inter-tumor heterogeneity.Research was funded by Ministry of Education and Science of Russia, grant number № 075-15-2020-809 (13.1902.21.0030) and by Russian Foundation for Basic Research, grants number №18-29-01047

DNA-Modified Plasmonic Sensor for the Direct Detection of Virus Biomarkers from the Blood.

The rapid spread of viral infections demands early detection strategies to minimize proliferation of the disease. Here, we demonstrate a plasmonic biosensor to detect Dengue virus, which was chosen as a model, via its nonstructural protein NS1 biomarker. The sensor is functionalized with a synthetic single-stranded DNA oligonucleotide and provides high affinity toward NS1 protein present in the virus genome. We demonstrate the detection of NS1 protein at a concentration of 0.1-10 μg/mL in bovine blood using an on-chip microfluidic plasma separator integrated with the plasmonic sensor which covers the clinical threshold of 0.6 μg/mL of high risk of developing Dengue hemorrhagic fever. The conceptual and practical demonstration shows the translation feasibility of these microfluidic optical biosensors for early detection of a wide range of viral infections, providing a rapid clinical diagnosis of infectious diseases directly from minimally processed biological samples at point of care locations.

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology.

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the ‘flanking effect’ in this work. This ‘flanking effect’ was more pronounced when nucleotides were added at the 5′-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5′-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

Raman Spectroscopy-Based Quality Control of "Silicon-On-Insulator" Nanowire Chips for the Detection of Brain Cancer-Associated MicroRNA in Plasma.

Application of micro-Raman spectroscopy for the monitoring of quality of nanowire sensor chips fabrication has been demonstrated. Nanowire chips have been fabricated on the basis of «silicon-on-insulator» (SOI) structures (SOI-NW chips). The fabrication of SOI-NW chips was performed by optical litography with gas-phase etching. The so-fabricated SOI-NW chips are intended for highly sensitive detection of brain cancer biomarkers in humans. In our present study, two series of experiments have been conducted. In the first experimental series, detection of a synthetic DNA oligonucleotide (oDNA) analogue of brain cancer-associated microRNA miRNA-363 in purified buffer solution has been performed in order to demonstrate the high detection sensitivity. The second experimental series has been performed in order to reveal miRNA-363 itself in real human plasma samples. To provide detection biospecificity, the SOI-NW chip surface was modified by covalent immobilization of probe oligonucleotides (oDNA probes) complementary to the target biomolecules. Using the SOI-NW sensor chips proposed herein, the concentration detection limit of the target biomolecules at the level of 3.3 × 10−17 M has been demonstrated. Thus, the approach employing the SOI-NW chips proposed herein represents an attractive tool in biomedical practice, aimed at the early revelation of oncological diseases in humans.

AS1411-functionalized delivery nanosystems for targeted cancer therapy.

Nucleolin (NCL) is a multifunctional nucleolar phosphoprotein harboring critical roles in cells such as cell proliferation, survival, and growth. The dysregulation and overexpression of NCL are related to various pathologic and oncological indications. These characteristics of NCL make it an ideal target for the treatment of various cancers. AS1411 is a synthetic quadruplex-forming nuclease-resistant DNA oligonucleotide aptamer which shows a considerably high affinity for NCL, therefore, being capable of inducing growth inhibition in a variety of tumor cells. The high affinity and specificity of AS1411 towards NCL make it a suitable targeting tool, which can be used for the functionalization of therapeutic payloaddelivery nanosystems to selectively target tumor cells. This review explores the advances in NCL-targeting cancer therapy through AS1411-functionalized delivery nanosystems for the selective delivery of a broad spectrum of therapeutic agents.

Genetically encoded RNA nanodevices for cellular imaging and regulation.

Nucleic acid-based nanodevices have been widely used in the fields of biosensing and nanomedicine. Traditionally, the majority of these nanodevices were first constructed in vitro using synthetic DNA or RNA oligonucleotides and then delivered into cells. Nowadays, the emergence of genetically encoded RNA nanodevices has provided a promising alternative approach for intracellular analysis and regulation. These genetically encoded RNA-based nanodevices can be directly transcribed and continuously produced inside living cells. A variety of highly precise and programmable nanodevices have been constructed in this way during the last decade. In this review, we will summarize the recent advances in the design and function of these artificial genetically encoded RNA nanodevices. In particular, we will focus on their applications in regulating cellular gene expression, imaging, logic operation, structural biology, and optogenetics. We believe these versatile RNA-based nanodevices will be broadly used in the near future to probe and program cells and other biological systems.

CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides

CRISPR-Cas12a (CRISPR-Cpf1) was reported to have multiple types of cleavage activities. Without the assistance of CRISPR RNA (crRNA), we investigated DNase activity and substrate specificity of Cas12a orthologs in the presence of diverse divalent metal ions. Cas12a from different species are capable of degrading single-stranded DNA (ssDNA) and/or double-stranded DNA (dsDNA), depending on the metal ions used. In spite of sharing high sequence similarity and functional domains among diverse Cas12a orthologs, only Acidaminococcus sp. Cas12a (AsCas12a) showed a predominant preference for cleaving ssDNA, but no detectable activity toward dsDNA substrate in the presence of magnesium (II) ions. In addition, we found that both AsCas12a and Francisella novicida Cas12a (FnCas12a) caused substantial dsDNA cleavage in the presence of manganese (II) ion. More importantly, the DNase activities can be inhibited by synthetic DNA oligonucleotides with phosphorothioate linkage modifications. Overall, ssDNase activity of the Cas12a orthologs uncovered a distinct approach for DNA cleavage compared with crRNA-guided dsDNA breaks, and provided insights into potential biological and therapeutic applications.

A Spontaneous Ring-Opening Reaction Leads to a Repair-Resistant Thymine Oxidation Product in Genomic DNA.

The alphabet of modified DNA bases goes beyond the conventional four letters, with biological roles being found for many such modifications. Herein, we describe the observation of a modified thymine base that arises from spontaneous N1 -C2 ring opening of the oxidation product 5-formyl uracil, after N3 deprotonation. We first observed this phenomenon in silico through ab initio calculations, followed by in vitro experiments to verify its formation at a mononucleoside level and in a synthetic DNA oligonucleotide context. We show that the new base modification (Trex , thymine ring expunged) can form under physiological conditions, and is resistant to the action of common repair machineries. Furthermore, we found cases of the natural existence of Trex while screening a number of human cell types and mESC (E14), thus suggesting potential biological relevance of this modification.