Denaturing Polyacrylamide Gel Electrophoresis Research Articles

Fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of RNases in vivo.

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5'-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure. In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5' ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Global PROTOMAP Profiling to Search for Biomarkers of Early-Recurrent Hepatocellular Carcinoma

This study used global protein expression profiling to search for biomarkers to predict early recurrent hepatocellular carcinoma (HCC). HCC tissues surgically resected from patients with or without recurrence within 2 years (early recurrent) after surgery were compared with adjacent nontumor tissue and with normal liver tissue. We used the PROTOMAP strategy for comparative profiling, which integrates denaturing polyacrylamide gel electrophoresis migratory rates and high-resolution, semiquantitative mass-spectrometry-based identification of in-gel-digested tryptic peptides. PROTOMAP allows examination of global changes in the size, topography, and abundance of proteins in complex tissue samples. This approach identified 8438 unique proteins from 45 708 nonredundant peptides and generated a proteome-wide map of changes in expression and proteolytic events potentially induced by intrinsic apoptotic/necrotic pathways. In the early recurrent HCC tissue, 87 proteins were differentially expressed (≥20-fold) relative to the other tissues, 46 of which were up-regulated or specifically proteolyzed and 41 of which were down-regulated. This data set consisted of proteins that fell into various functional categories, including signal transduction and cell organization and, notably, the major catalytic pathways responsible for liver function, such as the urea cycle and detoxification metabolism. We found that aberrant proteolysis appeared to occur frequently during recurrence of HCC in several key signal transducers, including STAT1 and δ-catenin. Further investigation of these proteins will facilitate the development of novel clinical applications.

Northern blots for small RNAs and microRNAs.

This protocol describes the detection of small RNAs (~10-200 nucleotides) by blot hybridization. The RNA samples, denatured in formamide, are separated by denaturing polyacrylamide gel electrophoresis. Because high-percentage polyacrylamide gels are required to separate RNAs in this size range, it is necessary to perform electrophoretic transfer to positively charged nylon membranes. After transfer, the immobilized RNAs are subjected to hybridization with a (32)P-radiolabeled DNA or RNA probe and detected by phosphorimaging or autoradiography. This procedure is commonly used to detect small, U-rich spliceosomal small nuclear RNAs (snRNAs) and miRNAs. It should be possible also to detect most miRNAs using high-percentage (e.g., 15%) urea-polyacrylamide gel electrophoresis.

Identification of a novel allergen from muscle and various organs in banana shrimp (Fenneropenaeus merguiensis)

BackgroundThe increasing consumption of shellfish can cause an increase in allergic symptoms. Shrimp allergy can be species specific, but specific allergies in different organs have not been studied. Identification of allergens in muscle and others organs of banana shrimp is necessary for improved diagnostics of allergies for shrimp and food safety control. ObjectiveTo identify the IgE-binding proteins in various organs of Fenneropenaeus merguiensis by immunoblotting and tandem mass spectrometry. MethodsProteomic methods were used to investigate the allergenic proteins from banana shrimp. Proteins from muscle and various organs were separated by denaturing polyacrylamide gel electrophoresis. Allergens were analyzed by immunoblotting with pooled sera from shrimp allergic patients (n = 21) and tandem mass spectrometry. ResultsThe important allergens in banana shrimp are arginine kinase, sarcoplasmic calcium-binding protein, myosin heavy chain, hemocyanin, enolase, and glyceraldehyde-3-phosphate dehydrogenase, which can be demonstrated by immunoblotting in muscle and shell. Moreover, vitellogenin, ovarian peritrophin 1 precursor, β-actin, and 14-3-3 protein were suggested as allergens in the ovary at different stages of ovarian development. ConclusionTen allergens were identified as allergens in various organs, and they are suggested as novel allergens in banana shrimp. The major allergen in muscle and shell from this shrimp is arginine kinase, whereas the major allergen in the ovary is vitellogenin.

New recombinant T. pallidum antigens Tp0453 and Tp0319 in the diagnostics of syphilis

A new technology for obtaining a recombinant version of a new dedicated protein T. pallidum Tp0319 for the serological diagnostics of syphilis was gene engineered. As a result, a homogeneous (according to denaturating polyacrylamide gel electrophoresis) recombinant version of T. pallidum Tp0319 protein was obtained. The molecular mass of recombinant T. pallidum Tp0319 protein according to electrophoretic mobility is about 37 kDa, which corresponds to the molecular mass of a fragment of Tp0319 protein without a signal peptide calculated based on the amino-acid sequence (37.3 kDa). According to the developed technology, purified recombinant T. pallidum Tp0319 and Tp0453 proteins were obtained. The proteins were used as antigens as a part of a solid-phase immunoadsorbent for detecting specific IgG to the study protein in the serum of patients suffering from different types of acquired syphilis. The use of the resulting recombinant protein enabled the authors to reveal T. pallidum antibodies in the blood serum in patients suffering from syphilis (primary, secondary, latent early and late stage syphilis). The examination of blood serum samples taken from healthy donors revealed no antibodies. Based on the study results, it is possible to make a conclusion about the possibility to use the resulting recombinant protein (Тр0319) as an extra antigen for diagnostics of syphilis. The introduction of the new antigen in the immunoadsorbent for test systems used for diagnosing syphilis (in the form of immune-enzyme assay, immunoblotting or immune chips) expands the potential of serological diagnostics of this disease due to the expansion of the range of T. pallidum antibodies to be revealed.

DNA-mediated oxidation of p53.

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs over long distances through the π-stacked bases and leads to the oxidative dissociation of p53. The extent of protein dissociation depends upon the redox potential of the DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using oligonucleotides containing both synthetic and human p53 consensus sequences with an appended photooxidant, anthraquinone. Greater p53 dissociation is observed from sequences containing low-redox potential purine regions, particularly guanine triplets. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites corresponding to sites of preferred electron hole localization were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets. Oxidative DNA damage is inhibited in the presence of p53, but only at sites in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress as well as possible biological implications have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

Detection of DNA mutations by PCR-TTGE method

In our study PCR-temporal temperature gelelectrophoresis (TTGE) and MeltINGENY bioinformatic program were used to analyse the mutations in the genes of melanocortin-1 receptor (MC1R) and pituitary adenylate-cyclase activating polypeptide (PACAP) in cattle. Amplification of target DNA by PCR was performed with GC-clamp primers and non-GC-clamp primers in simplex PCR reactions. The fragments were separated by denaturing polyacrylamide gelelectrophoresis (denaturing agents: high temperature, urea) after PCR reactions.MC1R homozygous individuals were used for the reaction.
 We concluded that MeltINGENY program makes the decision and detection system easier, and more simple as the melting profile of target sequence is determined by the software. In case of MC1R gene, PCR-TTGE method is appropriate for SNP detection, however PACAP gene polymorphism can not be identified by the method, because PACAP mutations are not included in melting domains, therefore PCR-TTGE cannot detect them.

Heterologous expression and characterization of laccase 2 from Coprinopsis cinerea capable of decolourizing different recalcitrant dyes

The gene (CcLcc2) encoding laccase from the basidiomycete Coprinopsis cinerea Okayama-7 #130 was synthesized by polymerase chain reaction-based two-step DNA synthesis, and heterologously expressed in Pichia pastoris. The recombinant protein was purified by ammonium sulphate precipitation and nickel nitrilotriacetic acid chromatography. The molecular mass of CcLcc2 was estimated to be 54 kDa by denaturing polyacrylamide gel electrophoresis. The optimum pH and temperature for laccase catalysis for the oxidation of 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) were 2.6 and 45 °C, respectively. The Km values of the enzyme towards the substrates ABTS, 2,6-dimethoxyphenol (2,6-DMP) and guaiacol were 0.93, 1.02 and 28.07 mmol·L−1, respectively. The decolourization of methyl orange, crystal violet and malachite green, commonly used in the textile industry, was assessed. The decolourization percentage of crystal violet and malachite green was 80% after 4 h of reaction, and that of methyl orange was 50% at 4 h. These results show that the CcLcc2 has enormous potential for the decolourization of highly stable triphenylmethane dyes.

Determinants of RNA Binding and Translational Repression by the Bicaudal-C Regulatory Protein

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Specific Alkylation of Human Telomere Repeat Sequences by a Tandem-Hairpin Motif of Pyrrole–Imidazole Polyamides with Indole-seco-CBI

We designed and synthesized a tandem-hairpin motif of pyrrole (P)-imidazole (I) polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates (1) that targets the human telomere repeat sequence 5'-d(CCCTAA)n-3'. As a control, conjugate 2 (hairpin PI polyamide with seco-CBI), which also targets the human telomere repeat sequence, was synthesized. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) using 5' Texas Red-labeled 219-bp DNA fragments revealed the outstandingly high sequence selectivity of 1, with no mismatch alkylation. Furthermore, an evaluation performed in human cancer cell lines demonstrated that conjugate 1 has low cytotoxicity compared with conjugate 2. In addition, a cell-staining analysis indicated that conjugate 1 induced apoptosis moderately by DNA damage. This study demonstrated that conjugate 1 can be used as an effective alkylator for telomere repeat sequences or as an apoptotic inducer.

KINETIC ASSESSMENT AND EFFECT ON DEVELOPMENTAL PHYSIOLOGY OF A TRYPSIN INHIBITOR FROM Eugenia jambolana (JAMBUL) SEEDS ON Helicoverpa armigera (HÜBNER)

A trypsin inhibitor was purified from the seeds of Eugenia jambolana (Jambul) with a fold purification of 14.28 and a yield recovery of 2.8%. Electrophoretic analysis of E. jambolana trypsin inhibitor (EjTI) revealed a molecular weight of approximately 17.4 kDa on 12% denaturing polyacrylamide gel electrophoresis with or without reduction. EjTI exhibited high stability over a wide range of temperatures (4-80 °C for 30 min) and pH (3.0-10.0) and inhibited trypsin-like activities of the midgut proteinases of fourth instar Helicoverpa armigera larvae by approximately 86%. Feeding assays containing 0.05, 0.15, and 0.45 (% w/w) EjTI on functionally important fourth-instar larvae indicated a dose-dependent downfall in the larval body weight as well as on extent of survival. The nutritional analysis suggests that EjTI exerts toxic effects on H. armigera. Dixon plot analysis revealed competitive inhibition of larval midgut proteinases by EjTI, with an inhibition constant (Ki ) of approximately 3.1 × 10(-9) M. However, inhibitor kinetics using double reciprocal plots for trypsin inhibition demonstrated a mixed inhibition pattern. These observations suggest the potential of E. jambolana trypsin inhibitor protein in insect pest management.

Purification of Radiolabeled RNA Products Using Denaturing Gel Electrophoresis

This unit discusses a basic method for purification of radiolabeled RNAs using denaturing polyacrylamide gel electrophoresis. The method consists of a number of experimental procedures, including total RNA preparation from yeast cells, isolation of a specific RNA from total yeast RNA, RNA 3'-terminal labeling using nucleotide (5' [(32) P]pCp) addition (via ligation), denaturing (8 M urea) polyacrylamide gel electrophoresis, and RNA extraction from the gel slice. Key points for achieving good electrophoretic separation of RNA are also discussed.

In vitro Assay for Cytidine Deaminase Activity of APOBEC3 Protein

Cytidine deaminases are enzymes that catalyze the removal of an amino group from cytidine, forming uridine. APOBEC3 (ApolipoproteinB mRNA editing enzyme, catalytic polypeptide like) proteins are cytidine deaminases that deaminate cytidines in polynucleotides (RNA/DNA), resulting in editing of their target substrates. Mammalian APOBEC3 proteins are an important element in cellular defenses against retrovirus replication, and this "restriction" of retroviral infections is partially due to the cytidine deaminase activity of the APOBEC3. The present protocol (Nair et al., 2014) describes the assay to detect the deaminase activity of mouse APOBEC3 protein, which targets cytidines present in TCC or TTC motifs in a single-stranded DNA substrate. In brief, the protein preparation to be assayed is incubated with a fluorophore-labeled oligodeoxynucleotide containing the deamination target motif (radiolabeled oligonucleotide substrates have also been successfully used by other groups). Cytidines in the oligonucleotide are deaminated to uridines; the addition of Uracil DNA Glycosylase (UDG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, generating an abasic (AB) site in the oligonucleotide. Mild alkali treatment cleaves the substrate oligonucleotide at the AB site; cleaved products are resolved from uncleaved substrate by denaturing polyacrylamide gel electrophoresis and visualized on a fluorescence scanner. The protocol described here is mainly adapted from that described by Iwatani et al. (2006) with modifications. The assay can, of course, be used to detect the activity of other APOBEC3 deaminases targeting DNA substrates, using oligonucleotides containing the cytidine-containing target sequence for the deaminase.

Screening of highly informative and representative microsatellite markers for genotyping of major cultivated cotton varieties.

We screened and assessed published cotton simple sequence repeat (SSR) primers to establish a set of core SSR markers suitable for cotton major cultivars in China and analyzed genetic diversity based on the core marker set. Using a stepwise screening strategy, 12 leading cultivars for preliminary screening and 96 cultivars for rescreening were evaluated. A total of 184 polymorphic SSR markers were initially screened from 3299 candidates, and a core set of 52 SSR markers with wide genome coverage (2 markers per chromosome) was obtained. Among 96 major cultivars, 273 amplification genotypes were generated using the core marker set. Polymorphism information content values ranged from 0.28-0.83, with an average value of 0.56. The core SSR marker set detected on denaturing polyacrylamide gel electrophoresis indicated that the band genotype was either a single or double band on conventional cultivars, while most were double bands (65.4%). Among 56 hybrids, the average heterozygosis rate was 35.8%, ranging from 7.1-55.4%. Eighteen of 96 cultivars had distinct band genotypes. The genetic diversity analyzed using the of NTSYS-pc V2.10 software indicated that the Yangtze River valley cotton region had the highest polymorphic level, followed by Xinjiang and then the Yellow River valley. The genetic basis of conventional cultivars was narrower than that of hybrids. The core marker set can be used for fingerprint construction, variety identification, and purity tests of major cotton cultivars in China.

Tertiary structure mapping of the pri-miRNA miR-17~92.

The understanding of RNA in regulating gene expression has exploded over the past 15 years. MicroRNAs (miRNAs) have vastly expanded the role of RNA in gene regulation beyond spliceosomal, ribosomal, and messenger RNAs. Approximately one half of miRNAs are polycistronic, where two or more miRNAs are encoded on a single pri-miRNA transcript, termed a miRNA cluster. The six miRNAs of the miR-17~92 cluster are contained within a ~800 nucleotide region within intron 3 of the cl13orf25 ~7 kb pri-miRNA transcript. We recently reported on the tertiary structured domain of miR-17~92 and its role in modulating miRNA biogenesis. The key finding was that the cluster structure explained the differential processing of the miRNA hairpins by Drosha. This work demonstrated the need to consider pri-miRNA tertiary structure in miRNA biogenesis. Since biochemical structure probing is typically performed on relatively short RNAs (≤200 nucleotides), we had to adapt these methodologies for application on large RNAs (~800 nucleotide miR-17~92 pri-miRNA). We present here our adaptation of a protection footprinting method using ribonucleases to probe the structure of the ~800 nucleotide miR-17~92 pri-miRNA. We outline the technical difficulties involved in probing large RNAs and data visualization using denaturing polyacrylamide gel electrophoresis and how we adapted the existing approaches to probe large RNAs. The methodology outlined here is generally applicable to large RNAs including long noncoding RNAs (lncRNA).

A Nonhost Peptidase Inhibitor of ~14 kDa from Butea monosperma (Lam.) Taub. Seeds Affects Negatively the Growth and Developmental Physiology of Helicoverpa armigera.

Helicoverpa armigera is one of the major devastating pests of crop plants. In this context a serine peptidase inhibitor purified from the seeds of Butea monosperma was evaluated for its effect on developmental physiology of H. armigera larvae. B. monosperma peptidase inhibitor on 12% denaturing polyacrylamide gel electrophoresis exhibited a single protein band of ~14 kDa with or without reduction. In vitro studies towards total gut proteolytic enzymes of H. armigera and bovine trypsin indicated measurable inhibitory activity. B. monosperma peptidase inhibitor dose for 50% mortality and weight reduction by 50% were 0.5% w/w and 0.10% w/w, respectively. The IC50 of B. monosperma peptidase inhibitor against total H. armigera gut proteinases activity was 2.0 µg/mL. The larval feeding assays suggested B. monosperma peptidase inhibitor to be toxic as reflected by its retarded growth and development, consequently affecting fertility and fecundity of pest and prolonging the larval-pupal duration of the insect life cycle of H. armigera. Supplementing B. monosperma peptidase inhibitor in artificial diet at 0.1% w/w, both the efficiencies of conversion of ingested as well as digested food were downregulated, whereas approximate digestibility and metabolic cost were enhanced. The efficacy of Butea monosperma peptidase inhibitor against progressive growth and development of H. armigera suggest its usefulness in insect pest management of food crops.

Negative effects of a nonhost proteinase inhibitor of ~19.8 kDa from Madhuca indica seeds on developmental physiology of Helicoverpa armigera (Hübner).

An affinity purified trypsin inhibitor from the seed flour extracts of Madhuca indica (MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases of Helicoverpa armigera larvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50 of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with a K i value of 4.1 × 10−10 M for Helicoverpa trypsin like midgut proteinases. In vivo experiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5% w/w) showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50) of larvae was 1.5% w/w and that to cause reduction in mass of larvae by 50% (ED50) was 1.0% w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development of H. armigera larvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants.

Synthesis of Building Blocks and Oligonucleotides Containing {T}O4‐Alkylene‐O4{T} Interstrand Cross‐Links

This protocol describes the preparation of O(4)-thymidine-alkylene-O(4)-thymidine dimer bis-phosphoramidites and precursors for incorporation into DNA sequences to produce site-specific DNA interstrand cross-links. Linkers are introduced at the 4-position of thymidine by reacting the sodium salt of a diol with a pyrimidinyl-convertible nucleoside to produce mono-adducts, which then undergo reaction with a stoichiometric equivalent of a pyrimidinyl-convertible nucleoside under basic conditions to form O(4)-thymidine-alkylene-O(4)-thymidine dimers. Bis-phosphoramidites are incorporated into oligonucleotides by solid-phase synthesis, and mild conditions for deprotection and cleavage from the solid support are employed to prevent degradation of the thymidine modifications. Purification of these cross-linked oligonucleotides is performed by denaturing polyacrylamide gel electrophoresis. This approach allows for the preparation of cross-linked DNA substrates in quantities and purity sufficient for a wide range of biophysical experiments and biochemical studies as substrates to investigate DNA repair pathways.

Purification and characterization of a trypsin inhibitor from Senna tora active against midgut protease of podborer

Proteinaceous protease inhibitors have potential application in medicines, agriculture and biotechnology. Present study was undertaken to purify and characterize a proteinaceous protease inhibitor from a medicinal plant, Senna tora syn. Cassia tora. The inhibitor was purified by ammonium sulphate precipitation, anion exchange (Q-sepharose), affinity (trypsin-sepharose) and molecular exclusion (sephadex G-75) chromatography. Zymography and denaturing polyacrylamide gel electrophoresis revealed a single band of ∼20kDa trypsin inhibitor. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Matrix-assisted laser desorption ionization (MALDI) analyses revealed the presence of 19,725Da (pI 4.60) and ∼19,900Da (pI 4.57) isoform proteins in purified inhibitor. Protein identification by MALDI-peptide mass fingerprinting did not reveal high MASCOT (Matrix science) scores matching with previously known inhibitors. N-terminal amino acid sequence suggested this protein as a previously unreported inhibitor. Its dissociation constant (0.23×10−9M) was indicative of a high affinity trypsin inhibitor. The inhibitor was stable over a broad range of pH (4–10) and temperature (30–60°C). The purified inhibitor effectively inhibited total protease and trypsin-like activities of podborer (Helicoverpa armigera) midgut preparation. Hence, the inhibitor and its gene(s) can find application in combating against pest and protease dependent pathogens.

Creation of DNA array structure equipped with heat resistance by ultrafast photocrosslinking

BACKGROUND The outstanding specificity of the A-T and G-C Watson–Crick hydrogen-bonding interaction allows various DNA nanostructures to be created, which has increased interest in using DNA as a bio- or nano-material. DNA nanostructures have already been used for many applications as well as to create a platform for nanopatterning of a variety of function molecules. However, these structures can be used only at low temperature of Tm value or less in solution because they melt beyond this temperature. This paper reports the creation of a DNA array structure held by photocrosslinking mediated 3-cyanovinylcarbazole (CNVK) which allows crosslinking by photoirradiation in a few seconds. RESULTS The thermal stability of DNA nanostructures containing CNVK were evaluated before and after 366 nm UV-exposure by denaturing polyacrylamide gel electrophoresis and the melting curve. When CNVK was arranged in a suitable position into a 2D DNA array, its photocrosslinked structure was stable under denaturing conditions. The solution-state melting profile of the photocrosslinked DNA array was expanded to >80 °C, which is at least a 40 °C increase and showed decreased hyperchromic effect when compared with a non-photocrosslinked structure. CONCLUSION The method of creating DNA structures equipped with thermal resistance by ultrafast photocrosslinking reaction provides practical DNA nanodevices and enables the stepwise assembly of DNA structures. © 2013 Society of Chemical Industry