Mismatched Base Pairs Research Articles

New Size-Expanded Fluorescent Thymine Analogue: Synthesis, Characterization, and Application.

Here, the synthesis, photophysical characterization, and application of a new size-expanded thymine nucleoside, diox T, is described. diox T has desirable qualities as a T surrogate, including excellent quantum yield (0.36) and high environmental sensitivity. When incorporated into single- and double-stranded DNA, diox T showed excellent photophysical characteristics including a high quantum yield (average 0.20), and unlike BgQ, demonstrated dependence on neighboring bases without significant destabilization of the duplex. Interestingly, the matched base pair of adenine (A) and diox T has the unique property that it exhibits higher fluorescence than mismatched base pairs, and diox T has self-quenching effects. As one example of the possible applications of these promising features, single nucleoside polymorphism typing is demonstrated for discrimination of A by using diox T. The results suggest that diox T can be used for a broad range of applications in chemical biology.

Base-Pair Mismatch Can Destabilize Small DNA Loops through Cooperative Kinking.

Base-pair mismatch can relieve mechanical stress in highly strained DNA molecules, but how it affects their kinetic stability is not known. Using single-molecule fluorescence resonance energy transfer, we measured the lifetimes of tightly bent DNA loops with and without base-pair mismatch. Surprisingly, for loops captured by stackable sticky ends which leave single-stranded DNA breaks (or nicks) upon annealing, the mismatch decreased the loop lifetime despite reducing the overall bending stress, and the decrease was largest when the mismatch was placed at the DNA midpoint. These findings suggest that base-pair mismatch increases bending stress at the opposite side of the loop through an allosteric mechanism known as cooperative kinking. Based on this mechanism, we present a three-state model that explains the apparent dichotomy between thermodynamic and kinetic stability.

Colorimetric detection of single base-pair mismatches based on the interactions of PNA and PNA/DNA complexes with unmodified gold nanoparticles

Rapid and sensitive single nucleotide polymorphisms (SNPs) genotyping is of particular important for early diagnosis, prevention, and treatment of specific human diseases. A simple and low-cost SNP detection method would be valuable for routine analysis in resource-limited settings. Here, we demonstrated a novel and convenient gold nanoparticle (AuNPs) based colorimetric approach for efficient screening of SNPs at room temperature without instrumentation. SNP detection is performed in a single tube with one set of unmodified AuNPs, a label-free peptide nucleic acid (PNA) probe, a single exonuclease (S1 nuclease), and the target to be tested. S1 nuclease could digest DNAs in DNA/PNA duplexes involving a mismatch into small fragments, while DNAs in the fully-matched DNA/PNA duplexes can be effectively protected by PNA from enzymatic degradation. This difference could be easily discriminated by color changes associated with gold aggregation. PNA oligomers can induce immediate AuNP aggregation even in the presence of nucleoside monophosphates (dNMPs), the digestion products of DNA. Whereas PNA/DNA duplexes can effectively stabilize unmodified AuNPs, and the stabilization effect of PNA/DNA is better than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Without the need of precise temperature control and extra salt addition, SNPs are detected with a detection limit of 2.3 nM in cell lysate. Moreover, this system can effectively discriminate a range of different mismatches even in spiked cell lysate, demonstrate the potential use of this biosensor for biological samples.

Modulating RNA secondary and tertiary structures by mismatch binding ligands

Much recent attention has been focused on small organic molecules binding to non-canonical structures of nucleic acids, especially, RNA. The Human Genome Project and the ENCODE (encyclopedia of DNA elements) project revealed that more than 75% of the human genome is transcribed into RNA, while only ∼3% of the human genome encodes a protein. These non-protein-coding RNAs are thought to play significant roles in many cellular processes and are promising targets for drug discovery. Emerging roles of the non-coding RNAs in a variety of diseases provides enormous opportunities for pharmaceutical research on developing drugs targeting undruggable and rare diseases. During the last two decades, our laboratory has focused attention on small molecules binding to non-canonical DNA and RNA structures, especially to mismatched base pairs. Mismatch binding ligands (MBLs) we have developed are synthetic molecules designed in silico based on the hypothesis of hydrogen-bonding and semi-intercalation to DNA and RNA. Most of MBLs consists of two heterocycles having hydrogen bonding surfaces fully or partially complementary to that of nucleotide bases. In our design, each heterocycle binds to one of the mismatched bases by hydrogen bonding to form pseudo-base pairs, which would be stacked with the adjacent base pairs. The hypothesis allows us in principle to design small molecules binding to any mismatched base pairs, but it turned out not to be the case in reality. However, we have so far succeeded in developing several MBLs binding to DNA and RNA motifs of biological significance. In this review, we shall describe the hypothesis of molecular design of MBLs and its outcome regarding RNA targeting.

Target gene identification and functional characterization of miR-1704 in chicken

MiRNAs are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. SNPs in miRNA genes may lead to phenotypic variation by altering miRNA expression and their targets. In this study, miR-1704 expression profiles in nine tissues at 1 d, 6 weeks and 16 weeks old Gushi chickens were detected. MiR-1704 was widely expressed in the detection of tissues. The expression in 1 d and 6 weeks old was low abundance, while its expression at 16 weeks was very high. An rs14668705 (C > G) SNP was detected within the pre-miR-1704 in an F2 resource population of Gushi chicken crossed with Anka broiler. Bioinformatic analysis indicated that the C > G mutation could introduce a base-pair mismatch and cause the change of free energy. Experiments further revealed that the rs14668705 in precursor miR-1704 could significantly affect mature miR-1704 biogenesis and was significantly associated with body weight at the age of 0, 6, 8, 10, and 12 weeks, shank circumference at 4, 8, and 12 weeks, carcass weight, and semi-evisceration weight (p < 0.05). Insulin receptor 2 (IRS2) gene, one of the potential targets of miR-1704 was identified and further confirmed. These data suggested that miR-1704 targeted IRS2 and have an effect on body weight in chicken.

Deciphering Off-Target Effects in CRISPR-Cas9 through Accelerated Molecular Dynamics.

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations—using Gaussian accelerated MD (GaMD)—are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to “lock” the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to “lock” the catalytic HNH domain in an inactive “conformational checkpoint” is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

The butterfly effect in viral infection: From a host DNA single nucleotide change to HBV episome steadiness

Chronic hepatitis B virus (HBV) infection is one of the major health care burdens throughout the world. The chronicity of HBV infection is supported by the intracellular persistence of a multicopy viral episome called covalently closed circular DNA (cccDNA), which is the bona fide viral transcription template and resistant to currently available antivirals. 1 Therefore, noncytolytic elimination of cccDNA is considered as the “holy grail” for a cure of hepatitis B, and the metabolism and transcription regulation of cccDNA have become a hot topic in basic, translational, and clinical HBV research. 2 In a recent publication, Zhou et al conducted a case control study and identified a single nucleotide variant (SNV), rs59391722, located at −1128 bp of the promoter region of ubiquitin-conjugating enzyme E2L3 gene (UBE2L3, also called UBCH7), which is associated with susceptibility to HBV infection in children. 3 The SNVs at this site were found to be GG as a major SNV, and CC as a minor one, with transitional CG in Chinese Han population. GG and CG SNVs are associated with a higher binding efficiency of transcription activator NF-E2, leading to UBE2L3 upregulation compared to that in CC. Furthermore, via overexpression and knockdown approaches, the authors demonstrated that UBE2L3 facilitates HBV replication and antigen production primarily through maintaining cccDNA stability. Mechanistic studies revealed that UBE2L3 catalytically promotes the ubiquitin-dependent proteasomal degradation of a cellular cytidine deaminase APOBEC3A (A3A), which has been shown to cause base-pair mismatch hypermutations in cccDNA for apurinic/apyrimidinic endonuclease (APE1)-dependent degradation. 4 In addition, considering that A3A is an interferon-stimulated gene (ISG), the authors showed that UBE2L3 silencing could potentiate the antiviral activity of alpha-interferon (IFN-α) in cell culture model. Taken together, this study has further validated the function of A3A in editing and removing cccDNA, and established a proviral role of rs59391722-UBE2L3-A3A nexus in cccDNA persistence (Fig. 1). Figure 1 The rs59391722-UBE2L3-APOBEC3A nexus regulates HBV cccDNA stability. HBV establishes and maintains persistent infection in hepatocyte via the viral episomal cccDNA, which serves as transcription template to produce progeny virus and viral antigens (HBeAg, HBsAg, HBx). IFN-α treatment induces cccDNA degradation through ISG APOBEC3A (A3A)-mediated cccDNA cytidine deamination and subsequent APE1 cleavage. Zhou et al 3 identified a SNV rs59391722(C→G) that upregulates the expression of UBE2L3, which ubiquitinates A3A for proteasomal degradation, thereby maintaining the persistence of cccDNA and HBV infection. Figure 1 One of the strong sides of the work is that the authors analyzed HBV-associated SNVs from a small cohort of pediatric patients, as most chronic hepatitis B (CHB) patients acquire the infection through vertical transmission or during early childhood. 5 Besides the immunological dysfunction, the SNVs, such as rs59391722(C→G), may also contribute to the development of chronic HBV infection. Previous genome-wide association studies (GWASs) have largely focused on adult cohort and identified HBV-associated SNVs at 8 loci, including 7 HLA-related genes and UBE2L3, but lacked functional validations.6, 7, 8 The study by Zhou et al, for the first time, established the positive correlation of UBE2L3 expression with the clinical outcome of HBV infection in both adult and children patients; and delineated the UBE2L3-A3A axis as a novel regulatory mechanism of cccDNA longevity in cell culture infection models. This study has opened a new venue for future HBV research. It would be interesting to perform an extended study with wider sampling of CHB patients with different age and ethnic background to further justify the link between rs59391722 SNVs and HBV persistence. The co-evolution of HBV subtypes and SNVs patterns could also be assessed along with estimate of evolutionary advantage of the rs59391722 haplotypes. The UBE2L3-A3A axis can be further explored to identify additional viral/host factors that regulate cccDNA metabolism and function. One question would be whether HBV enhances the expression of UBE2L3 for its benefits. The answer might be yes because the authors showed that IFN-α downregulated UBE2L3 when HBV was inhibited by the cytokine. UBE2L3 is an E2 ubiquitin-conjugating enzyme, and even though it has been reported to have an E3-independent reactivity with lysine, 9 whether a specific E3 ubiquitin ligase, such as the UBE2L3-interacting UBE3A/E6AP, 10 is required for UBE2L3-mediated ubiquitination of A3A awaits further investigation. From the translational medicine perspective, the rs59391722(C→G) SNV may represent a risk factor for neonatal and childhood HBV infection, and the serum UBE2L3 may serve as a novel biomarker for monitoring the progression of hepatitis B and for prediction of response to IFN-α therapy. However, these potential diagnostic and prognostic values of rs59391722 and UBE2L3 should be further evaluated in larger cohort of patients. Since both cccDNA persistence and eradication largely rely on host functions, this study suggests that UBE2L3, or ideally the more-specific E3 ubiquitin ligase, if any, may be exploited as an antiviral target to promote A3A-mediated cccDNA decay and more importantly, to synergize IFN-α therapy.

Label-free and amplified electrogenerated chemiluminescence biosensing for the detection of thymine DNA glycosylase activity using DNA-functionalized gold nanoparticles triggered hybridization chain reaction

Effective detection of thymine DNA glycosylase (TDG) activity is extremely crucial and urgent for epigenetic research. Herein, a novel label-free electrogenerated chemiluminescence (ECL) biosensing method was developed for the detection of TDG activity using DNA-functionalized gold nanoparticles (DNA-AuNPs) triggered hybridization chain reaction (HCR). In this assay, the thiol modified hairpin probe DNA (hp-DNA) with 5′ overhangs and one mismatched base pair of guanines: thymine (G: T) in the stem part was boned onto gold electrode. TDG specifically removed T base of the G: T mismatch to produce apyrimidinic (AP) sites through the N-glycosidic bond hydrolysis. The AP site was then cleaved by the catalysis of Endonuclease IV (EnIV) to generate dsDNA containing a free 3’ end in the long sequence, which serves as a complementary sequence to hybridize with the specific sequence (ssDNA1) of DNA-AuNPs. Then, the functionalized DNA-AuNPs with initiator strands (ssDNA2) could trigger HCR to form nicked double helices DNA polymer which can embed numerous ECL indicator, Ru(phen)32+, resulting in significantly increased ECL signal. The proposed strategy combined the amplification function of DNA-AuNPs triggered HCR and the inherent high sensitivity of the ECL technique, a detection limit of 1.1 × 10−5 U/μL (0.0028 ng/mL) for TDG determination was obtained. In addition, this method was successfully applied to evaluate TDG activity in cancer cell, which provides great possibility for TDG activity assay in related clinical diagnostics.

Molecular Mechanism of Off-Target Effects in CRISPR-Cas9

Author(s): Ricci, Clarisse; Chen, Janice; Miao, Yinglong; Jinek, Martin; Doudna, Jennifer; McCammon, Andrew; Palermo, Giulia | Abstract: Abstract CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Cellular Target of a Rhodium Metalloinsertor is the DNA Base Pair Mismatch.

Defects in DNA mismatch repair (MMR) are commonly found in various cancers, especially in colorectal cancers. Despite the high prevalence of MMR-deficient cancers, mismatch-targeted therapeutics are limited and diagnostic tools are indirect. Here, we examine the cytotoxic properties of a rhodium metalloinsertor, [Rh(phen)(chrysi)(PPO)]2+ (RhPPO) in 27 diverse colorectal cancer cell lines. Despite the low frequency of genomic mismatches and the non-covalent nature of the RhPPO-DNA lesion, RhPPO is on average five times more potent than cisplatin. Importantly, the biological target and profile for RhPPO differs from that of cisplatin. A fluorescent metalloinsertor, RhCy3, was used to demonstrate that the cellular target of RhPPO is the DNA mismatch. RhCy3 represents a direct probe for MMR-deficiency and correlates directly with the cytotoxicity of RhPPO across different cell lines. Overall, our studies clearly indicate that RhPPO and RhCy3 are promising anticancer and diagnostic probes for MMR-deficient cancers, respectively.

Plasmoelectronic-Based Ultrasensitive Assay of Tumor Suppressor microRNAs Directly in Patient Plasma: Design of Highly Specific Early Cancer Diagnostic Technology.

It is becoming understood that microRNAs hold great promise for noninvasive liquid biopsies for screening for different types of cancer, but current state-of-the-art RT-PCR and microarray techniques have sensitivity limitations that currently restrict their use. Herein, we report a new transduction mechanism involving delocalization of photoexcited conduction electrons wave function of gold triangular nanoprism (Au TNP) in the presence of -ssDNA/microRNA duplexes. This plasmoelectronic effect increases the electronic dimension of Au TNPs and substantially affects their localized surface plasmon resonance (LSPR) properties that together allow us to achieve a sensitivity for microRNA assay as low as 140 zeptomolar concentrations for our nanoplasmonic sensors. We show that the position of a single base-pair mismatch in the -ssDNA/microRNA duplex dramatically alters the LSPR properties and detection sensitivity. The unprecedentedly high sensitivity of nanoplasmonic sensors has allowed us to assay four different microRNAs (microRNA-10b, -182, -143, and -145) from bladder cancer patient plasma (50 μL/sample). For the first time, we demonstrate the utility of a label-free, nanoplasmonic sensor in quantification of tumor suppressor microRNAs, the level of tumor suppressor microRNAs goes down in a cancer patient as compared to normal healthy individuals, in metastatic and nonmetastatic bladder cancer patient plasma. Our statistical analysis of patient samples unequivocally suggests that the tumor suppressor microRNAs are more specific biomarkers ( p-value of <0.0001) than oncogenic microRNAs for differentiation between metastatic and nonmetastatic bladder cancer, and nonmetastatic cancer from healthy individuals. This work demonstrating the electron wave functions delocalization dependent ultrasensitive LSPR properties of noble metal nanoparticles has a great potential for fabrication of miniaturized and extremely powerful sensors to investigate microRNA properties in other cancers (for example breast, lung, and pancreatic) through liquid biopsy.

Highly sensitive and selective mercury sensor based on mismatched base pairing with dioxT.

A highly selective and sensitive sensor for mercury was designed based on a new fluorescent nucleobase, dioxT. Its metal-sensing ability was investigated using mismatched dioxT-T and dioxT-C base pairing. The sensor exhibited a high sensitivity (quenching efficiency, 80%, 1 : 1 binding mode) and selectivity upon the addition of mercury ions.

Editors' Choice—Electric Field-Induced DNA Melting with Detection by Square Wave Voltammetry

Here we demonstrate a purely electrochemical approach for monitoring the electric field-induced melting of surface-bound DNA duplexes tethered to gold surfaces using standard surface attachment chemistry, i.e. single thiol-Au bonds. The sensitivity of square wave voltammetry is combined with the electric fields generated during programmed sequences of chronoamperometric pulses to enable a method for DNA analysis that can be carried out at room temperature without need for parallel spectroscopic monitoring. Electrochemical melting curves are obtained using both scanning potential pulses and constant potential pulses, which are analyzed to assess duplex stability and the extent of thiol desorption. The melting behavior is found to depend on the pulse potential and pule time. Under optimized conditions, thiol reduction is minimized and DNA duplexes can be discriminated based on the presence of a single base pair mismatch. The method is found to be less sensitive to the duplex length, presumably due to the rapid decay of the electric field away from the electrode surface. Based on these results, a simple model is proposed in which maintenance of the double-layer charge by accumulation of ions from the bulk of solution competes with electric field-induced loss of the negatively-charged DNA at a given applied potential.

DNA Base Pair Mismatches Induce Structural Changes and Alter the Free-Energy Landscape of Base Flip

Double-stranded DNA may contain mismatched base pairs beyond the Watson-Crick pairs, guanine-cytosine and adenine-thymine. Such mismatches bear adverse consequences for human health. We utilize molecular dynamics and metadynamics computer simulations to study the structure and dynamics of both matched and mismatched base pairs. We discover significant differences between the matched and mismatched pairs in structure and base flip work profiles. Mismatched pairs shift more in the plane normal to the DNA strand and exhibit more noncanonical structures, including the e-motif. We discuss the potential implications on the mismatch repair enzymes' detection of DNA mismatches.

Rapid and sensitive detection of the IS6110 gene sequences of Mycobacterium tuberculosis based on hybridization chain reaction and reusable magnetic particles

Mycobacterium tuberculosis (MTB) is the infectious pathogenic bacterium of tuberculosis, which is one of the major public health problems in the world. Until now, there remains a great challenge in the rapid, cost-saving and sensitive detection of MTB due to the strong infectivity and slow proliferation of MTB. In this manuscript, a cost-saving, enzyme-free and sensitive detection platform for the MTB IS6110 gene sequences (MTB DNA) was demonstrated by combining the superior separation ability of magnetic beads with the excellent signal amplification of hybridization chain reaction (HCR). The hairpin structure of H1 is destroyed by the hybridization of MTB DNA and H1 coupled with magnetic beads (MBs-H1) in present of MTB DNA, and HCR reaction is trigged. During this process, numerous TAMRA molecules are attached on the formed double-helix structure, and the fluorescence of the solution decreased after magnetic separation. Under the optimal conditions, the new method shows good fluorescence responses to MTB DNA with a lowest detectable concentration down to 10 pM. Importantly, the methodology can be further applied on the discrimination of DNA with single base-pair mismatches, which has great potential in single nucleotide polymorphism (SNP) analysis.

Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

BackgroundA wide variety of DNA lesions interfere with replication and transcription, leading to mutations and cell death. DNA repair mechanisms act upon these DNA lesions present in the genomic DNA. To investigate a DNA repair mechanism elaborately, an in vitro DNA repair substrate containing DNA lesions at a specific site is required. Previously, to prepare the substrate, phagemid ssDNA and DNA lesion-harboring oligonucleotides were employed with considerable amounts of DNA polymerase and DNA ligase. However, preparing in vitro DNA repair substrate in general is difficult and labor intensive.ResultsHere, we modified the construction method of in vitro mismatch repair substrate using a nicking-endonuclease, which produces gap corresponding to the ssDNA in the plasmid DNA, and swaps DNA lesion-containing oligonucleotide upon addition of restriction enzyme and T5 exonuclease. This modified method is able to produce in vitro DNA repair substrates containing adenine:cytosine mismatch basepair, 8-oxoG, and uracil. The DNA repair enzyme, each Fpg, hOGG1 could cleave an 8-oxoG-containing DNA substrate, the mixture of UDG and APE1 could cleave a uracil-containing DNA substrate. Omitting a column purification step, DNA repair substrates were prepared by one-pot synthesis.ConclusionsWe were able to prepare in vitro DNA repair substrates using this simple method involving restriction enzymes and T5 exonuclease. It is anticipated that this method, termed as “Oligo Swapping Method”, will be valuable for understanding the DNA repair machinery.

338 - Mitochondrial DNA damage in diabetic retinopathy: role of epigenetics

Purpose Diabetes dysfunctions retinal mitochondria and increases base pair mismatches in mitochondrial DNA (mtDNA), and damaged mtDNA impairs the electron transport chain system. A mismatch repair system actively repairs the uncomplimentary base pairs, but in diabetes, the enzyme responsible for cutting the base-mismatches, MutL homolog 1 (Mlh1), is also compromised, which further adds to the damage of mtDNA. In diabetes, retinal DNA methylation machinery is also altered, and the activity of DNA methyl transferases (Dnmts) is significantly increased. Since Mlh1 promoter has over 50 CpG sites, making it a good target for DNA methylation, our aim was to investigate the role of DNA methylation in mtDNA damage in the development of diabetic retinopathy. Methods Retinal microvessels from streptozotocin-induced diabetic mice, with or without Dnmt inhibitor 5-azacytidine (Aza, 2.5mg/kg, intraperitoneal), were analyzed for methylated cytosine (5mC) levels using immuno-capture method and Mlh1 promoter- specific primers. Base-mismatch was quantified by digesting mtDNA amplicons using mismatch specific surveyor endonuclease, followed by DNA fragmentation analysis. Similar experiments were performed in retinal microvessels prepared from human donors with documented diabetic retinopathy. Results Compared to normal mice, retinal microvessels from diabetic mice had decreased Mlh1 expression and increased Dnmt1 and 5mC levels at Mlh1 promoter. In the same mice, base-mismatches in their mtDNA were also increased. Administration of Aza ameliorated diabetes-induced decrease in Mlh1 expression, and prevented increase in Dnmt1 and DNA methylation of Mlh1 promoter. Similarly, compared to age-matched non diabetic human donors, retinal microvessels from donors with established diabetic retinopathy had decreased levels of Mlh1 and increased levels of Dnmt1 and 5mC at Mlh1 promoter. Conclusions Thus, DNA methylation of Mlh1 promoter has a major role in impaired mtDNA damage. Therapies targeted to halt DNA methylation could have potential to prevent/halt mtDNA damage, and the development of diabetic retinopathy.

A Novel Tautomeric Polymorph of Anhydrous Guanine and Its Reversible Water Harvesting Property

Tautomeric isomerism is very important in DNA/RNA research, especially base-pair mismatches. However, the tautomeric polymorph of anhydrous guanine (AG) composed of a N7-G purine ring has never been reported in the literature. Herein, a novel crystal phase of anhydrous guanine consisting of a N9-G purine ring was obtained by dehydration of guanine monohydrate (GM) at 111 °C, termed dehydrated-GM, the first reported tautomeric polymorph of AG (N7-G). The dehydration/hydration transformation between GM and dehydrated-GM can be repeated more than 10 times without any observable phase damage. The dehydrated-GM can harvest 8 wt % water within 30 min at relative low humidity (below 20%). Furthermore, dehydrated-GM has long-term stability in a very dry environment when the temperature is in between RT and 150 °C. The channel structure of dehydrated-GM can enlighten the design of materials to harvest water from atmospheric air. The tautomeric polymorphism of N7-G and N9-G purine rings provides a new insight in nu...

Platinum(II) Complexes with Sterically Expansive Tetraarylethylene Ligands as Probes for Mismatched DNA

Deficiencies in DNA mismatch repair (MMR) machinery result in greater incidence of DNA base pair mismatches in many types of cancer cells relative to normal cells. Consequently, luminescent probes capable of signaling the presence of mismatched DNA hold promise as potential cancer diagnostic and therapeutic tools. In this study, a series of cyclometalated platinum(II) complexes with sterically expansive tetraarylethylene ligands were synthesized and examined for selective detection of mismatched DNA. Increased steric bulk of the tetraarylethylene ligands in these complexes was observed to correlate with greater preferential luminescence enhancement in the presence of hairpin DNA oligonucleotides containing a mismatched site compared to well-matched oligonucleotides, with the most effective complex displaying ∼14-fold higher emission upon binding CC mismatched oligonucleotides compared to well-matched oligonucleotides. The results indicate binding to mismatched sites in DNA oligonucleotides occurs through metalloinsertion, and the luminescence response increases as a function of thermodynamic destabilization of the mismatch. Luminescence quenching experiments with Cu(phen)22+ and NaI further indicate mismatch binding from the minor groove, consistent with metalloinsertion. Binding to CC mismatched oligonucleotides was also investigated by isothermal titration calorimetry and UV-melting studies. These results demonstrate the efficacy of tetraarylethylene-based platinum(II) complexes for detection of mismatched DNA and establish a new molecular platform for development of organometallic DNA binding agents.

Fabrication of a novel DNA affinity biosensor based on hybridisation induced current by electrostatic repulsion of silicotungstic acid as a redox indicator

We report on a novel DNA affinity biosensor which utilises the capture of a neutral charged single stranded (ss) morpholino DNA on a gold electrode to trigger an electrostatic repulsion of negatively charged silicotungstate anions and, in turn, enabled detection of the hybridisation of complementary base pairs. The repulsion of the anions, as a redox indicator, is reflected by a decrease in its electrochemical response with increasing target ss-DNA concentration. A theoretical framework for DNA detection by the affinity biosensor is proposed and verified by electrochemical measurements in the presence of the target ss-DNA by either dc cyclic voltammetry or Fourier transformed alternating current voltammetry (FTACV). The optimised conditions for the capture of the target ss-DNA and the electrochemical detection include 1 μM thiolated neutral morpholino oligo-nucleotide probe, hybridisation time of 10 min, 0.25 mM [α-SiW12O40]4-, and 25 mM phosphate buffer. In addition, the use of the 5th harmonic component of the FTACV gave the most sensitive response for the detection of the target ss-DNA. Under these conditions, the DNA affinity biosensor, based on FTACV detection, achieved a minimum detectable concentration of 0.1 pM ss-DNA and a linear concentration range of 0.1–1000 pM. The biosensor also successfully distinguished between some matched and mismatched base pairs.