Triplex Formation Research Articles

Antigene and Antiproliferative Effects of Triplex-Forming Oligonucleotide (TFO) Targeted on hmgb1 Gene in Human Hepatoma Cells.

The high mobility group box 1 (hmgb1) is one of the frequently over-expressed genes whose aberrant expression is reported in a number of human cancers. Various strategies are underway to inhibit hmgb1 expression in cancer cells having considerable therapeutic value. The present work involves selective transcriptional inhibition of the hmgb1 gene using selective DNA triplex structure-based gene technology. Here, the promoter region of the hmgb1 gene at position (-183 to -165) from the transcription start site as a target was selected using bioinformatic tools. The DNA triplex formation by the DNA of the target gene and TFO was confirmed using UV absorption spectroscopy, Circular Dichroism, and Isothermal Calorimetry. Treatment of HepG2 cell with specific Triplex-forming Oligonucleotide significantly downregulated HMGB1 expression level at mRNA and protein levels by 50%, while the classical anticancer drugs, actinomycin/ adriamycin as positive controls showed 65% and the combination of TFO and drug decreased by 70%. The anti-proliferative effects of TFO correlated well with the fact of accumulation of cells in the Go phase and apoptotic cell death. Further, the binding of anti-cancer drugs to hmgb1 is stronger in DNA triplex state as compared to hmgb1 alone, suggesting the combination therapy as a better option. Therefore, the ability of hmgb1 targeted triplex-forming oligonucleotide in combination with triplex selective anticancer drug holds promise in the treatment of malignancies associated with hmgb1 overexpression. The result obtained may open up new vistas to provide a basis for the rational drug design and searching for high-affinity ligands with a high triplex selectivity.

Single-cell profiling of long noncoding RNAs and their cell lineage commitment roles via RNA-DNA-DNA triplex formation in mammary epithelium.

Long noncoding RNAs (lncRNAs), which are crucial for organ development, exhibit cell-specific expression. Thus, transcriptomic analysis based on total tissue (bulk-seq) cannot accurately reflect the expression pattern of lncRNAs. Here, we used high-throughput single-cell RNA-seq data to investigate the role of lncRNAs using the hierarchical model of mammary epithelium. With our comprehensive annotation of the mammary epithelium, lncRNAs showed much greater cell-lineage specific expression than coding genes. The lineage-specific lncRNAs were functionally correlated with lineage commitment through the coding genes via the cis- and trans-effects of lncRNAs. For the working mechanism, lncRNAs formed a triplex structure with the DNA helix to regulate downstream lineage-specific marker genes. We used lncRNA-Carmn as an example to validate the above findings. Carmn, which is specifically expressed in mammary gland stem cells (MaSCs) and basal cells, positively regulated the Wnt signaling ligand Wnt10a through formation of a lncRNA-DNA-DNA triplex, and thus controlled the stemness of MaSCs. Our study suggests that lncRNAs play essential roles in cell-lineage commitment and provides an approach to decipher lncRNA functions based on single-cell RNA-seq data.

Substitution-Inert Polynuclear Platinum Complexes Inhibit Reverse Transcription Preferentially in RNA Triplex-Forming Templates.

RNA triplexes are significant tertiary structure motifs that are found in many functional RNAs. Hence, small molecules capable of recognition, binding, and stabilization of the triple-helical RNA structures are emerging as attractive potential molecular biology tools and therapeutic agents. Here, we utilize methods of molecular biology and biophysics to study the interactions of a series of antitumor substitution-inert polynuclear platinum complexes (SI-PPCs) with triple-helical RNA structures. We show that SI-PPCs recognize and stabilize RNA triplexes and inhibit reverse transcription preferentially in the RNA template prone to the triplex formation. These so far unexplored properties of SI-PPCs suggest that the targeting of triple-stranded regions in RNA might contribute to the biological effects of SI-PPCs.

Formation of a DNA triple helical structure at BOLF1 gene of human herpesvirus 4 (HH4) genome

Eukaryotic genomes contain a large number of pyrimidine-purine rich regions and such regions can assume varied DNA conformations, including triple-stranded structures. These structures have fascinated scientists because of their considerable therapeutic applications. These structures have also profound implications in the field of nanotechnology as they can be used to develop DNA-based nanostructures and materials. Therefore, for any application, it is important to understand the formation of triplex structures, both in quantitative and qualitative terms. A combination of gel electrophoresis, UV-thermal denaturation and circular dichroism (CD) spectroscopy was used to investigate the formation of inter- as well as intramolecular triplex, in pyrimidine motif at BOLF1 gene of human herpesvirus 4 (HH4) genome. This gene codes for inner tegument protein, which plays crucial roles in viral replication. The said oligopurine•oligopyrimidine duplex was targeted via a designed triple helix forming oligopyrimidine nucleotide (TFO) in intermolecular as well as intramolecular fashion. Our studies revealed that intramolecular triplex formation takes place at acidic as well as at neutral pH; whereas low pH is required for its intermolecular version. This comparative study between inter- and intramolecular triplex allowed us to demonstrate that intramolecular structure is more stable to its intermolecular counterpart. Numerous models for mono-, bi- and trimolecular structures adopted by these DNA sequences have been suggested. This report adds to our existing knowledge about DNA triple helical structures.

RETRACTED: Long non-coding RNA CDKN2B-AS1 contributes to atherosclerotic plaque formation by forming RNA-DNA triplex in the CDKN2B promoter

BackgroundAtherosclerosis involves a slow process of plaque formation on the walls of arteries, and comprises a leading cause of cardiovascular disease. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. In this study, we aim to explore the possible involvement of lncRNA ‘cyclin-dependent kinase inhibitor 2B antisense noncoding RNA’ (CDKN2B-AS1) and CDKN2B in the progression of atherosclerosis. MethodsInitially, we quantified the expression of CDKN2B-AS1 in atherosclerotic plaque tissues and, in THP-1 macrophage-derived, and human primary macrophage (HPM)-derived foam cells. Next, we established a mouse model of atherosclerosis using apolipoprotein E knockout (ApoE−/−) mice, where lipid uptake, lipid accumulation, and macrophage reverse cholesterol transport (mRCT) were assessed, in order to explore the contributory role of CDKN2B-AS1 to the progression of atherosclerosis. RIP and ChIP assays were used to identify interactions between CDKN2B-AS1, CCCTC-binding factor (CTCF), enhancer of zeste homologue 2 (EZH2), and CDKN2B. Triplex formation was determined by RNA-DNA pull-down and capture assay as well as EMSA experiment. FindingsCDKN2B-AS1 showed high expression levels in atherosclerosis, whereas CDKN2B showed low expression levels. CDKN2B-AS1 accelerated lipid uptake and intracellular lipid accumulation whilst attenuating mRCT in THP-1 macrophage-derived foam cells, HPM-derived foam cells, and in the mouse model. EZH2 and CTCF were found to bind to the CDKN2B promoter region. An RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B promoter was found to recruit EZH2 and CTCF in the CDKN2B promoter region and consequently inhibit CDKN2B transcription by accelerating histone methylation. InterpretationThe results demonstrated that CDKN2B-AS1 promotes atherosclerotic plaque formation and inhibits mRCT in atherosclerosis by regulating CDKN2B promoter, and thereby could be a potential therapeutic target for atherosclerosis.

Transcription of DNA duplex containing deoxypseudouridine and deoxypseudoisocytidine, and inhibition of transcription by triplex forming oligonucleotide that recognizes the modified duplex

We developed new DNA triplexes that contain four base triads T-A·T, A-ψ·CBr, G-PIC·YO, and C-G·Py+, where CBr, YO, Py, ψ, and PIC are 5-bromocytosine, 5-methyl-4-pyrimidone, 2-aminopyridine, the aglycons of deoxypseudouridine, and deoxypseudoisocytidine, respectively. DNA duplex incorporating T-A, A-ψ, G-PIC, and C-G, and triplex forming oligonucleotide incorporating T, CBr, YO, and Py formed the triplex as evaluated by Tm measurements. The triplex formation was successfully applied to the inhibition of transcription of the DNA duplex incorporating T7-promoter sequence modified by the above modified bases.

LncRNA CISAL Inhibits BRCA1 Transcription by Forming a Tertiary Structure at Its Promoter.

SummaryCisplatin-based neoadjuvant chemotherapy has been shown to improve survival in patients with squamous cell carcinoma (SCC), but clinical biomarkers to predict chemosensitivity remain elusive. Here, we show the long noncoding RNA (lncRNA) LINC01011, which we termed cisplatin-sensitivity-associated lncRNA (CISAL), controls mitochondrial fission and cisplatin sensitivity by inhibiting BRCA1 transcription in tongue SCC (TSCC) models. Mechanistically, we found CISAL directly binds the BRCA1 promoter and forms an RNA-DNA triplex structure, sequestering BRCA1 transcription factor-GABPA away from the downstream regulatory binding region. Importantly, the clinical relevance of these findings is suggested by the significant association of CISAL and BRCA1 expression levels in TSCC tumors with neoadjuvant chemosensitivity and overall survival. We propose a new model where lncRNAs are tethered at gene promoter by RNA-DNA triplex formation, spatially sequestering transcription factors away from DNA-binding sites. Our study uncovers the potential of CISAL-BRCA1 signaling as a potential target to predict or improve chemosensitivity.

Synthesis of Deoxypseudouridine 5'-Triphosphate Bearing the Photoremovable Protecting Group at the N1 Position Capable of Enzymatic Incorporation to DNA.

Enzymatic incorporation of deoxynucleoside 5'-triphosphate bearing the photocleavable protecting group is a useful method for the preparation of photocaged oligodeoxynucleotides. Here, we describe the synthesis of new photocaged deoxynucleoside triphosphates N1-(2-nitrobenzyl)-deoxypseudouridine triphosphate (dNBΨTP) and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine triphosphate (dNPOMΨTP). We successfully synthesized dNBΨTP and dNPOMΨTP and applied them to enzymatic synthesis of photocaged oligonucleotides. In addition, we also synthesized phosphoramidites of N1-(2-nitrobenzyl)- and N1-(6-nitropiperonyloxymethyl)-deoxypseudouridine to enable chemical synthesis of photocaged oligonucleotides incorporating them. The photocleavable 2-nitrobenzyl and 6-nitropiperonyloxymethyl in oligonucleotides were cleaved by irradiation at 365 nm for 30 and 10 s, respectively. We also studied the enzymatic incorporation of dNBΨTP and dNPOMΨTP using the Klenow fragment exo-. As a result, it was clarified that dNPOMΨTP could be incorporated to oligonucleotide 193 times more efficiently than dNBΨTP, as judged by Vmax/Km. We also performed the incorporation of at least eight dNPOMΨ residues in a 35-mer oligodeoxynucleotide. It has also been revealed that the oligodeoxynucleotides incorporating photocaged deoxypseudouridine were useful for photocontrol of DNA triplex formation.

Analyzing RNA-DNA Triplex Formation in Chromatin.

A significant fraction of non-coding RNAs (ncRNAs) is associated with chromatin, shown to regulate gene expression and to organize nuclear architecture. Mechanisms of direct and indirect RNA-chromatin interactions have been described, including the sequence-specific formation of triple helix structures. Triplexes are formed by the sequence-specific binding of RNA to the bases located in the major groove of DNA. We recently showed that triplexes do exist in the context of cellular chromatin and that these structures are stabilized by the histone H3 tail of adjacent nucleosomes. The in vitro characterization of the specificity and binding affinity of triplex sequences next to nucleosomes are essential parameters to identify potential sites of RNA-chromatin interaction in vivo. Here we provide a detailed protocol to determine the influence of nucleosome positioning on triple helix formation. This assay allows the comparative quantification of triplex formation and specificity for triplex targeting sequences relative to the spatial nucleosome position.

Parallel motif triplex formation via a new, bi-directional hydrogen bonding pattern incorporating a synthetic cyanuryl nucleoside into the sense chain.

This research presents the first example of the formation of a triplex via hydrogen bonding of a synthetic cyanuryl nucleoside (Cn) composed of a 6-membered ring compound (triazine-2,4,6-trione) which functions as a pyrimidine base. The Cn was able to form the triad via hydrogen bonding in two directions with two adenines, one in the antisense and one in the parallel chain. The thermal stability of the duplex between the antisense and sense chains was evaluated via its UV melting temperature. The melting curves of triplexes possessing the cyanuryl nucleoside (sense chain) and two adenines (antisense and parallel chains) were biphasic. To prove the formation of hydrogen bonding between the cyanuryl nucleoside and the adenine base toward the major groove, structural analysis via NMR was undertaken. A cyanuryl nucleoside containing three 15N in triazine-2,4,6-trione was synthesized first, and then the 15N cyanuryl nucleoside was incorporated into target sequences. The triplex containing the A·Cn Created by potrace 1.16, written by Peter Selinger 2001-2019 ]]> A triad was analyzed via1H NMR, coupled and decoupled with 15N. This triad has two imino proton signals derived from the cyanuryl nucleoside, split according to the 15N coupling condition, at low field. These results are evidence of the formation of hydrogen bonds between the Cn and adenosine. The solution structure of the triplex was analyzed via NOE information from the imino proton. The cyanuryl nucleoside-containing triplex forms a right-handed helical structure similar to natural triplex DNA, albeit DNA having an enhanced pyrimidine analog in the sense chain capable of bidirectional hydrogen bonding with high sequence selectivity.

Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation.

Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow for isolating DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element of interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex-forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element of interest), proteins that preferentially bind to the DNA element of interest are captured and identified by mass spectrometry. Similarly, CoIFI also uses a triplex-forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element of interest.

NMR studies on oligonucleotide – Methylene blue conjugates targeting double-helical nucleic acids

Methylene blue (MB) – nucleic acid interactions are of considerable interest due to the photosensitizing activity of the dye with potential applications in medicine and biotechnology. Covalent attachment of the MB to an oligonucleotide through a flexible heptamethylene linker enabled a positioning of the dye moiety to specific sites through triplex formation with a target duplex. NMR studies demonstrated interactions of MB with the nucleic acids. In sequences with the MB moiety facing the triplex-duplex junction with an alternating CG duplex overhang next to a T·A·T triple-helical tract, proton resonances experienced severe linebroadening upon MB binding and point to kinetically labile complexes with exchange among different binding modes. For sequences with the MB moiety facing a terminal T·A·T base triad of the triplex tract, structural heterogeneity decreased when compared to a triplex without MB attached to the third strand. Also, the thermal stability of the latter construct increased significantly in the presence of MB, indicating external end stacking as predominant binding mode. Without any obvious disruptions of sequential imino-imino NOE contacts within the triplex and duplex tracts, a most favorable intercalation between T·A·T base triples or CG base pairs is not supported by the present data under our experimental conditions.

Oligonucleotides Containing Phenoxazine Artificial Nucleobases: Triplex‐Forming Abilities and Fluorescence Properties

1,3-Diaza-2-oxophenoxazine ("phenoxazine"), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π-π stacking effects with adjacent bases in a duplex. Phenoxazine has been widely used for improving duplex-forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9-triaza-2-oxophenoxazine (9-TAP), could improve triplex-forming abilities. A triplex-forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double-stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9-TAP-containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9-TAP moiety was maintained even in triplex (9-TAP:AT) formation, so 9-TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation.

A lncRNA coordinates with Ezh2 to inhibit HIF-1α transcription and suppress cancer cell adaption to hypoxia.

Hypoxia is a salient feature of the tumor microenvironment. HIF-1α is a master regulator of hypoxic adaption. The polycomb repressor complex 2 (PRC2) molecule Ezh2 is known to play roles in essential cellular processes of cell fate decisions. However, how PRC2-mediated epigenetic dynamic changes take part in hypoxic adaption is not completely understood. Recently, we identified a long non-coding RNA (lncRNA) named HITT (HIF-1α inhibitor at translation levels) that plays roles in modulating hypoxia-mediated angiogenesis and tumor growth in vivo. In this study, we reveal an important activity of HITT in evading hypoxia-induced apoptosis by coordinating with PRC2 activity to regulate HIF-1α transcription. Genetic or chemical inhibition of PRC2 significantly elevates HIF-1α mRNA levels. The occupancy of Ezh2 and its substrate H3K27me3 on the HIF-1α promoter is detected under normoxia, and is reduced by hypoxia. Restoring hypoxia-inhibited HITT expression rescues the association between Ezh2/H3K27me3 and the HIF-1α promoter, which also simultaneously abrogates hypoxia-induced HIF-1α mRNA transcription. Further mechanistic studies revealed that HITT inhibits HIF-1α transcription by guiding Ezh2 through the formation of an RNA-DNA triplex with the HIF-1α promoter. Importantly, HITT/Ezh2-regulated HIF-1α transcription leads to alerted HIF-1α protein output and elicits a significant effect to evade hypoxia-induced apoptosis. Importantly, a close association between HIF-1α mRNA and HITT was further verified in human colon cancer tissues in vivo. Collectively, these findings suggest a model for the epigenetic regulation of hypoxia-induced HIF-1α transcription modulated by lncRNA HITT, which provides important insights into how tumor cells sense and adapt to hypoxic stress.

Cooperative recognition of a repetitive sequence through consecutive formation of triplex and duplex structures

Cooperative recognition of a repetitive sequence was performed with a short single DNA strand consisting of duplex- and triplex-forming regions modified with a ligand (benzoquinoquinoxaline) to stabilize a triplex structure. The former region was complementary with one unit of a repetitive sequence and the latter had a sequence that can bind with a cognate duplex formed by another DNA molecule bound on an adjacent site. The DNA binding to one unit of the repetitive sequence is expected to facilitate the second binding to an adjacent unit through cooperative triplex formation. The cooperativity was confirmed by evaluation of thermal stabilities of the complexes with a series of model repetitive sequences.

Tuning of structural and magnetic properties by intriguing radical-radical interaction by double electron oxidation in U-A-U′ triplex formation

The spin coupling properties of a series of two di-radical centers AU and AU′ in U-A-U′ triplex were investigated. These two series are considered by intriguing the radical-radical cation (A+U+ and A+U′+), radical-dehydrogenated (A+U(-H1), A(-H1)U+, A(-H1)U′+ and A+U′(-H1)), and dehydrogenated-dehydrogenated (A(-H1)U(-H1) and A(-H1)U′(-H1)) as RNA base couplers. The structural and electronic properties were calculated employing density functional theory (DFT). The di-radical character was identified by performing DFT and complete active space self-consistent (CASSCF) calculations. The calculated value of coupling constant revealed the presence of both types of magnetic interaction i.e. ferromagnetic (FM) and anti-ferromagnetic (AFM). A strong magnetic interaction is found for the radical-dehydrogenated A(-H1)U+ series whereas a weak magnetic interaction is observed for radical-radical cation, A+U+, A+U′+ and other di-radical series e.g. A+U(-H1), A+U′(-H1), and A(-H1)U′(-H1). The transformation of FM and AFM coupling with the change in dehydrogenation sites is explained with the help of distribution of spin density.

Rapid identification of the raccoon rabies virus variant using a real-time reverse-transcriptase polymerase chain reaction

The raccoon-associated variant of rabies virus (RRV) is enzootic throughout the eastern seaboard of the United States with frequent incursions into Canada. Many wildlife management agencies are actively engaged in control programmes targeting elimination of this disease and rapid identification of raccoon rabies cases is crucial to the success of these operations. This report documents the development of a reverse transcriptase real-time PCR (RT-qPCR) that specifically identifies this rabies virus variant (RRV RT-qPCR) and which can be readily multiplexed with a generic rabies virus RT-qPCR for use as a typing tool. Using a large collection of rabies virus samples representative of the variants circulating around the world, but with a focus on those occurring in the Americas, the RRV RT-qPCR was 100% sensitive and 99.31% specific. To further apply these assays for diagnostic purposes, addition of an RT-qPCR targeting the host β-actin mRNA, which serves as an internal amplification control, in a triplex format was shown to yield highly comparable results using a subset of our viral collection. Use of these assays for early and accurate identification of this viral variant will help to optimize the utilization of resources required for control of this disease.

Convergence in LINE-1 nucleotide variations can benefit redundantly forming triplexes with lncRNA in mammalian X-chromosome inactivation

BackgroundAssociations between X-inactive transcript (Xist)–long noncoding RNA (lncRNA) and chromatin are critical intermolecular interactions in the X-chromosome inactivation (XCI) process. Despite high-resolution analyses of the Xist RNA-binding sites, specific interaction sequences are yet to be identified. Based on elusive features of the association between Xist RNA and chromatin and the possible existence of multiple low-affinity binding sites in Xist RNA, we defined short motifs (≥5 nucleotides), termed as redundant UC/TC (r-UC/TC) or AG (r-AG) motifs, which may help in the mediation of triplex formation between the lncRNAs and duplex DNA.ResultsThe study showed that r-UC motifs are densely dispersed throughout mouse and human Xist/XIST RNAs, whereas r-AG motifs are even more densely dispersed along opossum RNA-on-the-silent X (Rsx) RNA, and also along both full-length and truncated long interspersed nuclear elements (LINE-1s, L1s) of the three species. Predicted secondary structures of the lncRNAs showed that the length range of these sequence motifs available for forming triplexes was even shorter, mainly 5- to 9-nucleotides long. Quartz crystal microbalance (QCM) measurements and Monte Carlo (MC) simulations indicated that minimum-length motifs can reinforce the binding state by increasing the copy number of the motifs in the same RNA or DNA molecule. Further, r-AG motifs in L1s had a similar length-distribution pattern, regardless of the similarities in the length or sequence of L1s across the three species; this also applies to high-frequency mutations in r-AG motifs, which suggests convergence in L1 sequence variations.ConclusionsMultiple short motifs in both RNA and duplex DNA molecules could be brought together to form triplexes with either Hoogsteen or reverse Hoogsteen hydrogen bonding, by which their associations are cooperatively enhanced. This novel triplex interaction could be involved in associations between lncRNA and chromatin in XCI, particularly at the sites of L1s. Potential binding of Xist/XIST/Rsx RNAs specifically at L1s is most likely preserved through the r-AG motifs conserved in mammalian L1s through convergence in L1 nucleotide variations and by maintaining a particular r-UC/r-AG motif ratio in each of these lncRNAs, irrespective of their poorly conserved sequences.

Incorporating 2-Thiouracil into Short Double-Stranded RNA-Binding Peptide Nucleic Acids for Enhanced Recognition of A-U Pairs and for Targeting a MicroRNA Hairpin Precursor.

Chemically modified short peptide nucleic acids (PNAs) recognize RNA duplexes under near physiological conditions by major-groove PNA·RNA-RNA triplex formation and show great promise for the development of RNA-targeting probes and therapeutics. Thymine (T) and uracil (U) are often incorporated into PNAs to recognize A-U pairs through major-groove T·A-U and U·A-U base triple formation. Incorporation of a modified nucleobase, 2-thiouracil (s2U), into triplex-forming oligonucleotides stabilizes both DNA and RNA triplexes. Thiolation of uracil causes a decrease in the dehydration energy penalty for triplex formation as well as a decrease in the pKa of the N3 atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions, similar to the previously reported thiolation effect of pseudoisocytosine (J to L substitution). Here, we incorporated s2U into short PNAs, followed by binding studies of a series of s2U-modified PNAs. We demonstrated by nondenaturing polyacrylamide gel electrophoresis and thermal melting experiments that s2U and L incorporated into dsRNA-binding PNAs (dbPNAs) enhance the recognition of A-U and G-C pairs, respectively, in RNA duplexes in a position-independent manner, with no appreciable binding to the DNA duplex. Combining s2U and L modifications in dbPNAs facilitates enhanced recognition of dsRNAs and maintains selective binding to dsRNAs over ssRNAs. We further demonstrated through a cell-free assay the application of the s2U- and L-modified dbPNAs (8-mer, with a molecular mass of ∼2.3 kDa) in the inhibition of the pre-microRNA-198 maturation in a substrate-specific manner. Thus, s2U-modified dbPNAs may be generally useful for the enhanced and selective recognition of RNA duplexes and for the regulation of RNA functions.

Comparing the effects of physiologically-based metal oxide nanoparticles on ribonucleic acid and RAS/RBD-targeted triplex and aptamer interactions

Physiological metals such as zinc, magnesium, and nickel facilitate nucleic acid and protein interactions and stability. In the nanoscale, the impact these have on nucleic acid structure-function is very poorly understood and was investigated here. Nanoparticles’ (NP) RNA precipitation efficiency was in the order; NiO > MgO > ZnO > CaO > CaCO3>Cu. Gel mobility shift was observed for MgO and especially ZnO NP. Loss of staining intensity was shown for Cu suggesting this NP may denature RNA supported by the UV- and CD-spectroscopy patterns, change in area-under-the-curve (AUC) and abs260 nm measurements. Aptamer and triplex-forming oligomer (TFO) sequences were designed targeting RAS/Ras binding domain (RBD) and the impact of the NP on target interaction investigated. MgO NP promotes aptamer:RBD interaction and preserves triplex formation whereas NiO NP effects duplex migration and intensifies staining of the triplex suggesting a novel mechanism of interaction and conformation. These data strongly support the role of MgO, ZnO and NiO NP for nucleic acid nanobio interaction and suggest potential biomedical application for such novel interfaces.