Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

Inhibition of protein synthesis through RNA-based tandem G-quadruplex formation.

We demonstrate that an RNA template containing eight GGG repeat sequences exhibits a unique tandem G-quadruplex structure in which two individual G-quadruplexes are aligned in close proximity. Because of their unexpected stability, tandem G-quadruplexes formed in the coding region of mRNA strands effectively inhibited in vitro protein synthesis.

Small molecule-induced trinucleotide repeat contractions during in vitro DNA synthesis.

We demonstrated that a synthetic ligand NA, which selectively binds to a 5'-CAG-3'/5'-CAG-3' triad, induced repeat contractions during DNA polymerase-mediated primer extension through the CAG repeat template. A thorough capillary electrophoresis and sequencing analysis revealed that the d(CAG)20 template gave shortened nascent strands mainly containing 3-6 CTG units in the presence of NA.

Structures and Catalytic Activities of Complexes between Heme and All Parallel-Stranded Monomeric G-Quadruplex DNAs.

Heme in its ferrous and ferric states [heme(Fe2+) and heme(Fe3+), respectively] binds selectively to the 3'-terminal G-quartet of all parallel-stranded monomeric G-quadruplex DNAs formed from inosine(I)-containing sequences, i.e., d(TAGGGTGGGTTGGGTGIG) DNA(18mer) and d(TAGGGTGGGTTGGGTGIGA) DNA(18mer/A), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet, to form 1:1 complexes [heme-DNA(18mer) and heme-DNA(18mer/A) complexes, respectively]. These complexes exhibited enhanced peroxidase activities, compared with that of heme(Fe3+) alone, and the activity of the heme(Fe3+)-DNA(18mer/A) complex was greater than that of the heme(Fe3+)-DNA(18mer) one, indicating that the 3'-terminal A of the DNA sequence acts as an acid-base catalyst that promotes the catalytic reaction. In the complexes, a water molecule (H2O) at the interface between the heme and G-quartet is coordinated to the heme Fe atom as an axial ligand and possibly acts as an electron-donating ligand that promotes heterolytic peroxide bond cleavage of hydrogen peroxide bound to the heme Fe atom, trans to the H2O, for the generation of an active species. The intermolecular nuclear Overhauser effects observed among heme, DNA, and Fe-bound H2O indicated that the H2O rotates about the H2O-Fe coordination bond with respect to both the heme and DNA in the complex. Thus, the H2O in the complex is unique in terms of not only its electronic properties but also its dynamic ones. These findings provide novel insights into the design of heme-deoxyribozymes and -ribozymes.

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.

G-Quadruplex DNA- and RNA-Specific-Binding Proteins Engineered from the RGG Domain of TLS/FUS.

Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation.

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 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.

Formation of a ligand-assisted complex of two RNA hairpin loops.

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.

Guanine-tethered antisense oligonucleotides as synthetic riboregulators.

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.

A synthetic riboswitch that operates using a rationally designed ligand-RNA pair.

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.

Activation of prokaryotic translation by antisense oligonucleotides binding to coding region of mRNA.

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.

Structure-activity studies on the fluorescent indicator in a displacement assay for the screening of small molecules binding to RNA.

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.

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.

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 host's 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.

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.

Unstable tandem repeats in promoters confer transcriptional evolvability.

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.