RNA Oligomers Research Articles

Synthesis of a Convertible Linker Containing a Disulfide Group for Oligonucleotide Functionalization

The synthesis and incorporation of a tosylated phosphoramidite linker containing a disulfide bond is described. Incorporation of the linker into short DNA and RNA oligomers proceeded efficiently using automated solid phase synthesis. Treatment of the support bound oligonucleotide followed by cleavage from the solid support provided a variety of common functional handles, expanding the strategies of bifunctional modification of synthetic oligonucleotides for conjugation applications.

RNANetMotif: Identifying sequence-structure RNA network motifs in RNA-protein binding sites.

RNA molecules can adopt stable secondary and tertiary structures, which are essential in mediating physical interactions with other partners such as RNA binding proteins (RBPs) and in carrying out their cellular functions. In vivo and in vitro experiments such as RNAcompete and eCLIP have revealed in vitro binding preferences of RBPs to RNA oligomers and in vivo binding sites in cells. Analysis of these binding data showed that the structure properties of the RNAs in these binding sites are important determinants of the binding events; however, it has been a challenge to incorporate the structure information into an interpretable model. Here we describe a new approach, RNANetMotif, which takes predicted secondary structure of thousands of RNA sequences bound by an RBP as input and uses a graph theory approach to recognize enriched subgraphs. These enriched subgraphs are in essence shared sequence-structure elements that are important in RBP-RNA binding. To validate our approach, we performed RNA structure modeling via coarse-grained molecular dynamics folding simulations for selected 4 RBPs, and RNA-protein docking for LIN28B. The simulation results, e.g., solvent accessibility and energetics, further support the biological relevance of the discovered network subgraphs.

Lyotropic isotropic to columnar phase transition in RNA solutions

ABSTRACT RNA oligomers exhibit lyotropic chiral nematic and columnar liquid crystal phases. The lyotropic chiral nematic and columnar phases with special emphasize on the isotropic to columnar phase transition in RNA solutions are described based on Landau theory. A free energy is constructed based on the order parameters to describe the chiral nematic and columnar liquid crystal phases and isotropic to columnar phase transition. The lyotropic isotropic to columnar phase transition is found to be always first order. The birefringence has been calculated in the isotropic phase of isotropic to columnar phase transition. Theoretical observation of the existence of the lyotropic isotropic to columnar phase transition in RNA solutions is confirmed with experimental results.

Generation of RNA with 2', 3'-Cyclic Phosphates by Deoxyribozyme Cleavage in Frozen Solutions.

The generation of terminal 2', 3'-cyclic phosphates on RNA oligomers is an important process in the study of tRNA splicing and repair, ribozyme catalysis, and RNA circularization. Here, we describe a simple method for producing 2', 3'-cyclic phosphate functionalized RNA by the deoxyribozyme-catalyzed cleavage of a short 3'-RNA overhang in frozen solution. This method avoids the nonspecific modification and degradation of RNA and attached functional groups (e.g., fluorophores) inherent in other methods, and the use of frozen conditions enables cleavage at very low divalent metal ion concentrations, limiting RNA hydrolysis.

An in vitro Assay of mRNA 3' end Using the E. coli Cell-free Expression System.

At the end of about 80% of the operon in Escherichia coli, translation terminationdecouples transcription, leading to Rho-dependent transcription termination (RDT). However, no in vitro or in vivo assay system has proven to be good enough to see the 3' end of the mRNA generated by RDT. Here, we present a cell-free assay system that could provide detailed information on the 3' end of a transcript RNA generated by RDT. Our protocol shows how to extract transcript RNA generated by transcription reactions from a cell-free extract, followed by an RNA oligomer ligation to the 3' end of a transcript RNA of interest. The 3' end of the RNA is amplified using RT-PCR. Its genetic location can be determined using a gene-specific primer extension reaction. The 3' ends of mRNA can be visualized and quantified by polyacrylamide gel electrophoresis. One significant advantage of a cell-free assay system is that factors involved in the generation of the 3' end, such as proteins and sRNA, can be directly assayed by exogenously adding factor(s) to the reaction. Graphic abstract: An illustration of the experimental methodology.

Atomistic Simulations of Thermal Unfolding.

This tutorial will provide a practical overview of the use of atomistic simulations to study thermal unfolding of biomolecules, in particular small proteins and RNA oligomers. The tutorial focuses on the use of atomistic, all atom simulations of biomolecules in explicit solvent, to study (reversible) thermal unfolding. The simulation methods described here have also been applied to study biomolecules using implicit solvent and coarse-grained models. We do not intend to provide an up-to-date review of the vast literature of biomolecular dynamics, enhanced sampling methods, force field developments, and applications of these methods. The purpose of this tutorial is to provide basic guidelines into the use of these methods to the starting scientist.

A simple electroanalysis of polyG RNA in mixtures with 3′,5′-cyclic guanosine monophosphate achieved by selective desorption of the monomers from the electrode surface

• A simple label-free technique for analysis of nucleic acid polymerization mixtures. • The technique is based on selective removal of interfering monomers from electrode. • Monomers are removed by treatment with a surfactant or water at elevated temperature. Previously it has been shown that cyclic nucleoside monophosphates can spontaneously polymerize to form RNA oligonucleotides under conditions simulating prebiotic conditions on Archean Earth. The most efficient polymerization was documented with 3′,5′-cyclic guanosine monophosphate (cGMP). In this work a method for fast detection of short polyG RNAs present in a large overabundance of cGMP, modeling conditions in the non-enzymatic nucleotide polymerization mixtures, is presented. The method is based on electrochemical measurements of guanine (G) oxidation signals yielded by RNA oligomers adsorbed onto the surface of a pyrolytic graphite electrode (PGE). To avoid false positive results arising from the G oxidation signals due to co-adsorbed cGMP, a method for selective removal of the monomers from the electrode surface has been devised. In the first step, both cGMP and RNAs are co-adsorbed onto the PGE surface. In the second step, the cGMP is selectively desorbed using treatments in solutions of different tested surfactants (SDS, Tween 20 or Triton X-100), or by washing in deionized water at elevated temperature. We show that this new approach is suitable for selective analysis of products of polymerization reactions from mixtures of their building blocks.

Depsipeptide Nucleic Acids: Prebiotic Formation, Oligomerization, and Self-Assembly of a New Proto-Nucleic Acid Candidate.

The mechanism by which informational polymers first formed on the early earth is currently unknown. The RNA world hypothesis implies that RNA oligomers were produced prebiotically, before the emergence of enzymes, but the demonstration of such a process remains challenging. Alternatively, RNA may have been preceded by an earlier ancestral polymer, or proto-RNA, that had a greater propensity for self-assembly than RNA, with the eventual transition to functionally superior RNA being the result of chemical or biological evolution. We report a new class of nucleic acid analog, depsipeptide nucleic acid (DepsiPNA), which displays several properties that are attractive as a candidate for proto-RNA. The monomers of depsipeptide nucleic acids can form under plausibly prebiotic conditions. These monomers oligomerize spontaneously when dried from aqueous solutions to form nucleobase-functionalized depsipeptides. Once formed, these DepsiPNA oligomers are capable of complementary self-assembly and are resistant to hydrolysis in the assembled state. These results suggest that the initial formation of primitive, self-assembling, informational polymers on the early earth may have been relatively facile if the constraints of an RNA-first scenario are relaxed.

Liposome-Assisted Delivery of Biomembrane-Impermeable Cargo to Primitive Cells

We report on a platform for targeted delivery, and incorporation of membrane-impermeable compounds into cell-like unilamellar lipid bilayer microcompartments. We perform the delivery by means of an open-space microfluidic device, which perfuses individual surface-adhered compartments (primitive model cells) with cargo-loaded small unilamellar liposomes, composed of cationic, i.e. fusogenic, lipids. We show specifically that fluorescently labelled RNA and DNA oligomers can be successfully internalized in this way.

Amino Acid Specific Nonenzymatic Montmorillonite‐Promoted RNA Polymerization

AbstractUnderstanding prebiotic RNA synthesis is essential to both the RNA world and RNA‐protein co‐evolution theories of the origin of life. Nonenzymatic templated RNA synthesis occurs in solution or by montmorillonite clay heterogenous catalysis but the high magnesium concentrations required are deleterious to protocell membranes. Here, we explore a multicomponent environmental system consisting of amino acids, RNA mononucleotides and montmorillonite at various Mg2+ concentrations. We show that specific alpha amino acids, especially those that were prebiotically most relevant, act as prebiotic coenzymes and further enhance montmorillonite‐catalyzed polymerization in a cooperative mechanism to produce even longer RNA oligomers. Significantly, and different from template‐directed nonenzymatic RNA polymerization by primer extension, added Mg2+ is not required for montmorillonite‐catalyzed polymerization, especially as enhanced by specific amino acids. Thus amino acid specific montmorillonite‐catalyzed RNA polymerization is compatible with protocell membranes and could occur in a wider variety of geochemical environments of various Mg2+ concentrations.

Terminal Mono- and Bis-Conjugates of Oligonucleotides with Closo-Dodecaborate: Synthesis and Physico-Chemical Properties.

Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, the closo-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5′-, 3′-, or both terminal positions of DNA, RNA, 2′-O-Me RNA, and 2′-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3′-end of all oligomers stabilized their complementary complexes, whereas analogous 5′-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2′-O-methyl and 2′-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.

Label-free RNA-based electrochemical nanobiosensor for detection of Hepatitis C

In this study, an ultrasensitive label-free nanobiosensor for detecting the Hepatitis C virus (HCV) RNA was fabricated, without the need for target denaturation, by using an RNA oligomer as a probe. The biosensor was prepared by electrodeposition of the copper oxide (Cu2O) and gold nanoparticles (AuNPs) on the nitrogen-doped graphene (NG) coated fluorine-doped tin oxide electrode (FTOE). The thiolated complementary probe of RNA virus was adsorbed on the AuNPS through monolayer self-assembly (SAM), after that, the RNA target of HCV virus binds to its complementary sequence which leads to a significant change in differential pulse voltammetry (DPV) response of the probe in the presence of Fe(CN)63−/4− as redox sensing probe. Various important parameters were optimized and at optimum conditions, the nanobiosensor had a wide dynamic range (1 × 10−15–1 × 10−6 mol. L−1) with a very low detection limit of 1 × 10−15 mol. L−1. The biosensor was successfully evaluated in the blood real samples and according to its good selectivity, wide linear range, acceptable reproducibility, and repeatability; the proposed biosensor has a suitable ability for detection of the HCV RNA in clinical applications.

Harnessing chemical energy for the activation and joining of prebiotic building blocks.

Life is an out-of-equilibrium system sustained by a continuous supply of energy. In extant biology, the generation of the primary energy currency, adenosine 5'-triphosphate and its use in the synthesis of biomolecules require enzymes. Before their emergence, alternative energy sources, perhaps assisted by simple catalysts, must have mediated the activation of carboxylates and phosphates for condensation reactions. Here, we show that the chemical energy inherent to isonitriles can be harnessed to activate nucleoside phosphates and carboxylic acids through catalysis by acid and 4,5-dicyanoimidazole under mild aqueous conditions. Simultaneous activation of carboxylates and phosphates provides multiple pathways for the generation of reactive intermediates, including mixed carboxylic acid-phosphoric acid anhydrides, for the synthesis of peptidyl-RNAs, peptides, RNA oligomers and primordial phospholipids. Our results indicate that unified prebiotic activation chemistry could have enabled the joining of building blocks in aqueous solution from a common pool and enabled the progression of a system towards higher complexity, foreshadowing today's encapsulated peptide-nucleic acid system.

Magnetic microbeads-based amperometric immunoplatform for the rapid and sensitive detection of N6-methyladenosine to assist in metastatic cancer cells discrimination

This work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5′-triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.

Designing of RNA aptamer against DNA binding domain of the glucocorticoid receptor: A response element-based in-silico approach

Virtual screening, a conventional in-silico approach to design an RNA aptamer against target proteins require huge RNA library containing 1010 to 1015 combination of RNA oligomers and high-performance computing systems. However, in the case of nuclear receptor proteins, screening can be narrowed down by using response element sequences rather than random RNA oligomer library. In this study, we used a novel method to design RNA aptamer against the DNA binding domain of the glucocorticoid receptor α (GRα). GRα plays a vital role in cancer metastasis such as colon, cervical and breast cancer by activating the S100A8 calcium-binding protein, which makes it a potential drug target for those cancers. We started the screening of 24 RNA aptamers (16 nucleotides long), all of which are glucocorticoid response elements (GRE) of S100A8. Among the aptamers screened, Apt-2, Apt-5, Apt-6 and Apt-15 are found to be most suitable by molecular docking and dynamic studies. The stability and compactness of the aptamer-protein complexes were assessed by GROMACS. The binding energies were rescored using the MM-PBSA method, which were −3679.581, −3690.892, −8246.052 and −3412.802 KJ/mol, respectively for Apt-2, Apt- 5, Apt-6 and Apt-15. The designed RNA aptamer may directly bind to the DNA binding domain of GR and prevent the trans-activation of the S100A8 gene by blocking the binding of GR to its response element. Thus, this novel approach of design the response elements-based RNA aptamer against GRα like nuclear receptor proteins will help to generate target-specific RNA aptamers with minimal efforts and cost. Communicated by Ramaswamy H. Sarma

Sustainability and Chaos in the Abiotic Polymerization of 3′,5′ Cyclic Guanosine Monophosphate: The Role of Aggregation

AbstractPolymerization of 3′,5′ cyclic guanosine monophosphate (3′,5′ cGMP) was previously reported to afford short RNA sequences from a plausible mildly activated prebiotic precursor in a non‐enzymatic and template‐free manner. In the current paper we analyze the reaction with PAGE as well as by infrared micro‐ and nanospectroscopy on time scales not considered before. We provide evidence that a preliminary oligomerization step in dry state allows for a continued synthesis of RNA oligomers even in the presence of subsequently added water. On a longer time scale, the oligomerization yield oscillates in an apparently chaotic fashion hinting at the importance of some non‐reversible phase‐separation processes on the experimentally observed outcome of the polymerization/degradation chemistries. The long‐lasting (hundreds of hours) intrinsic sustainability and resilience of the process gives a robust prebiotic potential to this unique oligomerization reaction, which could lead to the very first oligonucleotide sequences on the primordial Earth.

Synthesis of Nucleobase-Modified RNA Oligonucleotides by Post-Synthetic Approach.

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

Abstract B25: HNRNPH1-dependent splicing of the fusion oncogene EWS-FLI1 reveals a targetable RNA G-quadruplex interaction

Abstract The primary oncogenic event in ~85% of Ewing sarcomas is a chromosomal translocation that generates a fusion gene encoding an aberrant transcription factor EWS-FLI1. In a third of EWS-FLI1 driven tumors, translocations that retain exon 8 of EWSR1 must exclude this exon to synthesize a protein-encoding mRNA. We have previously demonstrated that the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) binds to G-rich sequences within EWSR1 exon 8 and that this binding event is required to express an in-frame EWS-FLI1 mature mRNA. G-rich nucleic acids may fold into stable tertiary structures, such as RNA guanine quadruplexes (rG4s). Our current studies are focused on the tertiary RNA structure of the G-rich regions within EWSR1 exon 8 and its role in HNRNPH1-dependent RNA processing. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of rare transcript variants of wild-type EWSR1. We demonstrate that guanine-rich sequences within EWSR1 exon 8 can fold into RNA G-quadruplex structures and that HNRNPH1 preferentially binds these RNAs when in a folded state. Critically, we also demonstrate that transfection of a single-stranded RNA oligomer corresponding to one of these G-rich sequences (rG1) reduces the expression of EWS-FLI1 mRNA and EWS-FLI1 protein and decreases its transcriptional activity. Small molecules that can displace HNRNPH1 binding may provide a therapeutic vulnerability in a subset of Ewing sarcoma. To evaluate if this protein-RNA interaction is amenable to small molecules, we performed displacement assays with HNRNPH1 protein and an EWSR1 exon 8 RNA oligomer (rG1) at varying concentrations of a G4-binding molecule, pyridostatin (PDS). We found that PDS disrupts the formation of the HNRNPH1-rG1 complex (IC50 of 7 micromolar). Furthermore, treatment with PDS selectivity inhibits the growth of Ewing sarcoma cell lines harboring EWSR1 exon 8 fusions, decreases EWS-FLI1 activity in cell-based reporter assays, and restores mRNA expression of EWS-FLI1 deregulated transcriptional targets. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a promising strategy for future therapeutics against this fusion oncogene expressed in a third of Ewing sarcoma. Citation Format: Carla Neckles, Robert E. Boer, Nicholas Aboreden, Allison M. Cross, Robert L. Walker, Bong-Hyun Kim, Suntae Kim, John S. Schneekloth, Natasha J. Caplen. HNRNPH1-dependent splicing of the fusion oncogene EWS-FLI1 reveals a targetable RNA G-quadruplex interaction [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B25.

Structural Insights into RNA Dimerization: Motifs, Interfaces and Functions.

In comparison with the pervasive use of protein dimers and multimers in all domains of life, functional RNA oligomers have so far rarely been observed in nature. Their diminished occurrence contrasts starkly with the robust intrinsic potential of RNA to multimerize through long-range base-pairing (“kissing”) interactions, self-annealing of palindromic or complementary sequences, and stable tertiary contact motifs, such as the GNRA tetraloop-receptors. To explore the general mechanics of RNA dimerization, we performed a meta-analysis of a collection of exemplary RNA homodimer structures consisting of viral genomic elements, ribozymes, riboswitches, etc., encompassing both functional and fortuitous dimers. Globally, we found that domain-swapped dimers and antiparallel, head-to-tail arrangements are predominant architectural themes. Locally, we observed that the same structural motifs, interfaces and forces that enable tertiary RNA folding also drive their higher-order assemblies. These feature prominently long-range kissing loops, pseudoknots, reciprocal base intercalations and A-minor interactions. We postulate that the scarcity of functional RNA multimers and limited diversity in multimerization motifs may reflect evolutionary constraints imposed by host antiviral immune surveillance and stress sensing. A deepening mechanistic understanding of RNA multimerization is expected to facilitate investigations into RNA and RNP assemblies, condensates, and granules and enable their potential therapeutical targeting.

Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides.

The spontaneous emergence of long RNA molecules on the early Earth, a phenomenon central to the RNA World hypothesis, continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic mononucleotides under neutral to alkaline conditions, albeit in fully dehydrated state. In this study, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, wherein starting reactants were subjected to repeated dehydration–rehydration (DH–RH) regimes. DH–RH conditions, a recurring geological theme that was prevalent on prebiotic Earth, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions. The polymerization of 2′–3′ and 3′–5′ cyclic nucleotides of a purine (adenosine) and a pyrimidine (cytidine) was investigated. Additionally, the effect of amphiphiles was also evaluated. Furthermore, to discern the effect of “realistic” conditions on this process, the reactions were also performed using a hot spring water sample from a candidate early Earth environment. Our study showed that the oligomerization of cyclic nucleotides under DH–RH conditions resulted in intact informational oligomers. Amphiphiles increased the stability of both the starting monomers and the resultant oligomers in selected reactions. In the hot spring reactions, both the oligomerization of nucleotides and the back hydrolysis of the resultant oligomers were pronounced. Altogether, this study demonstrates how nonenzymatic oligomerization of cyclic nucleotides, under both laboratory-simulated prebiotic conditions and in a candidate early Earth environment, could have resulted in RNA oligomers of a putative RNA World.