Depletion of the RNA binding protein HNRNPD impairs homologous recombination by inhibiting DNA-end resection and inducing R-loop accumulation.

DNA double strand break (DSB) repair through homologous recombination (HR) is crucial to maintain genome stability. DSB resection generates a single strand DNA intermediate, which is crucial for the HR process. We used a synthetic DNA structure, mimicking a resection intermediate, as a bait to identify proteins involved in this process. Among these, LC/MS analysis identified the RNA binding protein, HNRNPD. We found that HNRNPD binds chromatin, although this binding occurred independently of DNA damage. However, upon damage, HNRNPD re-localized to γH2Ax foci and its silencing impaired CHK1 S345 phosphorylation and the DNA end resection process. Indeed, HNRNPD silencing reduced: the ssDNA fraction upon camptothecin treatment; AsiSI-induced DSB resection; and RPA32 S4/8 phosphorylation. CRISPR/Cas9-mediated HNRNPD knockout impaired in vitro DNA resection and sensitized cells to camptothecin and olaparib treatment. We found that HNRNPD interacts with the heterogeneous nuclear ribonucleoprotein SAF-A previously associated with DNA damage repair. HNRNPD depletion resulted in an increased amount of RNA:DNA hybrids upon DNA damage. Both the expression of RNase H1 and RNA pol II inhibition recovered the ability to phosphorylate RPA32 S4/8 in HNRNPD knockout cells upon DNA damage, suggesting that RNA:DNA hybrid resolution likely rescues the defective DNA damage response of HNRNPD-depleted cells.

Molecular Glue for RNA: Regulating RNA Structure and Function through Synthetic RNA Binding Molecules.

We introduce the concept of molecular glues for RNA, in which specific RNA-binding small molecules induce designed structural changes in target functional RNAs, resulting in modulation of the functions. (Z)-NCTS is an RNA-mismatch-binding small molecule that recognizes 5'-r(XGG)-3'/5'-r(XGG)-3' sequences (X=U or A) and acts as a molecular glue for RNA. The binding of (Z)-NCTS brings two distinct 5'-r(XGG)-3' domains into contact with each other, and this can result in higher-order structural changes of target RNAs. We applied (Z)-NCTS to induce the formation of a proposed tertiary structure of a ribozyme together with activation of RNA-cleaving ability. The concept of a molecular glue could inspire new small-molecule-based strategies for regulating biological functions: a synthetic small molecule targeting functional RNAs could regulate the RNA structure and function.

An aptamer based fluorometric microcystin-LR assay using DNA strand-based competitive displacement.

The high-affinity region of a truncated aptamer was applied to the development of a sensitive method for the determination of microcystin-LR (MC-LR) using competitive displacement and molecular beacons. In this assay, the fluorophore and quencher labelled complementary sequences of the aptamer are hybridized with the truncated aptamer to form a fluorophore-quencher pair. In the presence of MC-LR, the aptamer duplex dissociates, and the fluorophore-quencher pair is separated. This turn leads to an increase in the yellow fluorescence which is best measured at excitation/emission wavelengths of 555/580 nm. One of the truncated aptamers showed a 50-fold increase in the affinity (0.93 nM) compared to the wild type aptamer (50 nM). The truncated sequence shows considerable cross-reactivity with L congeners but none with other congeners. The assay works in 0.5 to 200 nM MC-LR concentration range. It was applied to spiked tap water samples and gave recoveries around 95 ± 5%. Graphical abstract Schematic representation of a method for determination of microcystin-LR via fluorescence that is induced by competitive displacement of complementary DNA strands in a truncated dsDNA aptamer.

Unzipping and shearing DNA with electrophoresed nanoparticles in hydrogels.

We show electric control of unzipping and shearing dehybridization of a DNA duplex anchored to a hydrogel. Tensile force is applied by electrophoresing (25 V cm-1) gold nanoparticles pulling the DNA duplex. The pulled DNA strand is gradually released from the hydrogel. The unzipping release rate is faster than shearing; for example, 3-fold for a 15 base pair duplex, which helps to design electrically driven DNA devices.

A novel electrochemical biosensor for selective determination of mercury ions based on DNA hybridization.

An electrochemical biosensor was developed for Hg(2+) determination based on DNA hybridization. In the presence of Hg(2+), the target and probe DNAs with thymine-thymine (T-T) mismatches could hybridize by forming T-Hg(2+)-T complex. This induced DNA hybridization led to the decrease in reduction peak currents of ethyl green (EG) as electroactive label, which could be used for determination of Hg(2+). The difference in the value of the peak currents of EG before and after DNA hybridization (ΔI) was linear with the concentration of Hg(2+) in the range of 9.0 × 10(-11)-1.0 × 10(-9) M. The detection limit was 3.08 × 10(-11) M.

Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation.

The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg(2+), the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg(2+), which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way.

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences.

Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and with genomic instability. An important example is sequences containing G-rich homopurine-homopyrimidine stretches, for which unusual transcriptional behavior is implicated in regulation of immunogenesis and in other processes such as genomic translocations and telomere function. To elucidate the mechanism of the effect of these sequences on transcription we have studied T7 RNA polymerase transcription of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, length- and supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution conditions, and ribonucleotide substitutions, we conclude that transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation. These structures are likely responsible for transcription-dependent replication blockage by G-rich sequences in vivo.

Efficient stabilization of phosphodiester (PO), phosphorothioate (PS), and 2'-O-methoxy (2'-OMe) DNA·RNA hybrid duplexes by amino sugars.

Antisense strategies that target DNA·RNA hybrid structures offer potential for the development of new therapeutic drugs. The α-sarcin loop region of the 23S [corrected] rRNA domain has been shown to be a high value target for such strategies. Herein, aminoglycoside interaction with three RNA·DNA α-sarcin targeted duplexes (rR·dY, rR·S-dY, and rR·2'OMe-rY) have been investigated to determine the overall effect of aminoglycoside interaction on the stability, affinity, and conformation of these hybrid duplexes. To this end, UV thermal denaturation, circular dichroism spectroscopy, fluorescence intercalator displacement, and ITC as well as DSC calorimetry experiments were carried out. The results suggest the following. (1) Of all the aminoglycosides studied, neomycin confers the highest thermal stability on all three hybrid duplexes studied. (2) There is no appreciable difference in aminoglycoside-induced thermal stability between the unmodified rR·dY and phophorothioate modified rR·S-dY duplexes. (3) The rR·2'OMe-rY duplexes thermal stability is slightly less than the other two hybrids. (4) In all three duplexes, aminoglycoside-induced thermal stability decreased as the number of amino groups decreased. (5) CD scans revealed similar spectra for the rR·dY and rR·S-dY duplexes as well as a more pronounced A-form signal for the rR·2'OMe-rY duplex. (6) FID assays paralleled the CD results, yielding similar affinity values between the rR·dY and rR·S-dY duplexes and higher affinities with the rR·2'OMe-rY duplex. (7) The overall affinity trend between aminoglycosides and the three duplexes was determined to be neomycin > paromomycin > neamine > ribostamycin. (8) ITC K(a) values revealed similar binding constants for the rR·dY and rR·S-dY duplexes with rR·dY having a K(1) of (1.03 ± 0.58) × 10(7) M(-1) and K(2) of (1.13 ± 0.07) × 10(5) M(-1) while rR·S-dY produced a K(1) of (1.17 ± 0.54) × 10(7) M(-1) and K(2) of (1.27 ± 0.69) × 10(5) M(-1). (8) The rR·2'OMe-rY produced a slightly higher binding constant values with a K(1) of (1.25 ± 0.24) × 10(7) M(-1) and K(2) of (3.62 ± 0.18) × 10(5) M(-1). (9) The ΔT(m)-derived K(Tm) of 3.81 × 10(7) M(-1) for rR·S-dY was in relative agreement with the corresponding K(1) of 1.17 × 10(7) M(-1) derived constant from the fitted ITC. These results illustrate that the increased DNA·RNA hybrid duplex stability in the presence of aminoglycosides can help extend the roles of aminoglycosides in designing modified ODNs for targeting RNA.

Control of DNA hybridization by photoswitchable molecular glue.

Hybridization of DNA is one of the most intriguing events in molecular recognition and is essential for living matter to inherit life beyond generations. In addition to the function of DNA as genetic material, DNA hybridization is a key to control the function of DNA-based materials in nanoscience. Since the hybridization of two single stranded DNAs is a thermodynamically favorable process, dissociation of the once formed DNA duplex is normally unattainable under isothermal conditions. As the progress of DNA-based nanoscience, methodology to control the DNA hybridization process has become increasingly important. Besides many reports using the chemically modified DNA for the regulation of hybridization, we focused our attention on the use of a small ligand as the molecular glue for the DNA. In 2001, we reported the first designed molecule that strongly and specifically bound to the mismatched base pairs in double stranded DNA. Further studies on the mismatch binding molecules provided us a key discovery of a novel mode of the binding of a mismatch binding ligand that induced the base flipping. With these findings we proposed the concept of molecular glue for DNA for the unidirectional control of DNA hybridization and, eventually photoswitchable molecular glue for DNA, which enabled the bidirectional control of hybridization under photoirradiation. In this tutorial review, we describe in detail how we integrated the mismatch binding ligand into photoswitchable molecular glue for DNA, and the application and perspective in DNA-based nanoscience.

Sequence dependence of Pd(II)-mediated base pairing by palladacyclic nucleobase surrogates.

A C-nucleoside derivative of phenylpyridine or the respective palladacycle was incorporated at either 3'- or 5'-terminus of a short oligodeoxynucleotide. Hybridization properties of these modified oligonucleotides were studied in a fluorescence-based competition assay in addition to conventional UV melting temperature analysis and compared with those of a previously prepared analogue featuring the modified nucleoside in the middle of the sequence. With the unpalladated phenylpyridine oligonucleotides, UV melting temperature qualitatively correlated with the ability to displace a strand from a double helix in the competition assay, decreasing in the order 5' > 3' > middle. Corresponding results on the palladacyclic oligonucleotides were more difficult to interpret but both UV melting and competition experiments revealed a decrease in the duplex stability upon palladation in most cases. On the other hand, dependence of the UV melting temperature on the identity of the canonical nucleobase opposite to the modified nucleobase analogue was much more pronounced with the palladacyclic duplexes than with their unpalladated counterparts. Furthermore, UV melting profiles of the palladacyclic duplexes featured an additional transition at a temperature exceeding the melting temperature of the unmodified part of the duplex. Taken together, these results lend support to the idea of Pd(II)-mediated base pairs that are highly stable but incompatible with the geometry of a double helix.

Low-Noise Solid-State Nanopore Enhancing Direct Label-Free Analysis for Small Dimensional Assemblies Induced by Specific Molecular Binding.

Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.

Salt concentration effects on equilibrium melting curves from DNA microarrays.

DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions.

High-throughput microarray technology in diagnostics of enterobacteria based on genome-wide probe selection and regression analysis.

BACKGROUND: The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. RESULTS: We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. CONCLUSIONS: Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.

Cytogenetic characterization and gene expression profiling of the trastuzumab-resistant breast cancer cell line JIMT-1.

Resistance to the HER-2 targeting drug trastuzumab can be observed clinically, but the lack of suitable experimental models hampers studies of resistance mechanisms. We characterized a HER-2-positive carcinoma cell line (JIMT-1) derived from a 62-year-old breast cancer patient which was clinically resistant to trastuzumab. Multicolor fluorescence in situ hybridization revealed a complex hyperdiploid karyotype with numerous marker chromosomes and unbalanced translocations. Comparative genomic hybridization (CGH) revealed numerous regions of copy number aberration (CNA). Further analysis by array CGH identified 27 regions of CNA (16 amplified, 11 deleted). Thirty-eight percent of the genes in the amplified regions were overexpressed, compared to only 9% in regions of normal copy number ratios (CNR). Accordingly, 26% of the genes in the deleted regions were underexpressed, compared to 10% in regions of normal CNR. Most amplified and overexpressed genes were located on chromosome 1 as well as on 8q, 12q14.1, 17q11 approximately q21, and 20q13. In 17q11 approximately q21, we identified two separate amplicons, the HER-2 amplicon and a previously unreported amplicon at 17q21.31. Several aberrant genes are implicated in cancer development (e.g., JUN, CDK4, and SLUG protooncogenes, as well as the drug/hormone-metabolizing genes GSTM1 and CYP24). We conclude that cytogenetic and expression profiling of JIMT-1 revealed several new features that need further characterization and may shed light on trastuzumab resistance.

Integrated in silico and biological validation of the blocking effect of Cot-1 DNA on Microarray-CGH.

In array-CGH, various factors may act as variables influencing the result of experiments. Among them, Cot-1 DNA, which has been used as a repetitive sequence-blocking agent, may become an artifact-inducing factor in BAC array-CGH. To identify the effect of Cot-1 DNA on Microarray-CGH experiments, Cot-1 DNA was labeled directly and Microarray-CGH experiments were performed. The results confirmed that probes which hybridized more completely with Cot-1 DNA had a higher sequence similarity to the Alu element. Further, in the sex-mismatched Microarray-CGH experiments, the variation and intensity in the fluorescent signal were reduced in the high intensity probe group in which probes were better hybridized with Cot-1 DNA. Otherwise, those of the low intensity probe group showed no alterations regardless of Cot-1 DNA. These results confirmed by in silico methods that Cot-1 DNA could block repetitive sequences in gDNA and probes. In addition, it was confirmed biologically that the blocking effect of Cot-1 DNA could be presented via its repetitive sequences, especially Alu elements. Thus, in contrast to BAC-array CGH, the use of Cot-1 DNA is advantageous in controlling experimental variation in Microarray-CGH.

Development of the novel drug releasing system triggered by hybridization with target sequence.

We have already established the strategy of synchronous activation by hybridization, in which the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, can be generated from its stable precursors, the phenylsulfide derivatives, by a hybridization-promoted activation process with selectivity to cytosine. In this study, this in situ activation system was applied to the method for the drug releasing system triggered by hybridization with the target sequence.

Surface behavior and molecular recognition in DNA microarrays from N,N-dimethylacrylamide terpolymers with activated esters as linking groups.

A series of terpolymers made of DMA, NAS and MAPS were synthesized by free radical copolymerization and used as functional coatings for the fabrication of glass slide DNA microarrays. The surface properties of coated glass slides were investigated through contact angle measurements, ellipsometry and atomic force microscopy. The terpolymer molecular weight showed a moderate effect on surface tension (gamma(s) = 56-62 mN x m(-1)), but no clear effect on polymeric layer thickness (5-8 nm) and roughness. Hybridization experiments with amine-functionalized oligonucleotides gave the best fluorescence intensity results for microarrays coated with intermediate-molecular-weight terpolymers. Finally, an accelerated ageing test of the microarray in a humidity chamber showed a nice relationship between decay curves of contact angle against water and fluorescence intensity.

Minimising the secondary structure of DNA targets by incorporation of a modified deoxynucleoside: implications for nucleic acid analysis by hybridisation.

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. The secondary conformation of the target may be destabilised to assist its interaction with oligonucleotide probes. To achieve this, we modified a DNA target, which has self-complementary sequence able to form a hairpin loop, by replacing dC with N:4-ethyldeoxycytidine (d(4Et)C), which hybridises specifically with natural dG to give a G:(4Et)C base pair with reduced stability compared to the natural G:C base pair. Substitution by d(4Et)C greatly reduced formation of the target secondary structure. The lower level of secondary structure allowed hybridisation with complementary probes made with natural bases. We confirmed that hybridisation could be further enhanced by modifying the probes with intercalating groups which stabilise the duplex.

Effects of hydroxyurea on murine type C virus-specific DNA synthesis in newly infected cells.

Cell transformation and replication of the Rauscher pseudotype of Moloney murine sarcoma virus in mouse embryo fibroblasts were inhibited by hydroxyurea within a critical time period of 30 to 90 min postinfection. In cells infected by Rauscher leukemia virus, treatment with 1mM hydroxyurea during the critical time period resulted in the accumulation of minus-strand DNA (molecular weight, 3 x 10(6)) in association with the parental viral genoma RNA. This 5 to 6 x 10(6) dalton RNA:DNA hybrid was found in the cytoplasm. Positive-strand DNA of genomic or smaller size was not detected in the presence of hydroxyurea, but virus-specific DNA was found in the nucleus 30 min after removal of drug.