[Show abstract][Hide abstract] ABSTRACT: A study on binding of antitumor chelerythrine to human telomeric DNA/RNA G-quadruplexes was performed by using DNA polymerase stop assay, UV-melting, ESI-TOF-MS, UV-Vis absorption spectrophotometry and fluorescent triazole orange displacement assay. Chelerythrine selectively binds to and stabilizes the K(+)-form hybrid-type human telomeric DNA G-quadruplex of biological significance, compared with the Na(+)-form antiparallel-type DNA G-quadruplex. ESI-TOF-MS study showed that chelerythrine possesses a binding strength for DNA G-quadruplex comparable to that of TMPyP4 tetrachloride. Both 1:1 and 2:1 stoichiometries were observed for chelerythrine's binding with DNA and RNA G-quadruplexes. The binding strength of chelerythrine with RNA G-quadruplex is stronger than that with DNA G-quadruplex. Fluorescent triazole orange displacement assay revealed that chelerythrine interacts with human telomeric RNA/DNA G-quadruplexes by the mode of end- stacking. The relative binding strength of chelerythrine for human telomeric RNA and DNA G-quadruplexes obtained from ESI-TOF-MS experiments are respectively 6.0- and 2.5-fold tighter than that with human telomeric double-stranded hairpin DNA. The binding selectivity of chelerythrine for the biologically significant K(+)-form human telomeric DNA G-quadruplex over the Na(+)-form analogue, and binding specificity for human telomeric RNA G-quadruplex established it as a promising candidate in the structure-based design and development of G-quadruplex specific ligands.
[Show abstract][Hide abstract] ABSTRACT: The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)3 in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Förster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.
No preview · Article · Apr 2014 · Chemistry - A European Journal
[Show abstract][Hide abstract] ABSTRACT: Regulation of gene expression by short oligonucleotides (antisense oligonucleotides), which can modulate RNA structures and inhibit subsequent associations with the translation machinery, is a potential approach for gene therapy. This chapter describes an alternative antisense strategy using guanine-tethered antisense oligonucleotides (G-ASs) to introduce a DNA-RNA heteroquadruplex structure at a designated sequence on RNA targets. The feasibility of using G-ASs to modulate RNA conformation may allow control of RNA function by inducing biologically important quadruplex structures. This approach to manipulate quadruplex structures using G-ASs may expand the strategies for regulating RNA structures and the functions of short oligonucleotide riboregulators.
No preview · Article · Feb 2014 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: The construction of an artificial riboswitch is based on a ligand-RNA pair without any molecular biology-based selection processes. The ligand selectively and significantly stabilized an RNA duplex containing an r(XGG)/r(XGG) sequence (X=U, A, G). The integration of the ligand-binding sequences into the 5'-untranslated region of mRNA provided an artificial riboswitch that was responsive to Z-NCTS.
No preview · Article · Sep 2013 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: A small DNA rectangular parallelepiped block was prepared from a single stranded scaffold strand of 1152 nt long and 28 staple strands by DNA origami method. The scaffold strand, which was obtained by PCR using a phosphorylated primer followed by lambda exonuclease digestion, was folded into the designed structure. Subsequent assembly of the DNA block into higher order DNA nanostructures was achieved by slow annealing conditions in the presence of linker strands, giving both the tail-to-tail dimer and the tail-to-head one-dimensional oligomer with a choice of appropriate linker strands.
No preview · Article · Nov 2012 · Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: A few examples of translational activation by antisense small noncoding RNAs (sRNAs) have already been discovered in prokaryotic cells, and all of them are through a sense-antisense interaction at the 5'-untranslated region (5'-UTR) of target mRNAs. Here, we report a novel phenomenon of translational activation of prokaryotic gene expression with trans-acting antisense oligonucleotides targeting the coding region of mRNA. Screening of antisense oligonucleotides complementary to the coding sequences of GFP or ZsGreen identified antisense sequences that activate translation of the mRNAs in a concentration-dependent manner. We also found that the translational activation highly depends on the hybridization positions of the antisense strands. Translation-activating antisense oligonucleotides (TAOs) tended to bind to the 5'-region rather than the 3'-region of the mRNA coding region. RNA folding simulation suggested that TAOs may disrupt the structured elements around the translation initiation region (TIR) by pairing with complementary sequences in the mRNA coding region, resulting in an increase in translation efficiency. Further, we demonstrate that number and position of locked nucleic acid (LNA) bases in the antisense strands govern the tendency of up- or down-regulation. Our findings described here may lead to the discovery of a new class of antisense sRNA and the development of a tool for activating desired gene expression in the future.
No preview · Article · Oct 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small-molecule-RNA interactions. The RNA-binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7-bis(2-aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11-mer double-stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV-1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double-stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1' position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double-stranded region by a factor of 40%. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double-stranded and loop regions suggested that the N,N-dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE.
No preview · Article · Aug 2012 · Chemistry - A European Journal
[Show abstract][Hide abstract] ABSTRACT: Unusual expansion of trinucleotide repeats has been identified as a common mechanism of hereditary neurodegenerative diseases. Although the actual mechanism of repeat expansion remains uncertain, trinucleotide repeat instability may be related to the increased stability of an alternative DNA hairpin structure formed in the repeat sequences. Here we report that a synthetic ligand naphthyridine carbamate dimer (NCD) selectively bound to and stabilized an intra-stranded hairpin structure in CGG repeat sequences. The NCD-CGG hairpin complex was a stable structure that efficiently interfered with DNA replication by Taq DNA polymerase. Considering the sequence preference of NCD, the use of NCD would be valuable to investigate the genetic instabilities of CGG/CCG repeat sequences in human genomes.
No preview · Article · Mar 2012 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: Spanner in the works: The synthetic ligand, naphthyridine-azaquinolone (NA), can induce hairpin secondary structures on d(CAG)n repeats and efficiently interfere with DNA replication by Taq DNA polymerase as well as the human DNA polymerase α (see figure). Considering its unique sequence preference, NA would be a valuable probe to understand the features of the genetic instabilities of CAG/CTG repeat sequences in genomes.
[Show abstract][Hide abstract] ABSTRACT: Voller Schleifen: Die Wechselwirkung einer Reihe von fehlpaarenden Molekülen mit Haarnadelschleifen-DNA, die eine d(CGG)3-Sequenz in der Schleife enthält, wird beschrieben (siehe Bild). Native Polyacrylamid-Gelelektrophorese von Haarnadelschleifen-DNA zeigt, dass das neu synthetisierte fehlpaarende Molekül, eine tetramere Form von N-Methoxycarbonyl-1,8-naphthyridin, an der Bildung eines Schleifen-Schleifen-Komplexes zwischen zwei DNA-Haarnadelschleifen beteiligt ist.
No preview · Article · May 2011 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: Guanine quadruplex structures in DNA and RNA affect normal cellular processes such as replication, recombination, and translation. Thus, controlling guanine quadruplex structures could make it possible to manipulate the biological function of nucleic acids. Here, we report a novel antisense strategy using guanine-tethered antisense oligonucleotides (g-ASs) that introduces an RNA-DNA heteroquadruplex structure on RNA templates in a predictable and sequence-specific manner, which in practice effectively inhibited reverse transcription on a variety of RNA sequences, including the HIV-1 RNA genome. Reverse transcriptase-mediated enzymatic analysis, together with other biophysical analyses, elucidated a cooperative binding of duplex and quadruplex in g-AS-RNA complexes. The remarkable ability of g-ASs to inhibit reverse transcription could make possible the development of novel anti-retroviral gene therapies based on blocking the replication of RNA genomes to complementary DNA, which is a critical step for integration into the host's genome.
Preview · Article · Aug 2010 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Here, we developed a reverse transcriptase based method (RTase stop assay) to characterize quadruplex formations in guanine-rich RNAs with high sensitivity and specificity. By using the RTase stop assay, we also revealed a plausible structural polymorphism in biologically important RNAs. The RTase stop assay would provide helpful insight into RNA quadruplex structures and functions, together with other analytical methods, including various footprinting techniques.
No preview · Article · Feb 2010 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: A newly designed ligand, methylcarbamoylnaphthyridine dimer (MCND), was synthesized and characterized. Ligand binding to d(GAA)(10) was investigated by UV thermal denaturation, circular dichroism spectroscopy, surface plasmon resonance, and cold-spray-ionization time-of-flight mass spectrometry. The results indicated that MCND bound to the d(GAA)(n) repeat to form a stable hairpin structure with a major binding stoichiometry of 3:1. The most likely binding site was identified as the G-G mismatch in the AGA/AGA triad. The polymerase stop assay showed that MCND binding to the d(GAA)(n) repeat effectively interfered with the extension of the primer at the first two GAA sites on the template with both prokaryotic Taq DNA polymerase and human DNA polymerase alpha.
No preview · Article · Oct 2009 · Chemistry - A European Journal
[Show abstract][Hide abstract] ABSTRACT: Haarnadel-Sekundärstrukturen am 5′-Ende eines PCR-Primers gehen im Lauf der PCR in eine Doppelstrangform über. DANP-Farbstoffmoleküle können an die Cytosin-Ausbuchtung (C-bulge) der Haarnadelstrukturen binden und ihre charakteristische Fluoreszenz abgeben. Wird ein PCR-Primer mit C-bulge-Haarnadel-Markierung in Gegenwart von DANP verwendet, so nimmt die Fluoreszenz des DANP-C-bulge-Komplexes ab, während die PCR fortschreitet.
No preview · Article · Oct 2009 · Angewandte Chemie International Edition
[Show abstract][Hide abstract] ABSTRACT: Relative to most regions of the genome, tandemly repeated DNA sequences display a greater propensity to mutate. A search for tandem repeats in the Saccharomyces cerevisiae genome revealed that the nucleosome-free region directly upstream of genes (the promoter region) is enriched in repeats. As many as 25% of all gene promoters contain tandem repeat sequences. Genes driven by these repeat-containing promoters show significantly higher rates of transcriptional divergence. Variations in repeat length result in changes in expression and local nucleosome positioning. Tandem repeats are variable elements in promoters that may facilitate evolutionary tuning of gene expression by affecting local chromatin structure.
[Show abstract][Hide abstract] ABSTRACT: Modulation of biological networks assembled by diverse interactions among biologically active molecules has provided a platform for innovative biotechnologies. Here, we report RNA aptamers that bind to a photoresponsive peptide (KRAzR; Lys-Arg-azobenzene-Arg) containing azobenzene chromophore, which can change its structure by photoirradiation. Aptamers were identified after 10 cycles of an in vitro selection procedure starting with a DNA library containing a 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation to the SPR sensor chip. Upon irradiation of 360 nm on the KRAzR-immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm restored the aptamer binding to the surface. We also observed that direct photoirradiation of the aptamer-peptide complex on a gold surface actively promoted dissociation of the complex. Furthermore, a doped reselection method was applied to acquire structural and sequence information of aptamer 66. From a data analysis of the conserved region and the mutation frequency, we were able to select a plausible secondary structure among three candidates predicted by computational folding simulation.
No preview · Article · Jan 2009 · Chemistry - A European Journal
[Show abstract][Hide abstract] ABSTRACT: The beads-on-a-string structure of telomeric overhang, as illustrated here, was confirmed by studying the binding behaviours of the G-quadruplex binders sanguinarine and TMPyP4 to long human telomeric sequences by using a DNA polymerase stop assay. Additionally, a novel mode of ligand binding to long telomeric sequences was further suggested, in which ligands favourably bind between tandem G quadruplexes leading to a cooperative stacking interaction.
[Show abstract][Hide abstract] ABSTRACT: Personalized medical solutions: Simple, accurate, and cost effective methods of single nucleotide polymorphism typing would be necessary for personalized medicine. Towards this end, a number of SNP typing methods have been investigated and reported. We report herein, our chemical approach to practical SNP typing based on allele-specific PCR integrated with a new concept of DNA-labeling by ligand-inducible secondary structure.