Specific DNA Recognition Research Articles

CRISPR-Cas9 target-strand nicking provides phage resistance by inhibiting replication.

Cas endonucleases, like Cas9 and Cas12a, are RNA-guided immune effectors that provide bacterial defense against bacteriophages. Cas endonucleases rely on divalent metal ions for their enzymatic activities and to facilitate conformational changes that are required for specific recognition and cleavage of target DNA. While Cas endonucleases typically produce double-strand breaks (DSBs) in DNA targets, reduced, physiologically relevant Mg2+ concentrations and target mismatches can result in incomplete second-strand cleavage, resulting in the production of a nicked DNA. It remains poorly understood whether nicking by Cas endonucleases is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only one of two nuclease domains is catalytically active. By testing a large panel of guide RNAs, we find that target strand nicking can be sufficient to provide immunity, while non-target nicking does not provide any additional protection beyond Cas9 binding. Target-strand nicking inhibits phage replication and can reduce the susceptibility of Cas9 to viral escape when targeting non-essential regions of the genome. Cleavage of the non-target strand by the RuvC domain is strongly impaired at low Mg2+ concentrations. As a result, fluctuations in the concentration of other biomolecules that can compete for binding of free Mg2+ strongly influences the ability of Cas9 to form a DSB at targeted sites. Overall, our results suggest that Cas9 may only nick DNA during CRISPR-mediated immunity, especially under conditions of low Mg2+ availability in cells.

Selective Activation of Cascade Assembly Amplification for DNA Methyltransferase Detection Using a Double-Loop Interlocked DNA Circuit.

Precise and reliable monitoring of DNA adenine methyltransferase (Dam) activity is essential for disease diagnosis and biological analysis. However, existing techniques for detecting Dam activity often rely on specific DNA recognition probes that are susceptible to DNA degradation and exhibit limited target sensitivity and specificity. In this study, we designed and engineered a stable and dynamic DNA nanodevice called the double-loop interlocked DNA circuit (DOOR) that enables the sensitive and selective monitoring of Dam activity in complex biological environments. The DOOR incorporates two interlocked specialized sequences: a palindromic sequence for Dam identification and an initiator sequence for signal amplification. In the presence of Dam, the DOOR is cleaved by double-stranded DNA phosphodiesterase I endonuclease, generating massive double-stranded DNA (dsDNA) units. These units can self-assemble into a long dsDNA scaffold, thereby enhancing the subsequent reaction kinetics. The dsDNA scaffold further triggers a hyperbranched hybrid chain reaction to produce a fluorescent 3D DNA nanonet, enabling more precise monitoring of the Dam activity. The DOOR device exhibits excellent sensitivity, specificity, and stability, rendering it a powerful tool for studying DNA methylation in various biological processes and diseases.

Enhancing public health safety: Development and application of a MoS2@CNT-Chit electrochemical DNA biosensor for rapid and accurate detection of S. Typhi

This study introduces an advanced electrochemical biosensor that utilizes MoS2@CNT as an electrode material combined with a specific DNA probe to detect Salmonella Typhi rapidly and accurately. The sensor offers a broad detection range from 1.0 × 10−6 to 1.0 × 10−18 molL−1 and boasts an exceptionally low limit of detection (LOD) of 1.0 × 10−20 molL−1 for the target bacterium. It demonstrates a detection range from 1.0 × 104 to 1.0 × 1011 CFUml−1 in real samples, with a corresponding LOD of 1.0 × 104 CFUml−1. Rigorous testing against base mismatches and various bacterial strains confirms its specificity, ensuring reliable performance. Validated in real samples, the biosensor can accurately identify Salmonella Typhi in water and milk, achieving recoveries ranging from 92.95 % to 99.58 %. The exceptional performance of the biosensor is attributed to the MoS2@CNT electrode material and the specific DNA recognition probe, which enhance electron transfer and reduce steric impedance. These improvements contribute to the sensor's enhanced sensitivity and specificity, making it a significant advancement in public health safety by providing a rapid and accurate tool for detecting Salmonella Typhi in food samples.

K+-Specification with Flavone P0 Probe in a G-Quadruplex DNA.

G-quadruplex (G4) DNA is considered as a prospective therapeutic target due to its potential biological significance. To understand G4 biological roles and function, a G4-specific fluorescent probe is necessary. However, it is difficult for versatile G4 to precisely recognize without perturbing their folding dynamics. Herein, we reported that flavone P0 can be a fluorescent probe for G4 DNA-specific recognition and have developed a highly selective detection of K+ ion by dimeric G4/P0 system. When comparing various nucleic acid structures, including G4, i-motif, ss/ds-DNA, and triplex, an apparent fluorescence enhancement is observed in the presence of G4 DNA for 85-fold, but only 8-fold for non-G4 DNA. Furthermore, based on fluorescent probe of flavone P0 for G4 DNA screening, the noncovalent dimeric G4/P0 system is exploited as a K+ sensor, that selectively responds to K+ with a 513-fold fluorescence enhancement and a detection range for K+ ion concentration from 0 to 500 mM. This K+ sensor also has a remarkably anti-interference ability for other metal cations, especially for a high concentration of Na+. These results have demonstrated that flavone P0 is an efficient tool for monitoring G-quadruplex DNA and endows flavone P0 with bioanalytical and medicinal applications.

Erratum to: Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter.

The original version of this article (Weng et al., 2023) unfortunately contained a mistake. In Acknowledgments, the number (No. 226-2022-00213) of the Fundamental Research Funds for the Central Universities is wrong. The correct number should be No. 2022FZZX01-33.

Structural basis for specific DNA sequence recognition by the transcription factor NFIL3

The CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of pivotal transcription factors involved in tissue development, cellular function, proliferation, and differentiation. NFIL3, as one of them, plays an important role in regulating immune cell differentiation, circadian clock system and neural regeneration, yet its specific DNA recognition mechanism remains enigmatic. In this study, we showed by the ITC binding experiments that NFIL3 prefers to bind to the TTACGTAA DNA motif. Our structural studies revealed that the α-helical NFIL3 bZIP domain dimerizes through its leucine zipper region, and binds to DNA via its basic region. The two basic regions of the NFIL3 bZIP dimer were pushed apart upon binding to DNA, facilitating the snug accommodation of the two basic regions within the major grooves of the DNA. Remarkably, our binding and structural data also revealed that both NFIL3 and C/EBPα/β demonstrated a shared preference for the TTACGTAA sequence. Furthermore, our study revealed that disease-associated mutations within the NFIL3 bZIP domain resulted in either reduction or complete disruption of its DNA binding ability. These discoveries not only provide valuable insights into the DNA binding mechanisms of NFIL3 but also elucidate the causal role of NFIL3 mutations in disease pathogenesis.

Is Simultaneous Binding to DNA and Gyrase Important for the Antibacterial Activity of Cystobactamids?

Cystobactamids are aromatic oligoamides that exert their natural antibacterial properties by inhibition of bacterial gyrases. Such aromatic oligoamides were proposed to inhibit α-helix-mediated protein-protein interactions and may serve for specific recognition of DNA. Based on this suggestion, we designed new derivatives that have duplicated cystobactamid triarene units as model systems to decipher the specific binding mode of cystobactamids to double stranded DNA. Solution NMR analyses revealed that natural cystobactamids as well as their elongated analogues show an overall bent shape at their central aliphatic unit, with an average CX-CY-CZ angle of ~110 degrees. Our finding is corroborated by the target-bound structure of close analogues, as established by cryo-EM very recently. Cystobactamid CN-861-2 binds directly to the bacterial gyrase with an affinity of 9 μM, and also exhibits DNA-binding properties with specificity for AT-rich DNA. Elongation/dimerization of the triarene subunit of native cystobactamids is demonstrated to lead to an increase in DNA binding affinity. This implies that cystobactamids' gyrase inhibitory activity necessitates not just interaction with the gyrase itself, but also with DNA via their triarene unit.

Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration.

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.

Rationally Designed Dual Base Pair Mismatch Enables Toehold-Mediated Strand Displacement to Efficiently Recognize Single-Nucleotide Polymorphism without Enzymes.

The efficiency of the enzyme-free toehold-mediated strand displacement (TMSD) technique is often insufficient to detect single-nucleotide polymorphism (SNP) that possesses only single base pair mismatch discrimination. Here, we report a novel dual base pair mismatch strategy enabling TMSD biosensing for SNP detection under enzyme-free conditions when coupled with catalytic hairpin assembly (CHA) and fluorescence resonance energy transfer (FRET). The strategy is based on a competitive strand displacement reaction mechanism, affected by the thermodynamic stability originating from rationally designed dual base pair mismatch, for the specific recognition of mutant-type DNA. In particular, enzyme-free nucleic acid circuits, such as CHA, emerge as a powerful method for signal amplification. Eventually, the signal transduction of this proposed biosensor was determined by FRET between streptavidin-coated 605 nm emission quantum dots (605QDs, donor) and Cy5/biotin hybridization (acceptor, from CHA) when incubated with each other. The proposed biosensor displayed high sensitivity to the mutant target (MT) with a detection concentration down to 4.3 fM and led to high discrimination factors for all types of mismatches in multiple sequence contexts. As such, the application of this proposed biosensor to investigate mechanisms of the competitive strand displacement reaction further illustrates the versatility of our dual base pair mismatch strategy, which can be utilized for the creation of a new class of biosensors.

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.

Rapid Information Retrieval from DNA Storage with Microfluidic Very Large-Scale Integration Platform.

Due to its high information density, DNA is very attractive as a data storage system. However, a major obstacle is the high cost and long turnaround time for retrieving DNA data with next-generation sequencing. Herein, the use of a microfluidic very large-scale integration (mVLSI) platform is described to perform highly parallel and rapid readout of data stored in DNA. Additionally, it is demonstrated that multi-state data encoded in DNA can be deciphered with on-chip melt-curve analysis, thereby further increasing the data content that can be analyzed. The pairing of mVLSI network architecture with exquisitely specific DNA recognition gives rise to a scalable platform for rapid DNA data reading.

Synthesis of 2-Aminopyridine-Modified Peptide Nucleic Acids

AbstractTriplex-forming peptide nucleic acids (PNAs) require chemical modifications for efficient sequence-specific recognition of DNA and RNA at physiological pH. Our research groups have developed 2-aminopyridine (M) as an effective mimic of protonated cytosine in C+•G-C triplets. M-modified PNAs have a high binding affinity and sequence specificity as well as promising biological properties for improving PNA applications. This communication reports the optimization of synthetic procedures that give PNA M monomer in seven steps, with minimal need for column chromatography and in good yields and high purity. The optimized route uses inexpensive reagents and easily performed reactions, which will be useful for the broad community of nucleic acid chemists. Thought has also been given to the potential for future development of industrial syntheses of M monomers.

Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter.

This study aims to gain insight into the DNA-specific recognition mechanism of c-Myb transcription factor during the regulation of cell early differentiation and proliferation. Therefore, we chose the chicken myeloid gene, mitochondrial import protein 1 (mim-1), as a target to study the binding specificity between potential dual-Myb-binding sites. The c-Myb-binding site in mim-1 is a pseudo-palindromic sequence AACGGTT, which contains two AACNG consensuses. Simulation studies in different biological scenarios revealed that c-Myb binding with mim-1 in the forward strand (complex F) ismore stable than that inthereverse strand (complex R). The principal component analysis (PCA) dynamics trajectory analyses suggested an opening motion of the recognition helices of R2 and R3 (R2R3), resulting in the dissociation of DNA from c-Myb in complex R at 330 K, triggered by the reduced electrostatic potential on the surface of R2R3. Furthermore, the DNA confirmation and hydrogen-bond interaction analyses indicated that the major groove width of DNA increased in complex R, which affected on the hydrogen-bond formation ability between R2R3 and DNA, and directly resulted in the dissociation of DNA from R2R3. The steered molecular dynamics (SMD) simulation studies also suggested that the electrostatic potential, major groove width, and hydrogen bonds made major contribution to the DNA‍-specific recognition. In vitro trials confirmed the simulation results that c-Myb specifically bound to mim-1 in the forward strand. This study indicates that the three-dimensional (3D) structure features play an important role in the DNA-specific recognition mechanism by c-Myb besides the AACNG consensuses, which is beneficial to understanding the cell early differentiation and proliferation regulated by c-Myb, as well as the prediction of novel c-Myb-binding motifs in tumorigenesis.

Replication mechanisms of circular ssDNA plant viruses and their potential implication in viral gene expression regulation.

The replication of members of the two circular single-stranded DNA (ssDNA) virus families Geminiviridae and Nanoviridae, the only ssDNA viruses infecting plants, is believed to be processed by rolling-circle replication (RCR) and recombination-dependent replication (RDR) mechanisms. RCR is a ubiquitous replication mode for circular ssDNA viruses and involves a virus-encoded Replication-associated protein (Rep) which fulfills multiple functions in the replication mechanism. Two key genomic elements have been identified for RCR in Geminiviridae and Nanoviridae: (i) short iterative sequences called iterons which determine the specific recognition of the viral DNA by the Rep and (ii) a sequence enabling the formation of a stem-loop structure which contains a conserved motif and constitutes the origin of replication. In addition, studies in Geminiviridae provided evidence for a second replication mode, RDR, which has also been documented in some double-stranded DNA viruses. Here, we provide a synthesis of the current understanding of the two presumed replication modes of Geminiviridae and Nanoviridae, and we identify knowledge gaps and discuss the possibility that these replication mechanisms could regulate viral gene expression through modulation of gene copy number.

Protein-DNA recognition mechanisms and specificity.

The accumulated knowledge about the structure of protein-DNA complexes allowed us to understand the mechanisms of protein-DNA recognition and searching for a specific site on DNA. Obviously, the mechanism of specific DNA recognition by a protein must satisfy two requirements. First, the probability of incorrect binding should be very small. Second, the time to find the "correct" binding site should not be too long. If we assume that protein recognition of a precise site on DNA occurs at some distance from DNA and calculate global minima, we can avoid local minima at short distances. The only long-range interaction is the interaction of charges. The location of charges on DNA in three-dimensional space depends on the local conformation of DNA and thus reflects the DNA sequence and sets the spatial pattern for recognition. Various factors such as counter ion concentration, ionic strength, and pH can affect protein recognition of DNA. Nowadays, the theory of long-range interactions makes it possible to calculate the best mutual spatial arrangement of protein and DNA molecules by charged groups and avoid misplaced binding.

Harnessing endogenous transcription factors directly by small molecules for chemically induced pluripotency inception

Chemistry-alone approach has recently been applied for incepting pluripotency in somatic cells, representing a breakthrough in biology. However, chemical reprogramming is hampered by low efficiency, and the underlying molecular mechanisms remain unclear. Particularly, chemical compounds do not have specific DNA-recognition domains or transcription regulatory domains, and then how do small molecules work as a driving force for reinstating pluripotency in somatic cells? Furthermore, how to efficiently clear materials and structures of an old cell to prepare the rebuilding of a new one? Here, we show that small molecule CD3254 activates endogenous existing transcription factor RXRα to significantly promote mouse chemical reprogramming. Mechanistically, CD3254-RXRα axis can directly activate all the 11 RNA exosome component genes (Exosc1-10 and Dis3) at transcriptional level. Unexpectedly, rather than degrading mRNAs as its substrates, RNA exosome mainly modulates the degradation of transposable element (TE)-associated RNAs, particularly MMVL30, which is identified as a new barrier for cell-fate determination. In turn, MMVL30-mediated inflammation (IFN-γ and TNF-α pathways) is reduced, contributing to the promotion of successful reprogramming. Collectively, our study provides conceptual advances for translating environmental cues into pluripotency inception, particularly, identifies that CD3254-RXRα-RNA exosome axis can promote chemical reprogramming, and suggests modulation of TE-mediated inflammation via CD3254-inducible RNA exosome as important opportunities for controlling cell fates and regenerative medicine.

High-Efficiency Aluminum-Metal Organic Framework/HEPES Electrochemiluminescence System for Ultrasensitive Detection of HBV DNA.

In this work, a novel aluminum metal-organic framework (Al-MOF)/N-2-hydroxyethylpiperazine-N'-ethane-sulfonic acid (HEPES) system with an excellent electrochemiluminescence (ECL) property was developed. First, Al-MOF was successfully synthesized through a one-pot solvothermal method by using 9,10-di(p-carboxyphenyl)anthracene (DPA) as the organic luminescence ligand and Al3+ as the metal node. Compared with DPA, Al-MOF showed high ECL intensity and excellent stability without an additional coreactant in the HEPES buffer. The corresponding ECL mechanism was studied in detail, verifying HEPES was not only the buffer in the system but also the coreactant of Al-MOF. In particular, the system of Al-MOF/HEPES showed a high ECL efficiency of 30.0%, taking the Ru(bpy)32+ system as the standard. In addition, the ECL signal of Al-MOF was effectively quenched by dopamine (DA). The biosensor for HBV DNA detection was constructed through the ECL signal on-off-on mode of DNA specific recognition integrated with the DNA walker signal amplification strategy. The ultrasensitive detection for HBV DNA was achieved with a linear range of 100 aM to 10 pM and a limit of detection (LOD) of 62.1 aM. This work proposed a high-efficiency Al-MOF/HEPES system, providing a new viewpoint for a coreactant-free system in the field of ECL.

New Xanthone Derivatives as Potent G-Quadruplex Binders for Developing Anti-Cancer Therapeutics.

Xanthone is an important scaffold for various medicinally relevant compounds. However, it has received scant attention in the design of agents that are cytotoxic to cancer cells via targeting the stabilization of G-quadruplex (G4) nucleic acids. Specific G4 DNA recognition against double-stranded (ds) DNA is receiving epoch-making interest for the development of G4-mediated anticancer agents. Toward this goal, we have synthesized xanthone-based derivatives with various functionalized side-arm substituents that exhibited significant selectivity for G4 DNA as compared to dsDNA. The specific interaction has been demonstrated by performing various biophysical experiments. Based on the computational study as well as the competitive ligand binding assay, it is inferred that the potent compounds exhibit an end-stacking mode of binding with G4 DNA. Additionally, compound-induced conformational changes in the flanking nucleotides form the binding pocket for effective interaction. Selective action of the compounds on cancer cells suggests their effectiveness as potent anti-cancer agents. This study promotes the importance of structure-based screening approaches to get molecular insights for new scaffolds toward desired specific recognition of non-canonical G4 DNA structures.

Deciphering the Binding Insights of Novel Disubstituted Anthraquinone Derivatives with G-Quadruplex DNA to Exhibit Selective Cancer Cell Cytotoxicity.

Anthraquinone-based compounds are well-known as duplex DNA as well as G-quadruplex DNA binders. Implications of various anthraquinone derivatives for specific recognition of G-quadruplex DNA over duplex DNA is a 'challenging' research work that requires adequate experience with molecular design. To address this important issue, we designed and synthesized ten new 2,6-disubstituted anthraquinone-based derivatives with different functionalized piperazinyl side-chains. Among these, particular compounds with certain distant groups have shown selective and significant binding affinities toward the c-MYC and c-KIT G-quadruplex DNA over the duplex DNA, as noticed from various biophysical experiments. The structural difference of quadruplex and duplex DNA was utilized to probe these derivatives for the end-stacking mode of binding with G-quadruplex DNA. The ability of the ligands to halt DNA synthesis by stabilizing G-quadruplex structures is one of the crucial points to further apply them for quadruplex-mediated anti-cancer therapeutics. Interestingly, these ligands trigger apoptosis to exhibit selective cytotoxicity toward cancer cells over normal cells. This was further evidenced by ligand-induced cell cycle arrest as well as cellular apoptotic morphological changes. These blood-compatible ligands provided detailed structure-activity relationship approaches for the molecular design of anthraquinone-based G-quadruplex binders.

DNA functionalized plasmonic nanoassemblies as SERS sensors for environmental analysis

AbstractSurface‐enhanced Raman scattering (SERS) is among the most widely applied analytical techniques due to its easy execution and extreme sensitivity. Target molecules can be detected and distinguished based upon the fingerprint spectra that arise when absorbed on the SERS substrates surface, particularly on the SERS‐active hotspots. Thus, rational fabricating the enhancing substrates plays a key role in broadening SERS application. Programmable DNA functionalized plasmonic nanoassemblies, where DNA acts as both structure basis and functional unit, combine the specificity of DNA recognition, and modulate the assembly of plasmonic nanoparticles (NPs). Specifically designed DNA not only improves the selectivity to target molecules but also promotes the sensitivity of the optical signals through precisely regulating the distance between the molecule and the substrate. A variety of DNA‐functionalized SERS sensors have been reported and obtained well performance in the analysis of heavy metal ions in water, toxins, pesticide residues, antibiotics, hormones, illicit drugs, or other small molecules. This review places an emphasis on the design and sensing strategies of the DNA‐functionalized plasmonic nanoassemblies, as well as basic principles of Raman enhancement, and recent advances for environmental analysis. The current challenges and potential trends in the development of DNA‐functionalized SERS sensors for environmental pollutant monitoring in complicated scenarios are subsequently discussed.