An Efficient Bead‐captured Denaturation Method for Preparing Long Single‐stranded DNA

Previous methods to prepare single‐stranded DNA (ssDNA ) substrates are limited to short DNA lengths and inefficient. We have developed an efficient and rapid method to prepare long ssDNA substrates (up to 4000 nt) based on the denaturation of the bead‐captured DNA substrates, with the individual steps optimized. Immobilization of the targeted DNA substrates on the antibody‐modified beads allows easy separation of the denatured targeted ssDNA strand. This method also allows the recovery of the captured strand, making it possible to obtain two ssDNA strands from the same duplex DNA . Within 20 min, 80 nM of the 200 nt ssDNA strand could be obtained from its duplex DNA.     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

Sequence‐based separation of single‐stranded DNA at high salt concentrations in capillary zone electrophoresis

DNA separation by fragment length can be readily achieved using sieving gels in electrophoresis. Separation by sequence has not been as simple, generally requiring adequate differences in native or induced conformation between single or hybridized strands or differences in thermal or chemical stability of hybridized strands. Previously, it was shown that four single‐stranded DNA (ssDNA) 76‐mers that differ by only a few A‐G substitutions could be separated based solely on sequence by adding guanosine‐5’‐monophosphate to the running buffer in capillary zone electrophoresis (CZE). The separation was attributed to interactions of the ssDNA with self‐assembled guanine‐tetrad structures; however, subsequent studies of an expanded set of ten 76‐mers showed that the separation was a more general phenomenon that occurred at high salt concentrations. With the long‐term goal of using experimental and computational methods to provide insight into the basis of the separation, a set of ssDNA 15‐mers was designed including a poly(dT) 15‐mer and nine variants. Separations were performed using fluorescent‐labeled ssDNA in CZE with laser‐induced fluorescence detection. Results show that separation improves with increasing buffer concentration and decreasing temperature, due at least in part to longer separation times. Migration times increase with increasing purine content, with A having a much larger effect that G. Circular dichroism spectra of the mixtures of the strands suggest that the separation is not due to changes in conformation of the ssDNA at high salt concentrations.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

High‐Mass MALDI‐MS Analysis for the Investigation of Protein Encapsulation within an Engineered Capsid Forming Protein

Chemical cross‐linking combined with MALDI ‐MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (AaLS ‐13) was investigated that self‐assembles into a capsid‐like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI ‐MS and native ESI ‐MS , were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo‐electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross‐linking with glutaraldehyde followed by MALDI ‐MS . The same approach was used to study protein‐protein interactions during encapsulation of GFP (+36) by the AaLS ‐13 capsid. Heterocomplexes between GFP (+36) and AaLS ‐13 multimers were not observed when mixed at maximal loading capacity (AalS‐13 monomer:GFP (+36) 4:1). This is in agreement with the known fast encapsulation of GFP (+36) by the protein capsid, which essentially removes any free GFP (+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP (+36) results in aggregation.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Electrospray‐Ionization Mass Spectrometry for Screening the Specificity and Stability of Single‐Stranded‐DNA Templated Self‐Assemblies

Supramolecular complexes consisting of a single‐stranded oligothymine ( dTn ) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen‐bonding unit have been studied with electrospray‐ionization mass spectrometry (ESI‐MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn . By changing the hydrogen‐bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single‐stranded‐DNA (ssDNA)–guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20‐component self‐assembled objects. Gas‐phase breakdown experiments on single‐ and multiple‐guest–DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI‐MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.     

Efficient Preparation of Large‐Sized Rings of Single‐Stranded DNA through One‐Pot Ligation of Multiple Fragments

Circular single‐stranded DNA (c‐ssDNA) has significant applications in DNA detection, the development of nucleic acid medicine, and DNA nanotechnology because it shows highly unique features in mobility, dynamics, and topology. However, in most cases, the efficiency of c‐ssDNA preparation is very low because polymeric byproducts are easily formed due to intermolecular reaction. Herein, we report a one‐pot ligation method to efficiently prepare large c‐ssDNA. By ligating several short fragments of linear single‐stranded DNA (l‐ssDNA) in one‐pot by using T4 DNA ligase, longer l‐ssDNAs intermediates are formed and then rapidly consumed by the cyclization. Since the intramolecular cyclization reaction is much faster than intermolecular polymerization, the formation of polymeric products is suppressed and the dominance of intramolecular cyclization is promoted. With this simple approach, large‐sized single‐stranded c‐ssDNAs (e.g., 200‐nt) were successfully synthesized in high selectivity and yield.     

Voltammetric Studies on the Recognition of a Copper Complex to Single‐ and Double‐Stranded DNA and Its Application in Gene Biosensor

A mixed‐ligands copper complex [Cu(phendione)(DAP)]SO₄ (phendione=1,10‐phenanthroline‐5,6‐dione, DAP=2,3‐diaminophenazine) was synthesized. Cyclic voltammetry showed that the complex underwent an obvious decrease of redox peak currents and positive shift of formal potential after interaction with double‐stranded DNA (dsDNA), suggesting that the copper complex behaved as a typical metallointercalator for dsDNA, The recognition properties of the copper complex to single‐stranded DNA (ssDNA) and dsDNA were assessed using surface‐based electrochemical methods and the results suggested that the complex had obviously different redox signals at ssDNA and dsDNA modified electrodes. The copper complex was further used as an electroactive indicator for the detection of cauliflower mosaic virus (CaMV) 35S promoter gene.     

Adsorptive Stripping Voltammetric Detection of Single‐Stranded DNA at Electrochemically Modified Glassy Carbon Electrode

Electrochemically modified glassy carbon electrode (GCE) was used to study the electrochemical oxidation and detection of denatured single‐stranded (ss) DNA by means of adsorptive stripping voltammetry. The modification of GCE, by electrochemical oxidation at +1.75 V (vs.SCE) for 10 min and cyclic sweep between +0.3 V and ?1.3 V for 20 cycles in pH 5.0 phosphate buffer, results in 100‐fold improvement in sensitivity for ssDNA detection. We speculated that the modified GCE has a high affinity to single‐stranded DNA through hydrogen bond (specific static adsorption). Single‐stranded DNA can accumulate at the GCE surface at open circuit and produce a well‐defined oxidation peak corresponding to the guanine residues at about +0.80 V in pH 5.0 phosphate buffer, while the native DNA gives no signal under the same condition. The peak currents are proportional to the ssDNA concentration in the range of 0–18.0 μg mL^?1. The detection limit of denatured ssDNA is ca. 0.2 μg mL^?1 when the accumulation time is 8 min at open circuit. The accumulation mechanism of ssDNA on the modified GCE was discussed.     

Fragmentation behavior of a thiourea‐based reagent for protein structure analysis by collision‐induced dissociative chemical cross‐linking

The fragmentation behavior of a novel thiourea‐based cross‐linker molecule specifically designed for collision‐induced dissociation (CID) MS/MS experiments is described. The development of this cross‐linker is part of our ongoing efforts to synthesize novel reagents, which create either characteristic fragment ions or indicative constant neutral losses (CNLs) during tandem mass spectrometry allowing a selective and sensitive analysis of cross‐linked products. The new derivatizing reagent for chemical cross‐linking solely contains a thiourea moiety that is flanked by two amine‐reactive N‐hydroxy succinimide (NHS) ester moieties for reaction with lysines or free N‐termini in proteins. The new reagent offers simple synthetic access and easy structural variation of either length or functionalities at both ends. The thiourea moiety exhibits specifically tailored CID fragmentation capabilities—a characteristic CNL of 85 u—ensuring a reliable detection of derivatized peptides by both electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) tandem mass spectrometry and as such possesses a versatile applicability for chemical cross‐linking studies. A detailed examination of the CID behavior of the presented thiourea‐based reagent reveals that slight structural variations of the reagent will be necessary to ensure its comprehensive and efficient application for chemical cross‐linking of proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

A Novel pH/Light‐Triggered Surface for DNA Adsorption and Release

A simple strategy for the immobilization of Cy3‐labeled single strand DNA (Cy3‐ssDNA) on a Si(001) surface and its release under control of both light and pH stimuli is presented. In order to prepare a dual pH/light‐triggered surface, positively chargeable azobenzene molecules are self‐assembled on the Si(001) surface. The surface wettability of this substrate can be changed under influence of both light and pH conditions. The substrates can be positively charged under mildly acidic conditions. The pH‐sensitive behavior of the film allows binding of Cy3‐ssDNA on the functionalized Si(001) surface through e?ective electrostatic interactions with the negatively charged polynucleotide backbone. Moreover, irradiation of the film with UVA light induces trans–cis isomerization of the azobenzene units on the surface. As a result, the binding a?nity for DNA decreases due to the changing surface hydrophilicity. In order to understand and control the reversible photoswitchable mechanism of this surface, water contact angles are measured after UVA and visible light irradiation. The release of DNA from a dual pH/light‐sensitive sample is performed using fluorescence microscopy. The results show that irradiation of the film with UVA light induces trans–cis isomerization of the photoresponsive azobenzene units; this leads to significant changes in the surface hydrophilicity and reduces the binding affinity for DNA.     

DNA Terminal Mismatch‐Induced Stabilization of Polymer Micelles from RAFT‐Generated Poly(N‐isopropylacrylamide)‐DNA Block Copolymers

Temperature‐responsive diblock copolymers made of poly(N‐isopropylacrylamide) (PNIPAAm) generated by reversible addition‐fragmentation chain transfer (RAFT) polymerization and a single‐stranded DNA (ssDNA) self‐assembled into polymer micelles. The micelles consisted of the PNIPAAm core surrounded by the ssDNA corona with a hydrodynamic diameter up to 300 nm in an aqueous medium above the lower critical solution temperature. In a medium of high ionic strength, the formation of the fully matched duplex with the complementary ssDNA on the surface of the polymer micelles induced rapid and spontaneous aggregation. By contrast, the micelles remained dispersed under the identical conditions when single‐base‐substituted ssDNA was added to form the corresponding terminal‐mismatched duplex on the micellar surface. This highly sequence‐selective process took place irrespective of the size of the PNIPAAm core.     

Fragmentation features of intermolecular cross‐linked peptides using N‐hydroxy‐ succinimide esters by MALDI‐ and ESI‐MS/MS for use in structural proteomics

The use of mass spectrometry coupled with chemical cross‐linking of proteins has become one of the most useful tools for proteins structure and interactions studies. One of the challenges in these studies is the identification of the cross‐linked peptides. The interpretation of the MS/MS data generated in cross‐linking experiments using N‐hydroxy succinimide esters is not trivial once a new amide bond is formed allowing new fragmentation pathways, unlike linear peptides. Intermolecular cross‐linked peptides occur when two different peptides are connected by the cross‐linker and they yield information on the spatial proximity of different domains (within a protein) or proteins (within a complex). In this article, we report a detailed fragmentation study of intermolecular cross‐linked peptides, generated from a set of synthetic peptides, using both ESI and MALDI to generate the precursor ions. The fragmentation features observed here can be helpful in the interpretation and identification of cross‐linked peptides present in cross‐linking experiments and be further implemented in search engine's algorithms. Copyright © 2011 John Wiley & Sons, Ltd.     

Tetrahydrofuran Activates Fluorescence Resonant Energy Transfer from a Cationic Conjugated Polyelectrolyte to Fluorescein‐Labeled DNA in Aqueous Media

A cationic water‐soluble conjugated polyelectrolyte, poly[9,9‐bis(6′′‐(N,N,N‐trimethylammonium)hexyl)fluorene‐co‐alt‐2,5‐bis(6′‐(N,N,N‐trimethylammonium)hexyloxyphenylene) tetrabromide], was synthesized. Fluorescence resonant energy transfer (FRET) experiments between the polymer and fluorescein‐labeled single‐stranded DNA (ssDNA‐Fl) were conducted in aqueous buffer and THF/buffer mixtures. Weak fluorescence emission in aqueous buffer was observed upon excitation of the polymer, whereas addition of THF turned on the fluorescence. Fluorescence self‐quenching of ssDNA‐Fl in the ssDNA‐Fl/polymer complexes as well as electron transfer from the polymer to fluorescein may account for the low fluorescence emission in buffer. The improved sensitization of fluorescence by the polymer observed in THF/buffer could be attributed to the weaker binding between the polymer and ssDNA‐Fl and a decrease in dielectric constant of the solvent mixture, which disfavors electron transfer. THF‐assisted signal sensitization was also observed for the polymer and fluorescein‐labeled double‐stranded DNA (dsDNA‐Fl). These results indicate that the use of cosolvent provides a strategy to improve the detection sensitivity for biosensors based on the optical amplification provided by conjugated polymers.     

Heterobifunctional isotope‐labeled amine‐reactive photo‐cross‐linker for structural investigation of proteins by matrix‐assisted laser desorption/ionization tandem time‐of‐flight and electrospray ionization LTQ‐Orbitrap mass spectrometry

Chemical cross‐linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross‐linked products presents an alternative approach to assess low‐resolution protein structures. By covalently connecting pairs of functional groups within a protein or a protein complex a set of structurally defined interactions is built up. We synthesized the heterobifunctional amine‐reactive photo‐cross‐linker N‐succinimidyl p‐benzoyldihydrocinnamate as a non‐deuterated (SBC) and doubly deuterated derivative (SBDC). Applying a 1:1 mixture of SBC and SBDC for cross‐linking experiments aided the identification of cross‐linked amino acids in the mass spectra based on the characteristic isotope patterns of fragment ions. The cross‐linker was applied to the calcium‐binding protein calmodulin with a subsequent analysis of cross‐linked products by nano‐high‐performance liquid chromatography matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (nano‐HPLC/MALDI‐TOF/TOF‐MS) and nano‐HPLC/nano‐electrospray ionization (ESI)‐LTQ‐Orbitrap‐MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Cationic and Anionic Conjugated Polyelectrolytes: Aggregation‐Mediated Fluorescence Energy Transfer to Dye‐Labeled DNA

An electrostatic complex of water‐soluble conjugated polyelectrolytes (CPs) between anionic poly(9,9‐bis(4′‐sulfonatobutyl)fluorene‐co‐alt‐1,4‐phenylene) disodium salt (a‐PFP) and cationic poly(9,9‐bis((6′‐N,N,N,‐trimethylammonium)hexyl)fluorene‐co‐2,1,3‐bezothiadiazole) dibromide (85:15) (c‐PFB₁₅) was tested as a fluorescence resonance energy transfer (FRET) donor to Texas Red (TR)‐labeled single‐stranded DNA (ssDNA‐TR) via two‐step FRET processes. Electrostatic complexation of a‐PFP and c‐PFB₁₅ in water leads to aggregation of polymer chains, a concomitant reduction of intersegment distances, and energy transfer to the benzothiadiazole (BT) segments. The following complexation with ssDNA‐TR leads to energy transfer from BT to TR via two‐step FRET processes. This detection schematic shows an FRET‐induced signal amplification, which can be achieved by adjusting the charge ratio in the cationic/anionic CP complex and controlling the number density of the binding CPs around the acceptor, resulting in enhanced antenna effects and sensitivity in CP‐based FRET DNA detection assays.

     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.