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.

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.

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.

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.

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.

A proposed mechanism of the influence of gold nanoparticles on DNA hybridization.

A combination of gold nanoparticles (AuNPs) and nucleic acids has been used in biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Here, we measured the rate constants of the hybridization and dehybridization of DNA on gold nanoparticle surfaces to enable the determination of activation parameters using transition state theory. We show that the target bases need to be detached from the gold nanoparticle surfaces before zipping. This causes a shift of the rate-limiting step of hybridization to the mismatch-sensitive zipping step. Furthermore, our results propose that the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the duplex DNA stabilizes the bubbles and accelerates the dehybridization process. We employ the proposed mechanism of DNA hybridization/dehybridization to explain the ability of 5 nm diameter gold nanoparticles to help discriminate between single base-pair mismatched DNA molecules when performed in a NanoBioArray chip. The mechanistic insight into the DNA-gold nanoparticle hybridization/dehybridization process should lead to the development of new biosensors.

Identification of pathogenetically relevant genes in lymphomagenesis by shRNA library screens.

RNA interference (RNAi) is a conserved posttranscriptional gene silencing mechanism that has recently emerged as a breakthrough genetic tool in functional genomics and drug target discovery. An increasing number of studies applying RNAi in high-throughput screens have begun to unravel complex signaling networks underlying diverse cellular processes. This chapter describes an approach to construct a conditional small-hairpin (sh)RNA library and its application in human lymphoma cell lines. A library cloning procedure outlines the incorporation of shRNA sequences and random 60-mer "bar code" oligonucleotides, enabling rapid identification of the hairpin by microarrays. Lymphoma cell lines are optimized for efficient retroviral transduction and tetracycline inducibility. The shRNA library is suitable for identifying molecular targets in cancer, but also versatile for various screening strategies.

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.

Nucleic acid-mediated cleavage of M1 gene of influenza A virus is significantly augmented by antisense molecules targeted to hybridize close to the cleavage site.

Influenza A virus genome segment 7 encodes protein M1, which is the matrix protein playing crucial role in the virus life cycle. Any antiviral strategy that aims at reducing, in particular, the expression of this genome segment should, in principle, reduce the infectivity of the virus. We developed a specific antiviral approach at the molecular level and designed several novel 10-23 DNAzymes (Dz) and hammerhead ribozymes (Rz), specifically targeted to cleave at the conserved domains of the influenza virus M1 RNA. We sought to use antisense molecules with the hope that it will facilitate the ribozyme-mediated cleavage. We observed that the Mg(2+)-dependent sequence-specific cleavage of M1 RNA was achieved by both the Dz and Rz in a dose-dependent manner. This combination of catalytic Dz and Rz with antisense molecules, in principle, resulted in more effective gene suppression, inhibited the whole virus replication in host cell, and thus could be exploited for therapeutic purposes.

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.

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.

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.

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.

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.

Formation of stable BOBO-3 H-aggregate complexes hinders DNA hybridization.

In recent works, we have been studying the photophysics and binding properties of the trimethine cyanine homodimer dye BOBO-3, a DNA intercalative fluorophore that shows an important fluorescence enhancement upon binding to double-stranded DNA. During the course of studying the interactions of the dye with single-stranded homo-oligonucleotides we detected the apparition of an additional absorption band centered on 466 nm. The large hypsochromic effect and the fact that direct excitation of this band resulted in negligible fluorescence emission are characteristic properties of an H-type molecular aggregate. In this work we study the properties of this H-aggregate, and obtain by means of Principal Component Analysis the spectral shape and association constant of the complex. The H-aggregate complex shows very unique features. On one hand, the nucleotide bases cytosine or adenine are crucially involved in the formation of the aggregate. We describe here that at least six consecutive cytidine nucleosides are required to properly form the BOBO-3 H-aggregate complex. On the other hand, we demonstrate that the formation of such a stable complex prevents hybridization of the bases involved with their complementary strands. This phenomenon draws important conclusions on the anomalously high stability of the BOBO-3 H-aggregate complex. To the best of our knowledge, this is the first time such a stable H-aggregate of a dimeric cyanine dye facilitated by specific nucleotide bases in single strands has been reported.

Rapid monitoring of mRNA levels with a molecular beacon during microbial fermentation.

In the microbial fermentation bioreactor, the processes of mRNA transcription, protein translation, and enzyme-catalyzed biosynthesis remain as "black boxes" of industrial monitoring and process control. Monitoring the kinetics of these "black boxes" is very helpful for optimizing and controlling the microbial fermentation process. This study first applied a molecular beacon (MB) to monitor the changes in the mRNA level of the phzC gene during antibiotic phenazine-1-carboxylic acid fermentation. Seven typical MB hybridization buffers were compared, and the effect of formamide on MBs was also studied. The results showed that rapid monitoring of the mRNA level using MBs was feasible. The optimal hybridization buffer for phzC MB was 100 mM Tris, 1 mM MgCl(2), pH 8.0. The optimal hybridization temperature was 35 degrees C, and formamide proved unsuitable for MB hybridization. The limit of detection of phzC MB was 1.67 nM and MB hybridization was complete by 7 min. Given that the time for RNA extraction is 12 min, it is possible that monitoring of phzC mRNA can be completed in less than 20 min. Since production of most amine acids, organic acids, wines, antibiotics, and proteins relies on microbial fermentation, our method may have some potential for application in these other microbial industries.

Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes.

A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV-vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 x 10(-13) to 4.5 x 10(-6)mol/L. The detection limit (3 sigma) of this method has decreased to pico-molar level.