Mismatched Base Pairs Research Articles

Zn2+ ions improve the fidelity of metal-mediated primer extension while suppressing intrinsic and Mn2+-induced mutagenic effects by DNA polymerases.

While Mn2+ ions are well-established for reducing the fidelity of DNA polymerases, leading to the misincorporation of nucleotides, our investigation of the effects of metal ions revealed a contrasting role of Zn2+. Here, we demonstrate that Zn2+ ions enhance the fidelity of DNA polymerases (the 3' → 5' exonuclease-deficient Klenow fragment and Taq DNA polymerase) by suppressing misincorporation during primer extension reactions. Remarkably, Zn2+ ions inhibit both intrinsic misincorporation and Mn2+-induced misincorporation of nucleotides. Furthermore, Zn2+ ions also effectively suppressed misincorporation during metal-mediated primer extension reactions, which involved forming Ag+ and Hg2+ ion-mediated base pairs. These findings suggest that Zn2+ ions inhibit both intrinsic and Mn2+-induced mismatched base pair formation. Consequently, the combined use of Mn2+ and Zn2+ ions may offer a strategy for precisely regulating the fidelity of DNA polymerases. Remarkably, Zn2+ ions even suppress misincorporation in primer extension reactions that rely on metal-mediated base pairs, and conversely, this suggests that DNA polymerases recognize metal-mediated base pairs such as T-Hg2+-T, C-Ag+-A, and C-Ag+-T as relatively stable base pairs. These results imply that Zn2+ ions may also enhance the fidelity of DNA polymerases when incorporating non-canonical nucleobases, potentially paving the way for the expansion of the genetic alphabet.

Localization and discrimination of GG mismatch in duplex DNA by synthetic ligand-enhanced protein nanopore analysis.

Mismatched base pairs in DNA are the basis of single-nucleotide polymorphism, one of the major issues in genetic diseases. However, the changes of physical and chemical properties of DNA caused by single-site mismatches are often influenced by the sequence and the structural flexibility of the whole duplex, resulting in a challenge of direct detection of the types and location of mismatches sensitively. In this work, we proposed a synthetic ligand-enhanced protein nanopore analysis of GG mismatch on DNA fragment, inspired by in silico investigation of the specific binding of naphthyridine dimer (ND) on GG mismatch. We demonstrated that both the importing and unzipping processes of the ligand-bound DNA duplex can be efficiently slowed down in α-hemolysin nanopore. This ligand-binding induced slow-down effect of DNA in nanopore is also sensitive to the relative location of the mismatch on DNA duplex. Especially, the GG mismatch close to the end of a DNA fragment, which is hard to be detected by either routine nanopore analysis or tedious nanopore sequencing, can be well differentiated by our ND-enhanced nanopore experiment. These findings provide a promising strategy to localize and discriminate base mismatches in duplex form directly at the single-molecule level.

7-Deazapurine and Pyrimidine Nucleoside and Oligonucleotide Cycloadducts Formed by Inverse Diels-Alder Reactions with 3,6-Di(pyrid-2-yl)-1,2,4,5-tetrazine: Ethynylated and Vinylated Nucleobases for Functionalization and Impact of Pyridazine Adducts on DNA Base Pair Stability and Mismatch Discrimination.

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

Substrate stress relaxation regulates neural stem cell fate commitment

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.

Isomerization Pathways of a Mismatched Base Pair of A:8OG in Free Duplex DNA.

The A:8OG base pair (bp) is the outcome of DNA replication of the mismatched C:8OG bp. A high A:8OG bp population increases the C/G to A/T transversion mutation, which is responsible for various diseases. MutY is an important enzyme in the error-proof cycle and reverts A:8OG to C:8OG bp by cleaving adenine from the A:8OG bp. Several X-ray crystallography studies have determined the structure of MutY during the lesion scanning and lesion recognition stages. Interestingly, glycosidic bond (χ) angles of A:8OG bp in those two lesion recognition structures were found to differ, which implies that χ-torsion isomerization should occur during the lesion recognition process. In this study, as a first step to understanding this isomerization process, we characterized the intrinsic dynamic features of A:8OG in free DNAs by a free energy landscape simulation at the all-atom level. In this study, four isomerization states were assigned in the order of abundance: Aanti:8OGsyn > Aanti:8OGanti > Asyn:8OGanti ≈ Asyn:8OGsyn. Of these bp states, only 8OG in Asyn:8OGanti was located in the extrahelical space, whereas the purine bases (A and 8OG) in the other bp states remained inside the DNA helix. Also, free energy landscapes showed that the isomerization processes connecting these four bp states proceeded mostly in the intrahelical space via successive single glycosidic bond rotations of either A or 8OG.

Structural Studies of a Complex of a CAG/CTG Repeat Sequence-Specific Binding Molecule and A-A-Mismatch-Containing DNA.

Triplet repeat diseases are caused by the abnormal elongation of repeated sequences comprising three bases. In particular, the elongation of CAG/CTG repeat sequences is thought to result in conditions such as Huntington's disease and myotonic dystrophy type 1. Although the causes of these diseases are known, fundamental treatments have not been established, and specific drugs are expected to be developed. Pyrrole imidazole polyamide (PIP) is a class of molecules that binds to the minor groove of the DNA duplex in a sequence-specific manner; because of this property, it shows promise in drug discovery applications. Earlier, it was reported that PIP designed to bind CAG/CTG repeat sequences suppresses the genes that cause triplet repeat diseases. In this study, we performed an X-ray crystal structure analysis of a complex of double-stranded DNA containing A-A mismatched base pairs and a cyclic-PIP that binds specifically to CAG/CTG sequences. Furthermore, the validity and characteristics of this structure were analyzed using in silico molecular modeling, ab initio energy calculations, gel electrophoresis, and surface plasmon resonance. With our direct observation using atomic force microscopy and DNA origami, we revealed that the PIP caused structural changes in the DNA strands carrying the expanded CAG/CTG repeat. Overall, our study provides new insight into PIP from a structural perspective.

Specific binding of Ag+ to central C[sbnd]C mismatched base pair but not terminal C[sbnd]C pair in duplex DNA

Metal ion-nucleic acid interactions are important for their contribution in structure formation and their potential applications in nanotechnology. Hg2+ and Ag+ bind to T–T and CC mismatched base pairs, respectively, at the center of duplex DNA to form T–Hg–T and C–Ag–C. Although primer-extension by DNA polymerases with Hg2+ incorporated thymidine 5′-triphosphate to form T–Hg–T, the same reaction with Ag+ did not incorporate deoxycytidine 5′-triphosphate to form C–Ag–C. Here, isothermal titration calorimetric analyses to examine the effect of CC position in duplex DNA on Ag+ binding demonstrated that Ag+ did not bind to the terminal CC base pair in duplex, but it bound to the central CC base pair in duplex at 1:1 molar ratio with 9 × 105 M–1 binding constant. Ag+ did not bind to the terminal and central C–A, C–G, and C–T base pairs in duplex. These findings are useful for developing efficient metal-mediated base pair formation in nanotechnology.

Highly Sensitive Detection of T790 M with a Three-Level Characteristic Current by Thymine-Hg(II)-Thymine in the α-Hemolysin Nanopore.

Sensitive detection of resistance mutation T790 M is of great significance for early diagnosis and prognostic monitoring of non-small-cell lung cancer (NSCLC). In this paper, we showed a highly sensitive detection strategy for T790 M using a three-level characteristic current signal pattern in an α-hemolysin nanopore. A probe was designed that formed a C-T mismatched base pair with wild-type/P and a T-T mismatched with the T790M/P. The T790M/P produced a unique three-level characteristic current signal in the presence of mercury ions(II): first, T790M-Hg2+-P entering the vestibule of α-HL under the transmembrane potential and overhang of probe occupying the β-barrel, then probe unzipping from the T790M/P, T790 M temporally residing inside the nanocavity due to the interaction with Hg(II), and finally T790 M passing through the β-barrel. The blocking current distribution was concentrated with a small relative standard deviation of about 3%, and the signal peaks of T790 M and wild-type can be completely separated with a high separation resolution of more than 2.5, which achieved the highly sensitive detection of T790 M down to 0.001 pM (confidence level P 95%) with a linear range from 0.001 pM to 1 nM in human serum samples. This highly sensitive recognition strategy enables the detection of low abundance T790 M and provides a method for prognostic monitoring in NSCLC patients.

A portable label-free electrochemical DNA biosensor for rapid detection of Salmonella Typhi.

A highly accurate, rapid, portable, and robust platform for detecting Salmonella enterica serovar Typhi (S. Typhi) is crucial for early-stage diagnosis of typhoid to avert and control the outbreaks of this pathogen, which threaten global public health. This study presents a proof-of-concept for our developed label-free electrochemical DNA biosensor system for S. Typhi detection, which employs a printed circuit board gold electrode (PCBGE), integrated with a portable potentiostat reader. Initially, the functionalized DNA biosensor and target detection were characterized using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) methods using a benchtop potentiostat. Interestingly, the newly developed DNA biosensor can identify target single-stranded DNA concentrations ranging from 10 nM to 20 μM, achieving a detection limit of 7.6 nM within a brief 5 minute timeframe. Under optimal detection conditions, the DNA biosensor exhibits remarkable selectivity, capable of distinguishing a single mismatch base pair from the target single-stranded DNA sequence. We then evaluated the feasibility of the developed DNA biosensor system as a diagnostic tool by detecting S. Typhi in 50 clinical samples using a portable potentiostat reader based on the DPV technique. Remarkably, the developed biosensor can distinctly distinguish between positive and negative samples, indicating that the miniaturised DNA biosensor system is practical for detecting S. Typhi in real biological samples. The developed DNA biosensor device in this work proves to be a promising point-of-care (POC) device for Salmonella detection due to its swift detection time, uncomplicated design, and streamlined workflow detection system.

Investigating the Cytotoxicity of Ru(II) Polypyridyl Complexes by Changing the Electronic Structure of Salicylaldehyde Ligands.

A novel class of Ru(II)-based polypyridyl complexes with an auxiliary salicylaldehyde ligand [Ru(phen)2(X-Sal)]BF4 {X: H (1), 5-Cl (2), 5-Br (3), 3,5-Cl2 (4), 3,5-Br2 (5), 3-Br,5-Cl (6), 3,5-I2 (7), 5-NO2 (8), 5-Me (9), 4-Me (10), 4-OMe (11), and 4-DEA (12), has been synthesized and characterized by elemental analysis, FT-IR, and 1H/13C NMR spectroscopy. The molecular structure of 4, 6, 9, 10, and 11 was determined by single-crystal X-ray diffraction analysis which revealed structural similarities. DFT and TD-DFT calculations showed that they also possess similar electronic structures. Absorption/emission spectra were recorded for 2, 3, 10, and 11. All Ru-complexes, unlike the pure ligands and the complex lacking the salicylaldehyde component, displayed outstanding antiproliferative activity in the screening test (10 μM) against CCRF-CEM leukemia cells underlining the crucial role of the presence of the auxiliary ligand for the biological activity. The two most active derivatives, namely 7 and 10, were selected for continuous assays showing IC50 values in the submicromolar and micromolar range against drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells, respectively. These two compounds were investigated in silico for their potential binding to duplex DNA well-matched and mismatched base pairs, since they showed remarkable selectivity indexes (2.2 and 19.5 respectively) on PBMC cells.

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.

Ultrasensitive and Quantitative DNA Methylation Detection Method Based on the MutS Protein.

DNA methylation is closely related to cancer. It is generally accepted that DNA methylation detection is crucial in cancer diagnosis, prognosis, and treatment monitoring. Therefore, there is an urgent demand for developing a simple, rapid, highly sensitive, and highly specific methylation detection method to detect DNA methylation at specific sites quantitatively. In this work, we introduce a DNA methylation detection method based on MutS and methylation-specific PCR, named MutS-based methylation-specific PCR (MB-MSP), which has the advantages of simplicity, speed, high specificity, sensitivity, and broad applicability. Utilizing the MutS's ability to bind mismatched base pairs, we inhibit not only the amplification of unmethylated DNA but also nonspecific primer amplification. We achieved a detection sensitivity of 0.5% for the methylated genes of ACP1, CLEC11A, and SEPT9 by MB-MSP. It has a good linear relationship and a detection time of only 1.5 h. To validate the feasibility of the MB-MSP method in clinical application, we conducted methylation detection on plasma-circulating tumor DNA samples from 10 liver cancer patients and 5 healthy people, achieving a 100% accuracy rate. In conclusion, MB-MSP, as a novel and reliable DNA methylation detection tool, holds significant application value and potential for advancing early cancer diagnosis.

CRISPR-dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria.

Bacterial small RNAs (sRNAs) function in post-transcriptional regulatory responses to environmental changes. However, the lack of eukaryotic RNA interference-like machinery in bacteria has limited the systematic engineering of RNA repression. Here, we report the development of clustered regularly interspaced short palindromic repeats (CRISPR)-guided dead CRIPSR-associated protein 13a (dCas13a) ribonucleoprotein that utilizes programmable CRISPR RNAs (crRNAs) to repress trans-acting and cis-acting sRNA as the target, altering regulatory mechanisms and stress-related phenotypes. In addition, we implemented a modular loop engineering of the crRNA to promote modular repression of the target gene with 92% knockdown efficiency and a single base-pair mismatch specificity. With the engineered crRNAs, we achieved targetable single-gene repression in the polycistronic operon. For metabolic application, 102 crRNAs were constructed in the biofoundry and used for screening novel knockdown sRNA targets to improve lycopene (colored antioxidant) production in Escherichia coli. The CRISPR-dCas13a system will assist as a valuable systematic tool for the discovery of novel sRNAs and the fine-tuning of bacterial RNA repression in both scientific and industrial applications.

Equilibrium melting probabilities of a DNA molecule with a defect: An exact solution of the Poland-Scheraga model.

In this study we derive analytically the equilibrium melting probabilities for basepairs of a DNA molecule with a defect site. We assume that the defect is characterized by a change in the Watson-Crick basepair energy of the defect basepair, and in the associated two stacking energies for the defect, as compared to the remaining parts of the DNA. The defect site could, for instance, occur due to DNA basepair mismatching, cross-linking, or by the chemical modifications when attaching fluorescent labels, such as fluorescent-quencher pairs, to DNA. Our exact solution of the Poland-Scheraga model for DNA melting provides the probability that the labeled basepair, and its neighbors, are open at different temperatures. Our work is of direct importance, for instance, for studies where fluorophore-quencher pairs are used for studying single basepair fluctuations of designed DNA molecules.

Analysis of Recurrent DNA Base Pair Mismatches in Eye Cancer Patient Genomes

This research investigates potential genetic drivers for uveal melanoma (UM), the most frequent primary cancer of the eye, through a frequency and ontology analysis of recurrent genetic mutations in UM patients. Genome data of 32 patients, 23 primary and 9 metastatic, was acquired from the U.S. National Institute of Health (NIH) and obtained from samples that were surgically collected from eye enucleations or resected from liver metastases. It was hypothesized that chromosomes 15 and 16 would predominantly exhibit genetic alterations linked to UM due to their role in the expression of eye color. During analysis, DNA from cancerous tumor cells was compared to a reference DNA sequence (extracted from normal tissue) to identify nucleotide base pair mismatches. The locus of each mutation noted to determine what genes mutated. Pareto analysis of cross-patient data performed to identify chromosomes with the most genetic mutations along with any recurrent genetic mutations across patients. A gene ontology (GO) analysis conducted to study the functions of mutated genes and investigate possible links to cancer, such as anomalies in genes with a known role in tumor suppression. A total of 130 genetic mutations were identified (7 recurrent and 123 non-recurrent), with most mutations occurring in chromosomes 3 and X. Recurrent mutations varied from 8.7% to 17.39% occurrence in the UM patient sample. The recurring mutations observed as missense mutations in seven genes. These findings suggest that UM is a recessive heterogeneous disease with selected homozygous mutations. Notably, this study has potential wider significance because the seven genes targeted by recurrent mutations affected in other cancers. It concluded that immunotherapy is a highly promising treatment for Uveal Melanoma due to the disease’s heterogeneous nature

Acetylation of MLH1 by CBP increases cellular DNA mismatch repair activity.

The DNA mismatch repair (MMR) proteins recognize and repair DNA base pair mismatches and insertions/deletions of DNA that have occurred during DNA replication. Additionally, they are involved in regulation of the DNA damage response, including cell cycle checkpoints and apoptosis. Therefore, regulation of these proteins is essential for maintaining genomic integrity. It has been recognized that post-translational modifications, such as phosphorylation, ubiquitination, and acetylation, are being used as an important means to regulate the functions and stability of MMR proteins. Here, we report that a histone acetyltransferase CREB binding protein (CBP) interacts with and acetylates MLH1, a component of the MutLα complex (MLH1-PMS2). Moreover, CBP stabilizes MLH1 by preventing it from degradation via the ubiquitin-proteasome degradation pathway. Consistently, acetylation induced by a pan-histone deacetylase inhibitor, Trichostatin A, promotes the assembly between the MutSα (MSH2-MSH6) and MutLα complexes. Furthermore, overexpression of CBP enhances MMR activities in cells. Overall, our results suggest a novel role of CBP in prolonging MLH1 stability and enhancing MutSα-MutLα complex formation, leading to increased cellular MMR activity.

DSN enzyme recognition initiated rolling circle amplification (DiRCA) for accurate miRNA detection in immune response and immune repair after trauma

Recent studies have reported that miRNA plays an important role in immune response and immune repair after trauma. By regulating the expression of related target genes, miRNA regulates the production, proliferation, development and immune response of immune cells. Therefore, it is in urgent demand to develop an novel method for miRNA analysis. Rolling circle amplification (RCA), as an attractive isothermal signal amplification strategy, has been widely utilized in constructing miRNA detection assays. However, accurate and sensitive miRNA quantitative determination remains a huge challenge for RCA based approaches. Herein, we propose a DSN enzyme based signal cycle initiated Rolling Circle Amplification assay (DiRCA) for sensitive and accurate miRNA detection. In DiRCA, target miRNA unfolds hairpin structure probe in the detection scaffold, forming a RNA–DNA duplex. DSN enzyme is utilized to specifically digest the DNA sequence in RNA–DNA duplex, releasing miRNA to form a signal cycle; its capability to distinguish one base pair mismatch in RNA–DNA duplex endows DiRCA a high selectivity. Meanwhile, DSN enzyme based cleavage initiates RCA, transcribing G-rich sequences for signal generation. Based on the DSN assisted signal cycle and RCA, DiRCA shows a low limit of detection of 0.43 fM and a superior capability in selectively detecting mismatched miRNA sequences, showing a promising prospect in the early-diagnosis of disease.

Effect of Hydrophilic Changes in Base Pair Mismatches on Deamination of Cytosine Photo-crosslinked to 3-Cyanovinylcarbazole

In this study, to understand the effects of base-pair mismatch on the deamination of cytosine cross-linked with 3-cyanovinylcarbazole, the complementary base of the target cytosine was changed from guanine (G) to adenine (A), thymine (T), cytosine (C), 5-methylcytosine (mC), and uracil (U). Notably, it was found that the fastest deamination of C to U occurs when C-C mismatching occurs.

Pulling short DNA with mismatch base pairs.

Due to misincorporation during gene replication, the accuracy of the gene expression is often compromised. This results in a mismatch or defective pair in the DNA molecule (James etal. 2016). Here, we present our study of the stability of DNA with defects in the thermal and force ensembles. We consider DNA with a different number of defects from 2to16 and study how the denaturation process differs in both ensembles. Using a statistical model, we calculate the melting point of the DNA chain in both the ensemble. Our findings display different manifestations of DNA denaturation in thermal and force ensembles. While the DNA with defects denatures at a lower temperature than the intact DNA, the point from which the DNA is pulled is important in force ensemble.

Target‐Catalyzed Self‐Assembly of DNA‐Streptavidin Nanogel for Enzyme‐Free miRNA Assay (Adv. Healthcare Mater. 9/2023)

Base-Pair Mismatch In article 2202076, Yoon Sung Nam and co-workers develop a new miRNA detection platform based on miRNA-catalyzed toehold-mediated strand displacement and the sequential formation of DNA-streptavidin nanogel. The detection limit is lowered from the nanomolar level to the picomolar level by introducing a single base-pair mismatch near the toehold enhancing strand displacement and generating a 3D nanogel network to facilitate Förster resonance energy transfer.