Recognition Of Double-stranded DNA Research Articles

1H, 13C and 15N backbone resonance assignments of hepatocyte nuclear factor-1-beta (HNF1β) POUS and POUHD.

Hepatocyte nuclear factor 1β (HNF1β) is a transcription factor that plays a key role in the development and function of the liver, pancreas, and kidney. HNF1β plays a key role in early vertebrate development and the morphogenesis of these organs. In humans, heterozygous mutations in the HNF1B gene can result in organ dysplasia, making it the most common cause of developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. Pathogenic variants in the HNF1B gene are known to cause various diseases, including maturity-onset diabetes of the young and developmental renal diseases. This study presents the backbone resonance assignments of HNF1β POUS and POUHD domains, which are highly conserved domains required for the recognition of double-stranded DNA. Our data will be useful for NMR studies to verify the altered structures and functions of mutant HNF1B proteins that can induce developmental renal diseases, including renal cysts, renal malformations, and familial hypoplastic glomerular cystic kidney disease. This study will provide the structural basis for future studies to elucidate the molecular mechanisms underlying how mutations in HNF1β cause diseases.

DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts.

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes.

Factors Impacting Invader-Mediated Recognition of Double-Stranded DNA.

The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman's rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences.

Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms.

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR–Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR–Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex’s components of Cas9–RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9–RNA/DNA formation from the Cas9–RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR–Cas9 system.

Sequence-Specific Recognition of Double-Stranded DNA by Peptide Nucleic Acid Forming Double-Duplex Invasion Complex

Peptide nucleic acid (PNA) is an analog of natural nucleic acids, where the sugar-phosphate backbone of DNA is replaced by an electrostatically neutral N-(2-aminoethyl)glycine backbone. This unique peptide-based backbone enables PNAs to form a very stable duplex with the complementary nucleic acids via Watson–Crick base pairing since there is no electrostatic repulsion between PNA and DNA·RNA. With this high nucleic acid affinity, PNAs have been used in a wide range of fields, from biological applications such as gene targeting, to engineering applications such as probe and sensor developments. In addition to single-stranded DNA, PNA can also recognize double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is hard to achieve with other artificial nucleic acids and is expected to be a promising method to recognize dsDNA in cellula or in vivo since the invasion does not require the prior denaturation of dsDNA. In this paper, we provide basic knowledge of PNA and mainly focus on the research of PNA invasion.

Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA

Modified Outer Membrane Protein-G Nanopores with Expanded and Truncated β-Hairpins for Recognition of Double-Stranded DNA

Nicked Invader probes: multistranded and sequence-unrestricted recognition of double-stranded DNA.

Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for enabling tools for applications in molecular biology, diagnostics, and medicine. Towards this end, we have previously introduced Invader probes, i.e., short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. The individual strands of these labile probes display high affinity towards complementary DNA (cDNA), which drives sequence-unrestricted dsDNA-recognition. However, recognition of long targets is challenging due to the high stability of the corresponding probes. To address this, we recently introduced toehold Invader probes, i.e., Invader probes with 5'-single-stranded overhangs. The toehold architecture allows for shorter double-stranded segments to be used, which facilitates probe dissociation and dsDNA-recognition. As an extension thereof, we here report the biophysical and dsDNA-targeting properties of nicked Invader probes. In this probe architecture, the single-stranded overhangs of toehold Invader probes are hybridized to short intercalator-modified auxiliary strands, leading to formation of additional labile segments. The extra binding potential from the auxiliary strands imparts nicked Invader probes with greater dsDNA-affinity than the corresponding toehold or blunt-ended probes. Recognition of chromosomal DNA targets, refractory to recognition by conventional Invader probes, is demonstrated for nicked Invader probes in the context of non-denaturing FISH experiments, which highlights their utility as dsDNA-targeting tools.

Serine-γPNA, Invader probes, and chimeras thereof: three probe chemistries that enable sequence-unrestricted recognition of double-stranded DNA.

Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence in situ hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), i.e., single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with (R)-hydroxymethyl moieties, (ii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of 2'-O-(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, i.e., duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.

Recognition of double-stranded DNA by the Rhodobacter sphaeroides Argonaute protein

In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.

Structural basis of G-quadruplex DNA recognition by the yeast telomeric protein Rap1.

G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.

Impact of non-nucleotidic bulges on recognition of mixed-sequence dsDNA by pyrene-functionalized Invader probes.

Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, are energetically activated for sequence-unrestricted recognition of double-stranded DNA (dsDNA) as they are engineered to violate the neighbor exclusion principle, while displaying high affinity towards complementary DNA sequences. The impact on Invader-mediated dsDNA-recognition upon additional modification with different non-nucleotidic bulges is studied herein, based on the hypothesis that bulge-containing Invader probes will display additionally disrupted base-stacking, more extensive denaturation, and improved dsDNA-recognition efficiency. Indeed, Invader probes featuring a single central large bulge - e.g., a nonyl (C9) monomer - display improved recognition of model DNA hairpin targets vis-à-vis conventional Invader probes (C50 values ∼1.5 μM vs. ∼3.9 μM). In contrast, probes with two opposing central bulges display less favorable binding characteristics. Remarkably, C9-modified Invader probes display perfect discrimination between fully complementary dsDNA and dsDNA differing in only one of eighteen base-pairs, underscoring the high binding specificity of double-stranded probes. Cy3-labeled bulge-containing Invader probes are demonstrated to signal the presence of gender-specific DNA sequences in fluorescent in situ hybridization assays (FISH) performed under non-denaturing conditions, highlighting one potential application of dsDNA-targeting Invader probes.

Dual-Color Emissive AIEgen for Specific and Label-Free Double-Stranded DNA Recognition and Single-Nucleotide Polymorphisms Detection.

Simple, rapid, and sensitive assays of DNA sequence hold great importance in genetic analysis, clinical diagnosis, and molecular biology research. Most current methods for DNA detection, based on the complementary base pairing, require hybridization with intricately modified single-stranded DNA (ssDNA) probes or analytes. Herein, we have developed a powerful molecule with aggregation-induced emission (AIE) characteristic, namely, TPBT, which can specifically recognize double-stranded DNA (dsDNA) by emitting out a unique dual-color fluorescent signal of red (∼640 nm) and green (∼537 nm). The red-color emission at around 640 nm is observed when TPBT binds with dsDNA, ssDNA, proteins, and other polyanionic analytes. However, the green emission at around 537 nm is demonstrated to be the exclusive response of TPBT to dsDNA, which is closely related to the conformational change of TPBT upon groove binding. More strikingly, TPBT can distinguish single-nucleotide polymorphisms (SNPs) in a dsDNA sequence and detect the DNA damage suffered from UV light with ultrahigh sensitivity and specificity. This label-free, AIEgen-based dsDNA assay method is facile, robust, and universal, which will lead to major advances in genomic and disease diagnosis.

Analysis of human cGAS activity and structure.

Cyclic GMP-AMP synthase (cGAS) is an innate immune system enzyme responsible for recognition of double-stranded DNA aberrantly localized in the cell cytosol. cGAS binds DNA and is activated to catalyze production of the nucleotide second messenger 2'-5'/3'-5' cyclic GMP-AMP (2'3' cGAMP). In spite of a major role for cGAS in the cellular immune response, a complete understanding of cGAS biology has been limited by a lack of genetic tools to rapidly screen cGAS activity, instability of human cGAS-DNA interactions in vitro, and a previous absence of structural information for the human cGAS-DNA complex. Here we detail procedures to map the molecular determinants of cGAS activation and describe methods developed to prepare human cGAS-DNA crystals for structural analysis. Together with earlier systems established to study mammalian homologs of cGAS, these innovations provide a foundation to understand and therapeutically target human cGAS biology.

Molecular recognition of double-stranded DNA by a synthetic, homoaromatic tripeptide (YYY): The spectroscopic and calorimetric study

The intermolecular interactions of a homoaromatic tripeptide, H-Tyr-Tyr-Tyr-OH (YYY) with model double-stranded (ds) DNA (ct-DNA) have been investigated by isothermal titration calorimetric (ITC) method along with various biophysical techniques such as fluorescence, time correlated single photon counting (TCSPC) and circular dichroism (CD) spectroscopy. The binding affinity [log (K) at 25 °C] of the YYY to ct-DNA is calculated as ≈4.3. The binding mode of the YYY to ds-DNA is elucidated by fluorescence intercalator displacement (FID) assay, melting temperature analysis, viscosity measurement and salt-induced fluorescence quenching study. The studies establish that the YYY recognizes the groove of the ct-DNA. The temperature dependent ITC studies show that the binding interaction is thermodynamically favourable. The compensation between enthalpy and entropy leads to the overall Gibbs free energy change almost invariant. Finally, the generality of the YYY to recognize ds-DNA has been analyzed with other model ds-DNA, ds26, which reveals almost similar binding affinity of the YYY as ct-DNA. The studies elucidate both the spectroscopic and calorimetric insight of the interactions of a homoaromatic tripeptide with ds-DNA and hold the promise of future applications as DNA targeting drug.

A colorimetric method for the sequence-specific recognition of double-stranded DNA on the surface of a silver-coated glass slide

In this study, a colorimetric method for sequence-specific recognition of double-stranded DNA (dsDNA) was established on the surface of a silver-coated glass slide. Oligo-1 was assembled on the surface of a silver-coated glass slide through an Ag–S bond, and Oligo-2 as reporter was used to bind with streptavidin-horseradish peroxidase (SA–HRP). They could bind with target dsDNA that was composed of Oligo-3 and Oligo-4 on the surface of a silver-coated glass slide through triplex formation. The bound HRP could be moved into the solution by DNase I and catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Therefore, the concentration of target dsDNA could be determined with the colour change of TMB. Under the optimum conditions, the absorbance was proportional to the concentration of target dsDNA over the range of 100 pmol/L to 2.0 nmol/L, with a detection limit of 13 pmol/L. In addition, this method showed good sequence selectivity, enabling it to be further developed for the detection of other polymerase chain reaction (PCR) products.

Sensitive Fluorescent Sensor for Recognition of HIV-1 dsDNA by Using Glucose Oxidase and Triplex DNA.

A sensitive fluorescent sensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed on the surface of silver-coated glass slide (SCGS). Oligonucleotide-1 (Oligo-1) was designed to assemble on the surface of SCGS and act as capture DNA, and oligonucleotide-2 (Oligo-2) was designed as signal DNA. Upon addition of target HIV-1 dsDNA (Oligo-3•Oligo-4), signal DNA could bind on the surface of silver-coated glass because of the formation of C•GoC in parallel triplex DNA structure. Biotin-labeled glucose oxidase (biotin-GOx) could bind to signal DNA through the specific interaction of biotin-streptavidin, thereby GOx was attached to the surface of SCGS, which was dependent on the concentration of target HIV-1 dsDNA. GOx could catalyze the oxidation of glucose and yield H2O2, and the HPPA can be oxidized into a fluorescent product in the presence of HRP. Therefore, the concentration of target HIV-1 dsDNA could be estimated with fluorescence intensity. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of target HIV-1 dsDNA over the range of 10 pM to 1000 pM, the detection limit was 3 pM. Moreover, the sensor had good sequence selectivity and practicability and might be applied for the diagnosis of HIV disease in the future.

Hairpin probe for sequence-specific recognition of double-stranded DNA on simian virus 40

Simian virus 40(SV40) is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine·homopurine duplex DNA(dsDNA) of SV40(4424—4440, gp6) was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5′-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3′(HP) containing G-rich sequence and 17-bp trip-lex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structure to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.

Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

Fluorescence recognition of double-stranded DNA based on the quenching of gold nanoparticles to a fluorophore labeled DNA probe.

An ultrasensitive fluorescent platform for sequence-specific recognition of double-stranded DNA (dsDNA) based on the quenching of gold nanoparticles (AuNPs) to a fluorophore labeled DNA probe was developed. The target dsDNA could hybridize with the loop portion of the molecular beacon (MB) to form a triplex DNA structure and opened the "stem-loop" structure of the MB; such triplex DNA was used as an assistant probe (AP). Meanwhile, a fluorophore labeled DNA-AuNP probe that contained a specific enzyme cleavage site was introduced and its fluorescence signal was efficiently quenched due to the vicinity of the fluorophore to the AuNP surface. Such a DNA-AuNP probe could hybridize with the 5' stem portion of the MB in the AP to form duplex DNA strands that contained a specific enzyme cleavage site for the nicking enzyme assisted cleavage reaction, and resulted in the release of the fluorophore from the AuNP surface and the recovery of the fluorescence signal. Because the AP remains intact during such a cleavage process, it could be reused to hybridize with the next DNA-AuNP probe and trigger the nicking nuclease assisted signal amplification. Under optimal conditions, a low detection limit of 3.8 pM was obtained for dsDNA detection, and the assay has high sequence specificity for dsDNA detection.

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.