Masaki Hagihara

Recognition of Chelerythrine to Human Telomeric DNA and RNA G-quadruplexes

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 chelerythrines 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.

A Small Molecule Affecting the Replication of Trinucleotide Repeat d(GAA)n

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.

A Small Molecule That Represses Translation of G-Quadruplex-Containing mRNA

The G-quadruplexes form highly stable nucleic acid structures, which are implicated in various biological processes in both DNA and RNA. Although DNA G-quadruplexes have been studied in great detail, biological roles of RNA G-quadruplexes have received less attention. Here, a screening of a chemical library permitted identification of a small-molecule tool that binds selectively to RNA G-quadruplex structures. The polyaromatic molecule, RGB-1, stabilizes RNA G-quadruplex, but not DNA versions or other RNA structures. RGB-1 intensified the G-quadruplex-mediated inhibition of RNA translation in mammalian cells, decreased expression of the NRAS proto-oncogene in breast cancer cells, and permitted identification of a novel sequence that forms G-quadruplex in NRAS mRNA. RGB-1 may serve as a unique tool for understanding cellular roles of RNA G-quadruplex structures.

A small molecule regulates hairpin structures in d(CGG) trinucleotide repeats

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.

Detection of L-DNA-tagged PCR products by surface plasmon resonance imaging.

The employment of tags plays an increasingly important role in genomics, in which PCR products are arrayed at the microor nanoscale. PCR products have been labeled in many ways. One conventional method is to incorporate labeled nucleotides into the PCR products during the extension reaction by DNA polymerase. Another method is to use a PCR primer that is labeled at the 5’ end. PCR primers with fluorophore labels are used for DNA sequencing, whereas biotinylated primers are used for the isolation and detection of the PCR products due to their strong and specific binding to streptavidin. Here, we report on a mirror-image DNA (l-DNA)-tagged PCR (LT-PCR) that enables us to label the PCR products with a defined sequence of l-DNA tag. The important findings leading to LT-PCR were that the l-DNA tag did not interfere with the PCR and remained as a single strand even after PCR. Therefore, LT-PCR products could be precisely delivered onto the DNA microarray, where the l-DNA complementary to the tag sequence was immobilized. A large number of sequences available for lDNA tags would be suitable for such comprehensive microarray analysis. We demonstrated that the surface plasmon resonance imaging (SPR) array carrying the complementary l-DNA on the surface successfully detected the LT-PCR products without any purification or additional fluorescent labeling. l-DNA consisting of l-2’-deoxyriboses is an enantiomer of natural DNA (d-DNA) and has unique properties. l-DNA has been shown to be a poor substrate for the human endoand exo-nucleases, as it has the opposite chirality to their intrinsic target, and does not interact with single-stranded d-DNA, whereas it does bind sequence-selectively to complementary l-DNA. We anticipated that an l-DNA tag attached to a PCR primer at the 5’-end would not be recognized as a PCR template by DNA polymerase and would remain in a single-stranded form after amplification (Scheme 1). Besides l-DNA, a peptide nucleic acid (PNA) or d-DNA tag attached through a nonreplicable linker were conceivable for coding PCR products. However, the binding ability of PNA or d-DNA to complementary d-DNA might induce the formation of undesirable intraprimer hairpins or an interaction to an unexpected sequence of the template DNA. The intrinsic possibility of forming hairpin secondary structures and ambiguity in the effect on the PCR reactions thus makes them unsuitable for the labeling. Primers used in LT-PCR consist of three parts, d-DNA to function as a general PCR primer, a l-DNA unit as a molecular tag, and three l-dTs as a spacer between the d and l-DNA. The spacer was inserted to mitigate steric congestion that might be produced between the left-handed and the right-handed duplexes upon hybridization of LT-PCR products with a com-

Antisense-induced guanine quadruplexes inhibit reverse transcription by HIV-1 reverse transcriptase.

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 hosts genome.

A reverse transcriptase stop assay revealed diverse quadruplex formations in UTRs in mRNA

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.

Chemical approaches untangling sequence-specific DNA binding by proteins

Structure-based design of novel DNA-binding proteins provides an ultimate test of our understanding of protein-DNA interactions. A combination of synthetic, organic, biochemical and molecular biological approaches has been developed to study the principle of molecular recognition associated with the protein-DNA interactions. The strategies enabled a specific formation of noncovalent peptide dimers and determination of the preferential DNA-binding sequence of short peptides.

Small molecule modulates hairpin structures in CAG trinucleotide repeats.

Short tandem DNA repeats are widely observed from prokaryotic to eukaryotic genomes. In humans, unusual expansions of trinucleotide repeats cause severe neurodegenerative diseases. For examples, the d(CAG)n expansion mutation in the first exon of the huntingtin gene causes Huntington’s disease. Fragile X syndrome is caused by an expansion of the d(CGG)n repeat in the coding-sequence of the FMR1 (fragile X mental retardation 1) gene. Several cellular processes involving DNA replication, repair, and recombination are related to the instability of repeat sequences. Most mechanisms responsible for repeat instability assume a formation of unusual non-B DNA conformations, such as hairpins, triplexes, and quadruplexes in a parent or daughter strand during DNA synthesis. Consequently, an extension of the slipped structure would produce a repeat expansion or contraction in repeat tracts, although the actual mechanism still remains uncertain. Small ligands that can bind and stabilize particular structures in the repeat regions would help our understanding of an abnormal expansion of repeats at the molecular level. We have recently reported that a synthetic ligand, naphthyridine-azaquinolone (NA), stabilizes CAG/CAG-triad containing DNA (Scheme 1 A). Structural characteristics have revealed that the naphthyridine and azaquinolone moieties in NA exhibit complementary hydrogen bonding to guanine and adenine, respectively, and make two cytosine bases flip out from the CAG/CAG triad (Scheme 1 B). These features explain the remarkable selectivity of NA against the CAG/CAG triad. Here, we demonstrate that NA can induce hairpin secondary structures on d(CAG)n repeats, which then efficiently interfere with DNA replication by Taq DNA polymerase. First, the biophysical and biochemical properties of a d(CAG)10, 30-mer ten-repeats of CAG trinucleotides, were investigated by circular dichroism (CD) and UV analysis (Figure 1). In the absence of NA, d(CAG)10 showed a positive broad signal at around 270 nm and a negative signal at 255 nm (Figure 1 A). Upon addition of NA, distinct spectral changes depending on NA concentration were observed by a negative induced CD at around 320 nm and a strong positive peak at 250 nm. No significant spectral change was observed in d(CTG)10 repeats even at 50 mm NA (Figure S1 in the Supporting Information), which indicated that NA preferentially bound to a repetitive CAG strand. UV thermal denaturation studies showed that the thermal stability of the d(CAG)10 was largely enhanced with a concomitant increasing concentration of NA (Figure 1 B). As NA concentration increased, the melting temperature of NA-induced hairpins rose from 47.1( 0.7) 8C to 78.8( 0.1) 8C at 50 mm NA, surpassing the melting temperature of d(CTG)10 hairpins (54.4 0.3 8C; Figure S2 in the Supporting Information). Taken together, NA could modulate the stability of the CAG hairpins without affecting its complementary CTG hairpins. To further characterize NA-bound structures in CAG repeat sequences, a polymerase-stop assay was performed on the DNA templates containing repeat sequences (Figure 2 A). Stabilization of hairpin secondary structures in repeat sequences resulted in the production of truncated complementary DNA products due to the inhibition of DNA synthesis, which was detected by PAGE analysis. In the absence of NA, the 20-mer primer that hybridized to the 3’-end of the template was fully elongated by Taq DNA polymerase in the d(CAG)10-containing templates; this suggests that no stable structures were formed that efficiently inhibited DNA synthesis. In contrast, addition of NA effectively inhibited DNA elongation to produce distinct truncated products in a concentration-dependent manner in relation to NA (Figure 2 B); this suggests that NA-induced hairpins were stable enough to efficiently interfere with DNA synthesis by Taq polymerase. Selective binding of NA to CAG repeats was assessed by comparison with DNA templates conScheme 1. A) Hydrogen bonding between naphthyridine-azaquinolone (NA) and G–A mismatch. B) Schematic illustration of the NA–CAG/CAG triad complex confirmed by NMR spectroscopy. Black rectangles: 2-amino-1,8-naphthyridine moiety; gray rectangles: 8-azaquinolone moiety.

Stepwise Functionalization of Ribonucleopeptides: Optimization of the Response of Fluorescent Ribonucleopeptide Sensors for ATP

A stable complex of a peptide and RNA, ribonucleopeptide (RNP), provides a new framework to construct a macromolecular receptor for small molecules. The RNP receptor functionalized by a fluorophore-labeled Rev peptide exerts an optical signal associated with the ligand binding events. Replacing the Rev peptide of the ATP-binding RNP with a fluorophore-modified Rev peptide affords a fluorescent ATP sensor.