RNA Triplex Research Articles

RNA helicase DEAD-box-5 is involved in R-loop dynamics of preimplantation embryos.

R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

Studying the folding mechanism of RNA triplex model system using time-resolved SWAXS technique

Studying the folding mechanism of RNA triplex model system using time-resolved SWAXS technique

Unraveling the complexity: Advanced methods in analyzing DNA, RNA, and protein interactions.

Exploring the intricate interplay within and between nucleic acids, as well as their interactions with proteins, holds pivotal significance in unraveling the molecular complexities steering cancer initiation and progression. To investigate these interactions, a diverse array of highly specific and sensitive molecular techniques has been developed. The selection of a particular technique depends on the specific nature of the interactions. Typically, researchers employ an amalgamation of these different techniques to obtain a comprehensive and holistic understanding of inter- and intramolecular interactions involving DNA-DNA, RNA-RNA, DNA-RNA, or protein-DNA/RNA. Examining nucleic acid conformation reveals alternative secondary structures beyond conventional ones that have implications for cancer pathways. Mutational hotspots in cancer often lie within sequences prone to adopting these alternative structures, highlighting the importance of investigating intra-genomic and intra-transcriptomic interactions, especially in the context of mutations, to deepen our understanding of oncology. Beyond these intramolecular interactions, the interplay between DNA and RNA leads to formations like DNA:RNA hybrids (known as R-loops) or even DNA:DNA:RNA triplex structures, both influencing biological processes that ultimately impact cancer. Protein-nucleic acid interactions are intrinsic cellular phenomena crucial in both normal and pathological conditions. In particular, genetic mutations or single amino acid variations can alter a protein's structure, function, and binding affinity, thus influencing cancer progression. It is thus, imperative to understand the differences between wild-type (WT) and mutated (MT) genes, transcripts, and proteins. The review aims to summarize the frequently employed methods and techniques for investigating interactions involving nucleic acids and proteins, highlighting recent advancements and diverse adaptations of each technique.

Employing pH-responsive RNA triplex to control CRISPR/Cas9-mediated gene manipulation in mammalian cells

The clustered regularly interspersed short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is an RNA-guided platform for highly efficient and specific genome targeting in diverse organisms, which has been exploited for various applications in gene manipulation. Compared with the constantly active CRISPR/Cas9 function, conditional control of its activity can improve the performance of the system with reduced side effects and high spatiotemporal precision. The pH-responsive triplex RNA was successful used in CRISPR-derived RNA/trans-activating crRNA (crRNA/tracrRNA) of CRISPR/Cas9, thus affecting RNA/dead Cas9 (dCas9) complex to target DNA in vitro and in vivo. This design of triplex RNA opens a new window towards the broad involvement of eukaryotic cells for conditional control of CRISPR/Cas9 function.

Binding and stabilizating effect of RNA triplex poly(U)⋅poly(A)*poly(U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(dppx)]2.

Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. Spectrophotometric titrations and viscosity measurements suggested that enantiomers Λ-1 and Δ-1 bind with the triplex through intercalation, while the binding strengths of the two enantiomers toward the triplex differed only slightly from each other. Fluorescence titrations showed that although enantiomers Λ-1 and Δ-1 exhibited molecular "light switch" effects toward the triplex, the effect of Δ-1 was more marked. Furthermore, Furthermore, thermal denaturation showed that the two enantiomers have significantly different stabilizing effects on the triplex. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar to a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex. Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)⋅poly(A)*poly(U) have been investigated by various biophysical techniques. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar as a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex.

Binding properties of a molecular “light switch” ruthenium(II) polypyridyl complex toward double- and triple-helical forms of RNA

To further develop new luminescent probes for RNA, a new ruthenium(II) polypyridyl complex [Ru(dmb)2dppz-idzo]2+ (dmb = 4,4′-dimethyl-2,2′-bipyridine, dppz-idzo = dppz-imidazolone) has been synthesized and characterized in this study. Binding properties of [Ru(dmb)2dppz-idzo]2+ to RNA duplex poly(A) · poly(U) and triplex poly(U) · poly(A) ∗ poly(U) have been explored by spectroscopic techniques and viscometry experiments. The binding modes of [Ru(dmb)2dppz-idzo]2+ to RNA duplex and triplex are intercalation as revealed from spectral titrations and viscosity experiments, while the binding strength of this complex to duplex structure is significantly greater than that of triplex structure. Fluorescence titrations indicate that [Ru(dmb)2dppz-idzo]2+ can act as a molecular “light switch” for both duplex poly(A) · poly(U) and triplex poly(U) · poly(A) ∗ poly(U), while [Ru(dmb)2dppz-idzo]2+ is more sensitive to poly(A) · poly(U) compared to poly(U) · poly(A) ∗ poly(U) and poly(U). Therefore, this complex can distinguish between RNA duplex, triplex and poly(U), and can as luminescent probes for the three RNAs used in this study. In addition, thermal denaturation studies show that [Ru(dmb)2dppz-idzo]2+ is able to significantly increase the Stabilization of RNA duplex and triplex. The results obtained in this study may contribute to further understanding of the binding of Ru(II) complexes with different structural RNAs.

Binding and stabilizations effects of RNA triplex poly(U*A-U) by enantiomers of ruthenium(II) polypyridyl complex [Ru(bpy)2(10-OH-dppz)]2+

Two new chiral ruthenium (II) complexes, Δ-[Ru (bpy)2 (10-OH-dppz)]2+ (Δ-1; bpy = 2,2ʹ-bipyridine, 10-OH-dppz = 10-hydroxyl-dipyrido [3,2-a:2′,3′-c]phenazine) and Λ-[Ru (bpy)2 (10-OH-dppz)]2+ (Λ-1), have been synthesized and characterized in this work. The binding properties of the two enantiomers with an RNA triplex poly (U*A-U) [in text, dash (−) denotes the Watson-Crick base pairing and the asterisk (*) denotes the Hoogsteen base pairing] have been studied. Analysis of the absorption and fluorescence spectra suggest that the binding constant of enantiomer Λ-1 toward the triplex is somewhat smaller than that of enantiomer Δ-1, which is further supported by viscosity experiments. Viscosity experiments showed that although the two enantiomers were intercalated to the triplex, the Δ-enantiomer was a better intercalator than the Λ-enantiomer. Furthermore, thermal denaturation experiments demonstrate that although the two enantiomers can significantly increase the triplex stabilization, unlike enantiomer Λ-1, enantiomer Δ-1 stabilizes the Watson-Crick base-paired duplex and the Hoogsteen base-paired third strand of the triplex with obvious selectivity. In addition, the triplex stabilization by enantiomer Δ-1 was significantly stronger than that by Λ-1. The obtained results indicate that the binding ability of enantiomers Δ-1 and Λ-1 to the triplex is dependent on the chirality of enantiomers.

Computational Methods to Study DNA:DNA:RNA Triplex Formation by lncRNAs.

Long non-coding RNAs (lncRNAs) impact cell function via numerous mechanisms. In the nucleus, interactions between lncRNAs and DNA and the consequent formation of non-canonical nucleic acid structures seems to be particularly relevant. Along with interactions between single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), such as R-loops, ssRNA can also interact with double-stranded DNA (dsDNA) to form DNA:DNA:RNA triplexes. A major challenge in the study of DNA:DNA:RNA triplexes is the identification of the precise RNA component interacting with specific regions of the dsDNA. As this is a crucial step towards understanding lncRNA function, there exist several computational methods designed to predict these sequences. This review summarises the recent progress in the prediction of triplex formation and highlights important DNA:DNA:RNA triplexes. In particular, different prediction tools (Triplexator, LongTarget, TRIPLEXES, Triplex Domain Finder, TriplexFFP, TriplexAligner and Fasim-LongTarget) will be discussed and their use exemplified by selected lncRNAs, whose DNA:DNA:RNA triplex forming potential was validated experimentally. Collectively, these tools revealed that DNA:DNA:RNA triplexes are likely to be numerous and make important contributions to gene expression regulation.

Identification of Long Noncoding RNAs That Exert Transcriptional Regulation by Forming RNA-DNA Triplexes in Prostate Cancer.

Long noncoding RNAs (lncRNAs) are involved in transcriptional regulation, and their deregulation is associated with the development of various human cancers, including prostate cancer (PCa). However, their underlying mechanisms remain unclear. In this study, lncRNAs that interact with DNA and regulate mRNA transcription in PCa were screened and identified to promote PCa development. First, 4195 protein-coding genes (PCGs, mRNAs) were obtained from the The Cancer Genome Atlas (TCGA) database, in which 1148 lncRNAs were differentially expressed in PCa. Then, 44,270 pairs of co-expression relationships were calculated between 612 lncRNAs and 2742 mRNAs, of which 42,596 (96%) were positively correlated. Among the 612 lncRNAs, 392 had the potential to interact with the promoter region to form DNA:DNA:RNA triplexes, from which lncRNA AD000684.2(AC002128.1) was selected for further validation. AC002128.1 was highly expressed in PCa. Furthermore, AD000684.2 positively regulated the expression of the correlated genes. In addition, AD000684.2 formed RNA-DNA triplexes with the promoter region of the regulated genes. Functional assays also demonstrated that lncRNA AD000684.2 promotes cell proliferation and motility, as well as inhibits apoptosis, in PCa cell lines. The results suggest that AD000684.2 could positively regulate the transcription of target genes via triplex structures and serve as a candidate prognostic biomarker and target for new therapies in human PCa.

Rational design of hybrid DNA-RNA triplex structures as modulators of transcriptional activity in vitro.

Triplex nanostructures can be formed in vitro in the promoter region of DNA templates, and it is commonly accepted that these assemblies inhibit the transcription of the downstream genes. Herein, a proof of concept highlighting the possibility of the up- or downregulation of RNA transcription is presented. Hybrid DNA-RNA triplex nanostructures were rationally designed to produce bacterial transcription units with switchable promoters. The rate of RNA production was measured using the signal of a transcribed fluorescent RNA aptamer (i.e. Broccoli). Indeed, several designed bacterial promoters showed the ability of induced transcriptional inhibition, while other properly tailored sequences demonstrated switchable enhancement of transcriptional activity, representing an unprecedented feature to date. The use of RNA-regulated transcription units and fluorescent RNA aptamers as readouts will allow the realization of biocomputation circuits characterized by a strongly reduced set of components. Triplex forming RNA oligonucleotides are proposed as smart tools for transcriptional modulation and represent an alternative to current methods for producing logic gates using protein-based components.

HIF1α-AS1 is a DNA:DNA:RNA triplex-forming lncRNA interacting with the HUSH complex

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.

Multiassay Profiling of a Focused Small Molecule Library Reveals Predictive Bidirectional Modulation of the lncRNA MALAT1 Triplex Stability In Vitro.

The rapidly accelerating characterization of RNA tertiary structures has revealed their pervasiveness and active roles in human diseases. Small molecule-mediated modulation of RNA tertiary structures constitutes an attractive avenue for the development of tools for therapeutically targeting and/or uncovering the pathways associated with these RNA motifs. This potential has been highlighted by targeting of the triple helix present at the 3'-end of the noncoding RNA MALAT1, a transcript implicated in several human diseases. This triplex has been reported to decrease the susceptibility of the transcript to degradation and promote its cellular accumulation. While small molecules have been shown to bind to and impact the stability of the MALAT1 triple helix, the small molecule properties that lead to these structural modulations are not well understood. We designed a library utilizing the diminazene scaffold, which is underexplored but precedented for nucleic acid binding, to target the MALAT1 triple helix. We employed multiple assays to holistically assess what parameters, if any, could predict the small molecule affinity and effect on triplex stability. We designed and/or optimized competition, calorimetry, and thermal shift assays as well as an enzymatic degradation assay, the latter of which led to the discovery of bidirectional modulators of triple helix stability within the scaffold-centric library. Determination of quantitative structure-activity relationships afforded predictive models for both affinity- and stability-based assays. This work establishes a suite of powerful orthogonal biophysical tools for the evaluation of small molecule:RNA triplex interactions that generate predictive models and will allow small molecule interrogation of the growing body of disease-associated RNA triple helices.

Insight into achirality and chirality effects in interactions of an racemic ruthenium(II) polypyridyl complex and its Δ- and Λ-enantiomers with an RNA triplex

RNA triplexes have a variety of potential applications in molecular biology, diagnostics and therapeutics, while low stabilization of the third strand hinders their practical utilities under physiological conditions. In this regard, achieving the third-strand stabilization by binding small molecules is a promising strategy. Chirality is one of the basic properties of nature. To clarify achirality and chirality effects on the binding and stabilizing effects of RNA triplexes by small molecules, we report for the first time the RNA interactions of an racemic ruthenium(II) polypyridyl complex [Ru(bpy)2(11-CN-dppz)]2+ (rac-Ru1) and its two enantiomers Δ/Λ-[Ru(bpy)2(11-CN-dppz)]2+ (Δ/Λ-Ru1) with an RNA triplex poly(U-A*U) (where “-” represents Watson-Crick base pairing, and “*” denotes Hoogsteen base pairing, respectively) in this work. Research shows that although rac-Ru1 and its two enantiomers Δ/Λ-Ru1 bind to the RNA triplex through the same mode of intercalation, the binding affinity for enantiomer Δ-Ru1 is much higher than that for rac-Ru1 and enantiomer Λ-Ru1. However, compared to enantiomer Λ-Ru1, the binding affinity for rac-Ru1 does not show much of an advantage, which is slightly greater than that for the former. Thermal denaturation measurements reveal both rac-Ru1 and Δ-Ru1 to have a preference for stabilizing the third strand rather than the template duplex of the RNA triplex, while Λ-Ru1 stabilizes the RNA triplex without significant selectivity. Besides, the third-strand stabilizing effects by rac-Ru1 and Δ-Ru1 are not markedly different from each other, but more marked than that by Λ-Ru1. This work shows that the binding properties of the racemic Ru(II) polypyridyl complex with the RNA triplex are not simply an average of its two enantiomers, indicating potentially complicated binding events.

Binding and stabilization effect of arene ruthenium(Ⅱ) polypyridyl complexes toward the triple-helical RNA poly(U)•poly(A)⁎poly(U)

To determine the binding properties of η6‑arene Ru(Ⅱ) complexes with the RNA triplex poly(U)•poly(A)⁎poly(U), two arene Ru(Ⅱ) complexes, [(ŋ6-C6H6)Ru(m-npip)Cl]+ (Ru1) and [(ŋ6-C6H6)Ru(m-fpip)Cl]+ (Ru2), have been synthesized and characterized in this study. The binding of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)⁎poly(U) have been investigated by viscosity measurement and various spectroscopic methods. Results demonstrate that the RNA-binding behaviors of Ru1 and Ru2 are intercalation mode. From the analyses of electronic absorption spectra and fluorescence spectra, we observed that the binding affinity for Ru2 is remarkably stronger than that for Ru1. Furthermore, thermal denaturation studies reflect that Ru1 and Ru2 selectively stabilize the RNA triplex, among them, Ru1 can stabilize the Hoogsteen base-paired strand poly(U) (the third strand), while it is little effect on the Watson-Crick base-paired duplex poly(U)•poly(A) (the template duplex). Contrary to Ru1, Ru2 can stabilize both the template duplex and the third-strand, and Ru2 is more effective for stabilization of the third-strand. To our knowledge, this work is the first example of arene metal complexes interacting with triple-helical nucleic acids. The obtained results suggest that subtle differences of substituent on the intercalation ligand may be the important factor affecting on the binding properties of the both arene Ru(Ⅱ) complexes with the RNA triplex, when Ru1 and Ru2 contain the same ancillary ligands.

Insights into the structural stability of major groove RNA triplexes by WAXS-guided MD simulations

RNA triple helices are commonly observed tertiary motifs that are associated with critical biological functions, including signal transduction. Because the recognition of their biological importance is relatively recent, their full range of structural properties has not yet been elucidated. The integration of solution wide-angle X-ray scattering (WAXS) with molecular dynamics (MD) simulations, described here, provides a new way to capture the structures of major-groove RNA triplexes that evade crystallographic characterization. This method yields excellent agreement between measured and computed WAXS profiles and allows for an atomically detailed visualization of these motifs. Using correlation maps, the relationship between well-defined features in the scattering profiles and real space characteristics of RNA molecules is defined, including the subtle conformational variations in the double-stranded RNA upon the incorporation of a third strand by base triples. This readily applicable approach has the potential to provide insight into interactions that stabilize RNA tertiary structure that enables function.

A naked-eye colorimetric molecular “light switch” based on ruthenium(II) polypyridyl complex [Ru(phen)2ttbd]2+ as binder and stabilizer for RNA duplex and triplex

Binding of [Ru(phen)2ttbd]2+ (phen = 1,10-phenanthroline, ttbd = 4-(6-propenylpyrido-[3,2-a]- phenzain-10-yl-benzene-1,2-diamine) to the RNA triplex poly(U-A*U) (herein “-” and “*” refer to the Watson-Crick and Hoogsteen binding, respectively) and the duplex poly(A-U) have been investigated by spectral technology and viscosity method. Analysis of spectral titrations and viscosity experiments as well as melting measurements suggest that [Ru(phen)2ttbd]2+ binds to the studied RNA triplex and duplex through intercalation, while its binding constant toward the triplex is greater than the duplex. Luminescent titrations indicate that [Ru(phen)2ttbd]2+ can act as a molecular “light switch” for the two RNAs and the switch effect can be detected by the naked-eye. Moreover, the “light switch” can be repeatedly cycled off and on by adjusting the pH of the solution, whereas color change in the case of the triplex is more significant compared with the duplex. To our knowledge, [Ru(phen)2ttbd]2+ is the first small molecule capable of serving as a pH-controlled reversible visual molecular “light switch” for both the RNA triplex poly(U-A*U) and duplex poly(A-U). Thermal denaturation experiments suggest that [Ru(phen)2ttbd]2+ can obviously increase the triplex stabilization, while it stabilizing third-strand is more marked in comparison with the template duplex of the triplex, indicating this complex preferentially binds to third-strand. The obtained results may be useful for understanding the binding of Ru(II) polypyridyl complexes to RNAs.

Multi‐assay profiling of diminazene library reveals predictive bidirectional modulation of MALAT1 triplex stability in vitro

The rapidly increasing characterization of RNA tertiary structures has revealed their pervasiveness and active roles in human diseases. Therefore, small molecule‐mediated modulation of RNA tertiary structures constitutes an attractive avenue for the development of tools for both therapeutically targeting and/or uncovering the pathways associated with these RNA motifs. This potential has been highlighted by preliminary targeting of the triple helix present at the 3’‐end of the non‐coding RNA MALAT1, a transcript implicated in several human diseases. This triplex has been reported to decrease the transcript susceptibility to degradation and, ultimately, promote its cellular accumulation. While small molecules have been shown to bind and impact the stability of the MALAT1 triple helix, the molecular recognition properties behind these structural modulations are not well understood. To elucidate these properties, we designed a focused library utilizing the diminazene scaffold, which is underexplored but precedented for nucleic acid binding, to target the MALAT1 triple helix. To gain a holistic perspective on small molecule recognition, we evaluated this library through a multi‐dimensional approach to assess what parameters, if any, could predict small molecule affinity and effect on triplex stability. We designed and/or optimized competition, calorimetry, and thermal shift assays as well as a novel enzymatic degradation assay, which led to the discovery of bidirectional modulators of triple helix stability within the scaffold‐centric library. Furthermore, employment of quantitative structure activity relationship (QSAR) afforded predictive models for stability‐ and affinity‐based assays. Together, this work provides novel biophysical tools for the evaluation of small molecule: RNA triplex interactions and predictive models that can be applied to small molecule interrogation of the growing body of disease‐associated RNA triple helices.

Effect of substituents on the binding of Ru(II) polypyridyl complexes [Ru(bpy)2L]2+ (L = pnpip and mnpip) to poly(U-A•U) triplex

Two ruthenium(Ⅱ) polypyridyl complexes as triple-helical RNA binders, [Ru(bpy)2pnpip](PF6)2 (Ru1, bpy = 2,2′-bipyridine, pnpip = 2-(4′-nitrophenyl)imidazo[4,5-f] [1,10]phenanthroline) and [Ru(bpy)2mnpip](PF6)2 (Ru2, mnpip = 2-(3-methyl-4-nitropnenyl)imidazo[4,5-f] [1,10] phenanthroline)), were synthesized and characterized in this work. The binding behaviors of complexes Ru1 and Ru2 with the RNA triplex poly(U-A•U) were studied by various biophysical techniques. The obtained results indicate that although the two complexes bind to the triplex in an intercalative mode, the binding affinity of Ru1 with the triplex is stronger than that of Ru2. Furthermore, Ru1 destabilized the template duplex, while Ru2 did not affect the stabilization of the duplex. The obtained results will advance our knowledge on the binding of Ru(Ⅱ) complexes with RNA triplex.

Interaction of arene ruthenium(II) complexes [(η6-C6H6)Ru(L)Cl]PF6 (L = o-fpip and p-fpip) with the RNA triplex poly(U)*poly(A)•poly(U)

To comprehend the binding properties of η6-arene Ru(II) complexes with poly(U)*poly(A)•poly(U) triplex, two arene Ru(II) complexes with different fluorine substituent positions, [(η6-C6H6)Ru(o-fpip)Cl]PF6 (Ru1,η6-C6H6 = benzene ring, o-fpip = 2-(2′‑fluorine) imidazo [4,5-f] Biver et al. (2008), Gupta et al. (2012) [1, 10] phenanthroline) and [(η6-C6H6)Ru(p-fpip)Cl]PF6 (Ru2,η6-C6H6 = benzene ring, o-fpip = 2-(4′‑fluorine) imidazo [4,5-f] Biver et al. (2008), Gupta et al. (2012) [1, 10] phenanthroline), have been synthesized and characterized in this study. The binding of Ru1 and Ru2 with poly(U)*poly(A)•poly(U) triplex has been investigated by viscosity measurement and spectroscopic methods. Analysis of UV–Vis absorption spectral titrations suggests that Ru1 and Ru2 bind to the triplex through an intercalative mode, but the binding affinity of Ru2 is slightly higher than that of Ru1, which is also verified by viscosity and EB (ethidium bromide) competition measurements. Furthermore, the thermal denaturation experiment shows that Ru1 and Ru2 increase the third-strand stabilization to a similar extent. Interestingly, the two complexes have essentially no effect on the stabilization of the template duplex. Considering the structure of Ru1 and Ru2, conceivably besides the intercalation of ligand, the force stabilizing the triplex should also involve covalent binding and electrostatic interaction. The obtained results will contribute to our understanding of the interaction of arene Ru(II) complexes with the poly(U)*poly(A)•poly(U) triplex.

Structural Insights into Human Adenovirus Type 4 Virus-Associated RNA I.

RNA molecules can adopt specific RNA triplex structures to execute critical biological functions. Human adenoviruses (HAdVs) are abundant pathogens encoding the essential, noncoding virus-associated RNA I (VA RNAI). Here, we employ a triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinoxaline 1, 10-phenanthroline (BQQ–OP), to unravel a potential RNA triplex formation in VA RNAI. The BQQ–OP cleavage of the pathogenic HAdV type 4 (HAdV-4) VA RNAI indicates that a potential triplex is formed involving the highly conserved stem 4 of the central domain and side stem 7. Further, the integrity of the HAdV-4 VA RNAI side stem 7 contributes to a potential triplex formation in vitro and virus growth in vivo. Collectively, we propose that the HAdV-4 VA RNAI can potentially form a biologically relevant triplex structure.