Recognition Of dsDNA Research Articles

Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations.

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

Elucidation of the potential disease-promoting influence of IgM apoptotic cell-reactive antibodies in lupus.

The undigested remnants of apoptosis are believed to stimulate the generation of autoantibodies in lupus. The biological properties of initiator, disease-specific IgM antibodies that specifically recognize apoptotic cells, readily detected in the sera of lupus patients, remain unclear. Apoptotic cell-reactive IgM monoclonal antibodies (generated from lupus-prone mice), as opposed to control IgM, preferentially stimulated maturation of bone marrow-derived dendritic cells (BMDCs) derived from such mice, relative to BMDCs derived from healthy mice. An influence of both antibody specificity and cell genotype was also apparent in the secretion of signature inflammatory cytokines. Immunization of such antibodies in lupus-prone animals induced increases in total serum IgG levels, with the elicited antibodies also preferentially recognizing moieties on dying cells. An expanded specificity was apparent both upon Western blot on cellular lysate and from the enhanced recognition of dsDNA, Ro60, RNP68 and Sm; the antibody most efficient in mediating autoreactive diversity, while being germline encoded, also induced the highest degree of phenotypic changes on BMDCs. Apoptotic cell-reactive IgM antibodies may therefore be potentially capable of influencing the course of systemic autoimmune disease by affecting both innate and adaptive immunity.

Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes.

Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′-aminouridine and 2′-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms.

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles.

Enhanced sequence-specific recognition of double-stranded DNA (dsDNA) was realized by using hybridization chain reaction (HCR) and positively charged gold nanoparticles ((+)AuNPs) dual signal amplification. To construct such a sensor, capture probe was initially assembled onto gold electrode surface. Upon addition of dsDNA, sandwiched DNA complex was formed between the capture probe and the detection probe, then another exposed part of the detection probe opened two alternating DNA hairpins (H1 and H2) in turn and initiated HCR to form a double-helix. Meantime, (+)AuNPs were electrostatically adsorbed onto such double-helix to amplify the electrochemical signal. Upon optimal conditions, the electronic signals of ferrocene (Fc) that modified on H1 and H2 increased linearly with increasing dsDNA concentration over the range from 15 pM to 1.0 nM, with a detection limit of 2.6 pM. Moreover, the proposed method showed good sequence specificity for dsDNA recognition.

Ligand binding and signaling by IFI16 (INM8P.400)

Abstract IFI16 plays important roles in inflammatory immune responses to viral infections as well as in autoimmune disorders such as lupus. It has been implicated in interferon induction through a STING-mediated pathway, as well as in inflammasome activation during recognition of viral DNA in the nucleus. Despite recent progress that highlighted the mechanisms of dsDNA binding by the IFI16 HINb domain, there are major gaps in our understanding of the ligand-induced structural changes, the polymerization of IFI16 mediated by dsDNA and its PYD domain, its interaction with downstream signaling partners, the regulation of IFI16 by host and microbe-derived molecules, and the cell-type specific immune responses mediated by IFI16. We have characterized the key structural determinants in both the recognition of dsDNA by IFI16 and the potential downstream signaling events. We will outline the implications of our findings and discuss future directions in clarifying the structure and function of IFI16 in infectious diseases and autoimmune disorders.

Dynamic tuning of DNA-nanoparticle superlattices by molecular intercalation of double helix.

Nanoparticle (NP) assembly using DNA recognition has emerged as a powerful tool for the fabrication of 3D superlattices. In addition to the vast structural diversity, this approach provides an avenue for dynamic 3D NP assembly, which is promising for the modulation of interparticle distances and, hence, for example, for in situ tuning of optical properties. While several approaches have been explored for changing NP separations in the lattices using responsiveness of single-stranded DNA (ss-DNA), far less work has been done for the manipulation of most abundant double-stranded DNA (ds-DNA) motifs. Here, we present a novel strategy for modulation of interparticle distances in DNA linked 3D self-assembled NP lattices by molecular intercalator. We utilize ethidium bromide (EtBr) as a model intercalator to demonstrate selective and isotropic lattice expansion for three superlattice types (bcc, fcc, and AlB2) due to the intercalation of ds-DNA linking NPs. We further show the reversibility of the lattice parameter using n-butanol as a retrieving agent as well as an increased lattice thermal stability by 12-14 °C due to the inclusion of EtBr. The proposed intercalator-based strategy permits the creation of reconfigurable and thermally stable superlattices, which could lead to tunable and functionally responsive materials.

Bulged Invader probes: activated duplexes for mixed-sequence dsDNA recognition with improved thermodynamic and kinetic profiles.

Double-stranded oligonucleotides with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides are energetically activated for recognition of mixed-sequence double-stranded DNA. Incorporation of nonyl (C9) bulges at specific positions of these probes, results in more highly affine (>5-fold), faster (>4-fold) and more persistent dsDNA recognition relative to conventional Invader probes.

Cytosolic RNA:DNA hybrids activate the cGAS-STING axis.

Intracellular recognition of non-self and also self-nucleic acids can result in the initiation of potent pro-inflammatory and antiviral cytokine responses. Most recently, cGAS was shown to be critical for the recognition of cytoplasmic dsDNA. Binding of dsDNA to cGAS results in the synthesis of cGAMP(2'-5'), which then binds to the endoplasmic reticulum resident protein STING. This initiates a signaling cascade that triggers the induction of an antiviral immune response. While most studies on intracellular nucleic acids have focused on dsRNA or dsDNA, it has remained unexplored whether cytosolic RNA:DNA hybrids are also sensed by the innate immune system. Studying synthetic RNA:DNA hybrids, we indeed observed a strong type I interferon response upon cytosolic delivery of this class of molecule. Studies in THP-1 knockout cells revealed that the recognition of RNA:DNA hybrids is completely attributable to the cGAS-STING pathway. Moreover, in vitro studies showed that recombinant cGAS produced cGAMP upon RNA:DNA hybrid recognition. Altogether, our results introduce RNA:DNA hybrids as a novel class of intracellular PAMP molecules and describe an alternative cGAS ligand next to dsDNA.

Electrochemical molecular beacon biosensor for sequence-specific recognition of double-stranded DNA

Direct recognition of double-stranded DNA (dsDNA) was crucial to disease diagnosis and gene therapy, because DNA in its natural state is double stranded. Here, a novel sensor for the sequence-specific recognition of dsDNA was developed based on the structure change of ferrocene (Fc) redox probe modified molecular beacon (MB). For constructing such a sensor, gold nanoparticles (AuNPs) were initially electrochemical-deposited onto glass carbon electrode (GCE) surface to immobilize thiolated MB in their folded states with Au–S bond. Hybridization of MB with target dsDNA induced the formation of parallel triplex DNA and opened the stem-loop structure of it, which resulted in the redox probe (Fc) away from the electrode and triggered the decrease of current signals. Under optimal conditions, dsDNA detection could be realized in the range from 350pM to 25nM, with a detection limit of 275pM. Moreover, the proposed method has good sequence-specificity for target dsDNA compared with single base pair mismatch and two base pairs mismatches.

Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines for Invaders Based on 2′-O-(Pyren-1-yl)methyl-RNA Monomers

The development of agents that recognize mixed-sequence double-stranded DNA (dsDNA) is desirable because of their potential as tools for detection, regulation, and modification of genes. Despite progress with triplex-forming oligonucleotides, peptide nucleic acids, polyamides, and other approaches, recognition of mixed-sequence dsDNA targets remains challenging. Our laboratory studies Invaders as an alternative approach toward this end. These double-stranded oligonucleotide probes are activated for recognition of mixed-sequence dsDNA through modification with +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl-RNA monomers and have recently been shown to recognize linear dsDNA, DNA hairpins, and chromosomal DNA. In the present work, we systematically studied the influence that the nucleobase moieties of the 2'-O-(pyren-1-yl)methyl-RNA monomers have on the recognition efficiency of Invader duplexes. Results from thermal denaturation, binding energy, and recognition experiments using Invader duplexes with different +1 interstrand zippers of the four canonical 2'-O-(pyren-1-yl)methyl-RNA A/C/G/U monomers show that incorporation of these motifs is a general strategy for activation of probes for recognition of dsDNA. Probe duplexes with interstrand zippers comprising C and/or U monomers result in the most efficient recognition of dsDNA. The insight gained from this study will drive the design of efficient Invaders for applications in molecular biology, nucleic acid diagnostics, and biotechnology.

Regulating Bioactivity of Cu2+ Bis-1,10-phenanthroline Artificial Metallonucleases with Sterically Functionalized Pendant Carboxylates

The synthetic chemical nuclease, [Cu(1,10-phenanthroline)2](2+), has stimulated research within metallonuclease development and in the area of cytotoxic metallodrug design. Our analysis reveals, however, that this agent is "promiscuous" as it binds both dsDNA and protein biomolecules, without specificity, and induces general toxicity to a diversity of cell lineages. Here, we describe the synthesis and characterization of small-molecule metallonucleases containing the redox-active cation, [Cu(RCOO)(1,10-phen)2](+), where 1,10-phen = 1,10-phenanthroline and R = -H, -CH3, -C2H5, -CH(CH3)2, and -C(CH3)3. The presence of coordinated carboxylate groups in the complex cation functions to enhance dsDNA recognition, reduce serum albumin binding, and offer control of toxicity toward human cancer cells, Gram positive and negative bacteria, and fungal pathogens. The induction of genomic dsDNA breaks (DSBs) were identified in ovarian adenocarcinoma cells using immunodetection of γ-H2AX. Formate, acetate, and pivalate functionalized complexes induced DSBs in a higher percentage of cells compared with [Cu(1,10-phen)2](2+), which supports the importance of inner-sphere modification toward enhancing targeted biological application.

Identification and characterization of second-generation invader locked nucleic acids (LNAs) for mixed-sequence recognition of double-stranded DNA.

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with "+1 interstrand zippers" of 2'-N-(pyren-1-yl)methyl-2'-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2'-O-(pyren-1-yl)methyl-RNA or 2'-N-methyl-2'-N-(pyren-1-yl)methyl-2'-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics.

DNA damage sensor MRE11 recognizes cytosolic double-stranded DNA and induces type I interferon by regulating STING trafficking

Double-stranded DNA (dsDNA) derived from pathogen- or host-damaged cells triggers innate immune responses when exposed to cytoplasm. However, the machinery underlying the primary recognition of intracellular dsDNA is obscure. Here we show that the DNA damage sensor, meiotic recombination 11 homolog A (MRE11), serves as a cytosolic sensor for dsDNA. Cells with a mutation of MRE11 gene derived from a patient with ataxia-telangiectasia-like disorder, and cells in which Mre11 was knocked down, had defects in dsDNA-induced type I IFN production. MRE11 physically interacted with dsDNA in the cytoplasm and was required for activation of stimulator of IFN genes (STING) and IRF3. RAD50, a binding protein to MRE11, was also required for dsDNA responses, whereas NBS1, another binding protein to MRE11, was dispensable. Collectively, our results suggest that the MRE11-RAD50 complex plays important roles in recognition of dsDNA and initiation of STING-dependent signaling, in addition to its role in DNA-damage responses.

Label-free electrochemical impedance biosensor for sequence-specific recognition of double-stranded DNA

A simple and label-free DNA electrochemical impedance biosensor has been developed for the sequence-specific recognition of double-stranded DNA (ds-DNA). The probe triplex-forming oligonucleotide (TFO) is immobilized on the gold electrode through Au–S interaction. In the presence of the target ds-DNA, the probe TFO interacted with the bases in the major groove of the ds-DNA to form a rigid triplex structure via Hoogsteen hydrogen bonds, leading to an increase of the electron-transfer resistance. It is found that the change of impedance has a linear relationship with the concentration of the target in the range of 0.1–40 nmol L−1, and the detection limit is as low as 0.04 nmol L−1 (S/N = 3).

Metal–organic frameworks-based biosensor for sequence-specific recognition of double-stranded DNA

A simple, cost-efficient, sensitive and selective fluorescence sensor is developed for sequence-specific recognition of duplex DNA (ds-DNA) in vitro using metal-organic framework (MOF) as the sensing platform. N,N-Bis(2-hydroxy-ethyl)dithiooxamidatocopper(II) (H(2)dtoaCu) was chosen as the example MOF, because it strongly chemisorbs the dye-labeled probe TFO (triplex-forming oligonucleotide), and quenches fluorescence from the dye. In the presence of target ds-DNA (the PPT of HIV RNA, a 16-bp ds-DNA sequence), the TFO could interact with the major groove in ds-DNA (via Hoogsteen hydrogen bonding) to form a rigid triplex structure, resulting in fluorescence recovery. The enhanced fluorescence signal has a relationship with the ds-DNA concentration, the detection limit is as low as 1.3 nmol L(-1) (S/N = 3) with good selectivity, which is lower than that based on a graphene oxide platform and electrochemical-DNA sensor.

Sequence-specific recognition of double-stranded DNA with molecular beacon with the aid of Ag+ under neutral pH environment

A sensitive fluorescent sensor for sequence-specific recognition of dsDNA was established with a molecular beacon (MB) based upon the formation of parallel triplex DNA in the presence of Ag(+) under neutral pH environment.

A novel quantum dots-based OFF–ON fluorescent biosensor for highly selective and sensitive detection of double-strand DNA

Abstract We have developed a novel biosensor for the rapid determination of double-strand (ds) DNA based on the photoinduced electron transfer (PET) from glutathione (GSH) capped CdTe quantum dots (QDs) to praseodymium(III)–rutin (Pr 3+ –rutin) complex. The biosensor was based on a fluorescence “OFF–ON” mode. To provide the platform for dsDNA detection, the Pr 3+ –rutin complex was first employed as an effective fluorescence quencher to QDs through PET. Because of its strong binding affinity with Pr 3+ –rutin complex, dsDNA can break up the low fluoresced ionic ensemble, set free the luminescent QDs. Thus, the recognition of dsDNA by Pr 3+ –rutin complex can be realized through the restoration of QDs fluorescence. The relative recovered fluorescence intensity is directly proportional to the concentration of herring sperm (hs) DNA between 0.0874 μg mL −1 and 20 μg mL −1 , and the detection limit (3 δ / K ) is 0.0262 μg mL −1 . The entire experimental process does not require any chemical modification and coupling of CdTe QDs and dsDNA, greatly simplifying the experimental process. Some possible reaction mechanisms are discussed.

Evaluation of damage to DNA induced by UV-C radiation and chemical agents using electrochemical biosensor based on low molecular weight DNA and screen-printed carbon electrode

There is great interest and need to detect and evaluate damage to DNA by environmental factors. In the present paper, simple electrochemical DNA biosensors composed of commercially available screen-printed carbon electrode (SPCE) and low molecular weight double-stranded DNA (dsDNA) recognition layer are reported and applied to the detection of damage to DNA by UV-C radiation and reactive oxygen species produced by the Fenton type reaction in model as well as mineral water samples with additives. Complex DNA biosensor response is based on square-wave voltammetric intrinsic signal of the guanine moiety as well as that of the intercalative indicator thioridazine, cyclic voltammetric response of the [Fe(CN)6]3−/4− indicator in solution and on electrochemical impedance spectroscopy when the measurements can be performed in the same solution. For the last two types of measurements, the biosensor was also used with an interface between the SPCE and DNA formed by a composite of carboxylated single-walled carbon nanotubes and chitosan to enhance the transducer conductivity. Individual electrochemical/electrical signals depend on the time of biosensor incubation in a cleavage medium and their profiles characterize process of deep DNA degradation.

Electrochemical detection of different types of single-base mismatches in DNA using copper-phthalocyanine tetrasulfonic acid

In this manuscript, an electrochemical DNA hybridization biosensor which exploits long range charge transfer through double stranded DNA (ds-DNA) to copper-phthalocyanine tetrasulfonic acid – tetra sodium salt (CuPcS 4) is described. Four negative charges of CuPcS 4 make it a good choice to overcome two main problems of the biosensors: direct electron transfer with the electrode surface instead of charge transfer through DNA and positioning of the redox reporter before the mismatch position. The DNA recognition layer consisted of a self assembled monolayer of amino modified single stranded DNA (ss-DNA) on the surface of glassy carbon electrode (GCE). A significant increase of the peak current for CuPcS 4 upon the hybridization process is observed. This biosensor can differentiate complementary target from non-complementary and also different types of single-base mismatch targets even thermodynamically stable G–A and G–T targets through diminution of voltammetric signal of CuPcS 4. In addition, the position of the mismatch does not affect on the capability of the biosensor.

Corrigendum: Structural Effects on ss‐ and dsDNA Recognition by a β‐Hairpin Peptide

Corrigendum: Structural Effects on ss‐ and dsDNA Recognition by a β‐Hairpin Peptide