Uracil-selective Cross-linking in RNA and Inhibition of miRNA Function by 2-Amino-6-vinyl-7-deazapurine Deoxynucleosides.

MicroRNAs (miRNAs) regulate gene expression through RNA interference. Consequently, miRNA inhibitors, such as anti-miRNA oligonucleotides (AMOs), have attracted attention for treating miRNA overexpression. To achieve efficient inhibition, we developed 2-amino-6-vinylpurine (AVP) nucleosides that form covalent bonds with uridine counterparts in RNA. We demonstrated that mRNA cross-linked with AVP-conjugated antisense oligonucleotides with AVP were protected from gene silencing by exogenous miRNA. However, endogenous miRNA function could not be inhibited in cells, probably because of slow cross-linking kinetics. We recently developed ADpVP, an AVP derivative bearing a 7-propynyl group-which boasts faster reaction rate than the original AVP. Here, we synthesized dADpVP-a deoxy analog of ADpVP-through a simplified synthesis protocol. Evaluation of the cross-linking reaction revealed that the reaction kinetics of dADpVP were comparable to those of ADpVP. In addition, structural analysis of the cross-linked adduct discovered N3 linkage against uridine. Incorporating dADpVP into two types of miRNA inhibitors revealed a marginal impact on AMO efficacy yet improved the performance of target site blockers. These results indicate the potential of cross-linking nucleosides for indirect miRNA function inhibition.

Michael addition-activated alkylation of G-quadruplex DNA with methylamine-protected vinyl-quinazolinone derivatives.

The role of G-quadruplex (G4) in cellular processes can be investigated by the covalent modification of G4-DNA using alkylating reagents. Controllable alkylating reagents activated by external stimuli can react elegantly and selectively. Herein, we report a chemical activation system that can significantly boost the reaction rate of methylamine-protected vinyl-quinazolinone (VQ) derivative for the alkylation of G4-DNA. The two screened activators can transform low-reactive VQ-NHR' to highly reactive intermediates following the Michael addition mechanism. This approach expands the toolbox of activable G4 alkylating reagents.

Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries.

The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule-RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule-RNA interactions and facilitates the discovery of novel RNA-binding molecules.

Water-Dispersible Carbon Nano-Test Tubes as a Container for Concentrated DNA Molecules.

Invited for the cover of this issue are Takashi Kyotani, Tetsuji Itoh and co-workers at Tohoku University, Gunma University and AIST. The image depicts the synthesis of water-dispersible carbon nano-test tubes by using a template technique and the selective adsorption of DNA into the inner space of these test tubes. Read the full text of the article at 10.1002/chem.202301422.

Water-Dispersible Carbon Nano-Test Tubes as a Container for Concentrated DNA Molecules.

Water-dispersible carbon nano-test tubes (CNTTs) with an inner and outer diameter of about 25 and 35 nm, respectively, were prepared by the template technique and then their inner carbon surface was selectively oxidized to introduce carboxy groups. The adsorption behavior of DNA molecules on the oxidized CNTTs (Ox-CNTTs) was examined in the presence of Ca2+ cations. Many DNA molecules are attracted to the inner space of Ox-CNTTs based on the Ca2+ -mediated electrostatic interaction between DNA phosphate groups and carboxylate anions on the inner carbon surface. Moreover, the total net charge of the DNA adsorbed was found to be equal to the total charge of the carboxylate anions. This selective adsorption into the interior of Ox-CNTTs can be explained from the fact that the electrostatic interaction onto the inner concave surface is much stronger than that on the outer convex surface. On the other hand, the desorption of DNA easily occurs whenever Ca2+ cations are removed by washing with deionized water. Thus, each of Ox-CNTTs works well as a nano-container for a large amount of DNA molecules, thereby resulting in the occurrence of DNA enrichment in the nanospace.

Formation of Direction-Controllable Pseudorotaxane and Catenane Using Chemically Cyclized Oligodeoxynucleotides and Their Noncovalent RNA Labeling.

The formation of interlocked structures, such as rotaxane and catenane, enables noncovalent conjugations. We previously confirmed that the chemically cyclized pseudorotaxane-forming oligodeoxynucleotides (prfODNs) with double-tailed parts formed a pseudorotaxane structure with the target DNA and RNA via the slipping process. Here, we report the one-step synthesis of cyclized prfODNs from alkyne-modified ODNs, after which we investigated the properties and mechanism of the slipping process and performed noncovalent RNA labeling with prfODNs. Additionally, the catenane structure was formed by the combination of pseudorotaxane formation with a 5'-end-phosphorylated RNA and enzymatic ligation. The newly synthesized prfODN represents a new tool for achieving the noncovalent conjugation of various functional moieties to RNAs.

Synthesis of 6-Alkynylated Purine-Containing DNA via On-Column Sonogashira Coupling and Investigation of Their Base-Pairing Properties.

Synthetic unnatural base pairs have been proven to be attractive tools for the development of DNA-based biotechnology. Our group has very recently reported on alkynylated purine-pyridazine pairs, which exhibit selective and stable base-pairing via hydrogen bond formation between pseudo-nucleobases in the major groove of duplex DNA. In this study, we attempted to develop an on-column synthesis methodology of oligodeoxynucleotides (ODNs) containing alkynylated purine derivatives to systematically explore the relationship between the structure and the corresponding base-pairing ability. Through Sonogashira coupling of the ethynyl pseudo-nucleobases and CPG-bound ODNs containing 6-iodopurine, we have demonstrated the synthesis of the ODNs containing three NPu derivatives (NPu1, NPu2, NPu3) as well as three OPu derivatives (OPu1, OPu2, OPu3). The base-pairing properties of each alkynylated purine derivative revealed that the structures of pseudo-nucleobases influence the base pair stability and selectivity. Notably, we found that OPu1 bearing 2-pyrimidinone exhibits higher stability to the complementary NPz than the original OPu, thereby demonstrating the potential of the on-column strategy for convenient screening of the alkynylated purine derivatives with superior pairing ability.

o-Nitrobenzyl Oxime Ethers Enable Photoinduced Cyclization Reaction to Provide Phenanthridines under Aqueous Conditions.

In this paper, we describe a novel N-O photolysis of o-nitrobenzyl oxime ethers that enables the synthesis of phenanthridines via intramolecular cyclization reactions. Without the use of additional photocatalysts or photosensitizers, the process proceeds with an efficiency of ≤96% upon exposure of the sample to near-visible light (405 nm) under aqueous conditions. Through the photoinduced production of a fluorescent phenanthridine derivative in HeLa cells, the progress of the reaction under biological conditions was demonstrated. This photoinduced cyclization reaction could be used as a different photochemical instrument to control biological processes by inducing the production of bioactive molecules.

Selective Chemical Modification to the Higher-Order Structures of Nucleic Acids.

DNA and RNA can adopt a variety of stable higher-order structural motifs, including G-quadruplex (G4 s), mismatches, and bulges. Many of these secondary structures are closely related to the regulation of gene expression. Therefore, the higher-order structure of nucleic acids is one of the candidate therapeutic targets, and the development of binding molecules targeting the higher-order structure of nucleic acids has been pursued vigorously. Furthermore, as one of the methodologies for detecting the higher-order structures of these nucleic acids, developing techniques for the selective chemical modification of the higher-order structures of nucleic acids is also underway. In this personal account, we focus on the following higher-order structures of nucleic acids, double-stranded DNA containing the abasic site, T-T/U-U mismatch structure, and G-quadruplex structure, and describe the development of molecules that bind to and chemically modify these structures.

Reactivity Modulation of Reactive OFF-ON Type G-Quadruplex Alkylating Agents.

Alkylating agents for nucleic acids have been widely used in cancer chemotherapy, as well as in chemical biology for strong inhibitors and tagging methods. We provide a series of reactive OFF-ON type alkylating agents which enable the reactivity modulation toward G-quadruplex (G4) DNA and RNA. Due to the protonation-accelerated process and equilibrium elimination method, the amine leaving groups show highly reactive and storable properties in an extensive investigation of vinyl quinazolinone (VQ) precursors with different leaving groups.

Hybridization-specific chemical reactions to create interstrand crosslinking and threaded structures of nucleic acids.

The interstrand crosslinking and threaded structures of nucleic acids have high potential in oligonucleotide therapeutics, chemical biology, and nanotechnology. For example, properly designed crosslinking structures provide high activity and nuclease resistance for anti-miRNAs. The noncovalent labeling and modification by the threaded structures are useful as new chemical biology tools. Photoreversible crosslinking creates smart materials, such as reversible photoresponsive gels and DNA origami objects. This review introduces the creation of interstrand crosslinking and threaded structures, such as catenanes and rotaxanes, based on hybridization-specific chemical reactions and their functions and perspectives.

Selective and stable base pairing by alkynylated nucleosides featuring a spatially-separated recognition interface.

Unnatural base pairs (UBPs) which exhibit a selectivity against pairing with canonical nucleobases provide a powerful tool for the development of nucleic acid-based technologies. As an alternative strategy to the conventional UBP designs, which involve utility of different recognition modes at the Watson-Crick interface, we now report that the exclusive base pairing can be achieved through the spatial separation of recognition units. The design concept was demonstrated with the alkynylated purine (NPu, OPu) and pyridazine (NPz, OPz) nucleosides endowed with nucleobase-like 2-aminopyrimidine or 2-pyridone ('pseudo-nucleobases') on their major groove side. These alkynylated purines and pyridazines exhibited exclusive and stable pairing properties by the formation of complementary hydrogen bonds between the pseudo-nucleobases in the DNA major groove as revealed by comprehensive Tm measurements, 2D-NMR analyses, and MD simulations. Moreover, the alkynylated purine-pyridazine pairs enabled dramatic stabilization of the DNA duplex upon consecutive incorporation while maintaining a high sequence-specificity. The present study showcases the separation of the recognition interface as a promising strategy for developing new types of UBPs.

Development of a selective ligand for G-G mismatches of CGG repeat RNA inducing the RNA structural conversion from the G-quadruplex into a hairpin-like structure.

The trinucleotide CGG repeat is located in the 5'-UTR of FMR1 and its abnormal expansion and formation of a noncanonical RNA structure causes fetal genetic diseases. In this study, a small molecular dimer-type ligand consisting of dual G-clamp units for the recognition of two neighboring guanines was synthesized, and the binding properties for the r(CGG) repeats were investigated. Compound 2 was confirmed to bind to the mismatch guanines in the stem region of the r(CGG) repeat hairpin. In addition, the RNase T1 assay demonstrated that 2 induced the structural conversion of the r(CGG)8 repeat from the G-quadruplex into a hairpin-like structure.

Synthesis of Crosslinked 2'-OMe RNA Duplexes Using 2-Amino-6-Vinylpurine and Their Application for Effective Inhibition of miRNA Function.

Crosslinking reactions to nucleic acids are an effective way to prepare stable complexes formed by covalent bonding. We demonstrated that fully 2'-O-methylated (2'-OMe) RNAs having a 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. Recently, we reported the preparation of crosslinked 2'-OMe RNA duplexes using AVP and the anti-miRNA oligonucleotides (AMOs) containing crosslinked duplexes at the terminal positions. These AMOs exhibited efficient microRNA (miRNA) inhibition at very low concentrations. In this article, we describe the chemical synthesis of 2'-OMe oligonucleotides containing AVP and preparation of the AMOs bearing crosslinked 2'-OMe RNA duplexes using AVP. In addition, we describe in detail the miRNA inhibition assay using these AMOs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphoramidite of 2-amino-6-vinylguanosine derivative Basic Protocol 2: Synthesis of AVP-2'-OMe RNA Basic Protocol 3: Evaluation of the crosslink reactivity of CFO containing AVP to the 2'-OMe RNA and preparation of AMOs containing crosslinked duplex Basic Protocol 4: miRNA inhibition assays.

Rapid Alkene-Alkene Photo-Cross-Linking on the Base-Flipping-Out Field in Duplex DNA.

Specific chemical reactions by enzymes acting on a nucleobase are realized by flipping the target base out of the helix. Similarly, artificial oligodeoxynucleotides (ODNs) can also induce the base flipping and a specific chemical reaction. We now report an easily prepared and unique structure-providing photo-cross-linking reaction by taking advantage of the base-flipping-out field formed by alkene-type base-flipping-inducing artificial bases. Two 3-arylethenyl-5-methyl-2-pyridone nucleosides with the Ph or An group were synthesized and incorporated into the ODNs. We found that the two Ph derivatives provided the cross-linked product in a high yield only by a 10 s photoirradiation when their alkenes overlap each other in the duplex DNA. The highly efficient reaction enabled forming a cross-linked product even when using the duplex with a low Tm value.

Synthesis of crosslinked 2'-OMe RNA duplexes and their application for effective inhibition of miRNA function.

The interstrand crosslinking of nucleic acids is one of the strategies to create the stable complex between an oligonucleotide and RNA by covalent bond formation. We previously reported that fully 2'-O-methylated (2'-OMe) RNAs having the 2-amino-6-vinylpurine (AVP) exhibited an efficient crosslinking to uracil in the target RNA. In this study, we established a chemical method to efficiently synthesize the crosslinked 2'-OMe RNA duplexes using AVP and prepared the anti-miRNA oligonucleotides (AMOs) containing the antisense targeting miR-21 and crosslinked duplex at the terminal sequences. These AMOs showed a markedly higher anti miRNA activity than that of the commercially-available miR-21 inhibitor which has locked nucleic acid (LNA) residues.

Selective alkylation of parallel G-quadruplex structure.

The selective alkylation of nucleic acids is important for a medicinal approach and biological study. We now report a novel selective alkylation of the parallel G-quadruplex structure using the conjugate of the macrocyclic hexaoxazole L2G2-6OTD-1M1PA and vinyl-quinazolinone-S(O)Me (6OTD-VQ-S(O)Me).

Liquid-like droplet formation by tumor suppressor p53 induced by multivalent electrostatic interactions between two disordered domains.

Early in vivo studies demonstrated the involvement of a tumor-suppressing transcription factor, p53, into cellular droplets such as Cajal and promyelocytic leukemia protein bodies, suggesting that the liquid-liquid phase separation (LLPS) might be involved in the cellular functions of p53. To examine this possibility, we conducted extensive investigations on the droplet formation of p53 in vitro. First, p53 itself was found to form liquid-like droplets at neutral and slightly acidic pH and at low salt concentrations. Truncated p53 mutants modulated droplet formation, suggesting the importance of multivalent electrostatic interactions among the N-terminal and C-terminal domains. Second, FRET efficiency measurements for the dimer mutants of p53 revealed that distances between the core domains and between the C-terminal domains were modulated in an opposite manner within the droplets. Third, the molecular crowding agents were found to promote droplet formation, whereas ssDNA, dsDNA, and ATP, to suppress it. Finally, the p53 mutant mimicking posttranslational phosphorylation did not form the droplets. We conclude that p53 itself has a potential to form droplets that can be controlled by cellular molecules and by posttranslational modifications, suggesting that LLPS might be involved in p53 function.

Reactive OFF-ON type alkylating agents for higher-ordered structures of nucleic acids.

Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these structures are involved in genetic control and diseases. Selective alkylation of these higher-order structures is challenging due to the chemical instability of the alkylating agent and side-reactions with the single- or double-strand DNA and RNA. We now report the reactive OFF-ON type alkylating agents, vinyl-quinazolinone (VQ) precursors with a sulfoxide, thiophenyl or thiomethyl group for the OFF-ON control of the vinyl reactivity. The stable VQ precursors conjugated with aminoacridine, which bind to the G-4 DNA, selectively reacted with a T base on the G-4 DNA in contrast to the single- and double-strand DNA. Additionally, the VQ precursor reacted with the T or U base in the AP-site, G-4 RNA and T-T mismatch structures. These VQ precursors would be a new candidate for the T or U specific alkylation in the higher-ordered structures of nucleic acids.