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.

Reversible Control of Gene Expression by Guest-Modified Adenosines in a Cell-Free System via Host-Guest Interaction.

Gene expression technology has become an indispensable tool for elucidating biological processes and developing biotechnology. Cell-free gene expression (CFE) systems offer a fundamental platform for gene expression-based technology, in which the reversible and programmable control of transcription can expand its use in synthetic biology and medicine. This study shows that CFE can be controlled via the host-guest interaction of cucurbit[7]uril (CB[7]) with N6-guest-modified adenosines. These adenosine derivatives were conveniently incorporated into the DNA strand using a post-synthetic approach and formed a selective and stable base pair with complementary thymidine in DNA. Meanwhile, alternate addition of CB[7] and the exchanging guest molecule induced the reversible formation of a duplex structure through the formation and dissociation of a bulky complex on DNA. The kinetics of the reversibility was fine-tuned by changing the size of the modified guest moieties. When incorporated into a specific region of the T7 promoter sequence, the guest-modified adenosines enabled tight and reversible control of in vitro transcription and protein expression in the CFE system. This study marks the first utility of the host-guest interaction for gene expression control in the CFE system, opening new avenues for developing DNA-based technology, particularly for precise gene therapy and DNA nanotechnology.

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 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.

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.

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.

Synthesis of C-nucleoside analogues based on the pyrimidine skeleton for the formation of anti-parallel-type triplex DNA with a CG mismatch site.

The triplex DNA forming method is an attractive tool as a gene-targeting agent. Using artificial nucleoside analogues based on C-nucleoside, stable and selective triplex DNA can be formed in a specific region of duplex DNA, and its biotechnology applications will greatly expand. In this study, we designed and synthesized novel C-nucleoside analogues based on the pyrimidine skeleton, 3MeAP-d(Y-Cl) and 3MeAP-d(Y-H), capable of recognizing a CG mismatch site that is not recognized by natural nucleosides. After incorporating them into the oligonucleotides, their triplex forming abilities were evaluated by gel-shift assay. Although it was only one sequence, the 3'-GZG-5' sequence, the stability of the CG mismatch site recognition was greatly improved compared with previous nucleoside analogues.

Enhancements in the utilization of antigene oligonucleotides in the nucleus by booster oligonucleotides.

We recently found the translocation of double-stranded DNA into the nucleus. We herein describe the concept of novel booster oligodeoxynucleotides including 2'-deoxy uridine, which release antigene oligonucleotides in the nucleus by enzymatic digestion. This system exhibited stronger hTERT mRNA expression inhibitory activity than single-stranded antigene oligonucleotides.

Development of novel C-nucleoside analogues for the formation of antiparallel-type triplex DNA with duplex DNA that includes TA and dUA base pairs.

Expansion of the triplex DNA forming sequence is required in the genomic targeting fields. Basically, triplex DNA is formed by the interaction between the triplex-forming oligonucleotides and homo-purine region with the target duplex DNA. The presence of the base pair conversion sites hampers stable triplex formation. To overcome this limitation, it is necessary to develop an artificial nucleic acid to recognize the base conversion sites, and the CG and TA base pairs. We describe the synthesis of C-nucleoside analogues and an evaluation of the ability of triplex formation. Consequently, the combined use of the novel C-nucleoside analogues, AY - AY-d(Y-NH2), AY-d(Y-Cl) and IAP-d(Y-Cl), is capable of recognizing duplex DNA including the TA or dUA base pair.

Proto-Urea-RNA (Wöhler RNA) Containing Unusually Stable Urea Nucleosides.

The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information-carrying RNA oligomers able to self-replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto-RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis-urea (biuret) and tris-urea (triuret) are able to directly react with ribose. The urea-ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides' base-pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea-containing RNA bases are good candidates for a proto-RNA world.

Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides.

Theories about the origin of life require chemical pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.

Synthesis of 2'-deoxy-4-aminopyridinylpseudocytidine Derivatives for Incorporation Into Triplex Forming Oligonucleotides.

This unit describes the detailed synthetic protocol for the preparation of the phosphoramidite units of the 2'-deoxy-4-aminopyridinylpseudocytidine derivatives. These C-nucleoside derivatives are useful units for the incorporation into triplex forming oligonucleotides (TFOs) to form the stable triplex DNA containing the CG interrupting sites. Commercially available 1-methyl-2'-deoxypseudouridine is prepared from thymidine and 5-iodo-uracil by a simple method, that is, coupling of glycal and 5-iodo-1-methyluracil by the Heck reaction, followed by desilylation and diastereoselective reduction. The carbonyl group at the 4 position of the pseudouridine derivative is activated by 3-nitorotriazole and treated with the corresponding aromatic amine compounds to produce the 2'-deoxy-4-aminopyridinylpseudocytidine derivatives. These derivatives are then successfully converted to the phosphoramidite units and incorporated into the oligodeoxynucleotides. © 2019 by John Wiley & Sons, Inc.

A one-pot, water compatible synthesis of pyrimidine nucleobases under plausible prebiotic conditions.

Herein, we report a new prebiotically plausible pathway towards a pyrimidine nucleobase in continuous manner. The route involves simultaneous methylation and carbamoylation of cyanoacetylene-derived α,ß-unsaturated thioamide with N-methyl-N-nitrosourea (MNU) in aqueous media. This provides S-methylpyrimidinone in one-pot, which can be converted into a variety of 4-substituted pyrimidine nucleobases including cytosine and uracil.

A Click-Chemistry Linked 2'3'-cGAMP Analogue.

2'3'-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3'-5' and a unique 2'-5' linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogues with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, the synthesis of a cGAMP analogue with one amide and one triazole linkage is reported. The molecule is best prepared via a first CuI -catalyzed click reaction, which establishes the triazole, while the cyclization is achieved by macrolactamization.

Modification of the aminopyridine unit of 2'-deoxyaminopyridinyl-pseudocytidine allowing triplex formation at CG interruptions in homopurine sequences.

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.

Stable and Selective Antiparallel Type Triplex DNA Formation by Targeting a GC Base Pair with the TFO Containing One N2-Phenyl-2'-deoxyguanosine.

The antiparallel triplex DNA is formed by the interaction between purine-rich triplex forming oligonucleotides (TFOs) and the homo-purine region within a duplex DNA. The formation of such a structure with the genome DNA promises to control the gene expression in a living cell. In this study, in an attempt to enhance the stability of the triplex DNAs, we have designed the N2-arylated deoxyguanosine derivatives. Among these analogues, we found that the TFOs containing N2-phenyl-2'-deoxyguanosine (PhdG) showed a stable and selective triplex DNA formation with the GC base pair as compared to the natural dG/GC triplet. However, the multiple incorporation of PhdG into the TFOs hampered the stable triplex DNA, instead, showed a tendency to form a higher order structure. Therefore, we concluded that the stable and selective triplex DNA formation is expected by the replacement of dG by PhdG in the purine-rich TFO sequence.

Noncanonical RNA Nucleosides as Molecular Fossils of an Early Earth-Generation by Prebiotic Methylations and Carbamoylations.

The RNA-world hypothesis assumes that life on Earth started with small RNA molecules that catalyzed their own formation. Vital to this hypothesis is the need for prebiotic routes towards RNA. Contemporary RNA, however, is not only constructed from the four canonical nucleobases (A, C, G, and U), it also contains many chemically modified (noncanonical) bases. A still open question is whether these noncanonical bases were formed in parallel to the canonical bases (chemical origin) or later, when life demanded higher functional diversity (biological origin). Here we show that isocyanates in combination with sodium nitrite establish methylating and carbamoylating reactivity compatible with early Earth conditions. These reactions lead to the formation of methylated and amino acid modified nucleosides that are still extant. Our data provide a plausible scenario for the chemical origin of certain noncanonical bases, which suggests that they are fossils of an early Earth.

Wet-dry cycles enable the parallel origin of canonical and non-canonical nucleosides by continuous synthesis.

The molecules of life were created by a continuous physicochemical process on an early Earth. In this hadean environment, chemical transformations were driven by fluctuations of the naturally given physical parameters established for example by wet-dry cycles. These conditions might have allowed for the formation of (self)-replicating RNA as the fundamental biopolymer during chemical evolution. The question of how a complex multistep chemical synthesis of RNA building blocks was possible in such an environment remains unanswered. Here we report that geothermal fields could provide the right setup for establishing wet-dry cycles that allow for the synthesis of RNA nucleosides by continuous synthesis. Our model provides both the canonical and many ubiquitous non-canonical purine nucleosides in parallel by simple changes of physical parameters such as temperature, pH and concentration. The data show that modified nucleosides were potentially formed as competitor molecules. They could in this sense be considered as molecular fossils.

Effect of the 3-halo substitution of the 2'-deoxy aminopyridinyl-pseudocytidine derivatives on the selectivity and stability of antiparallel triplex DNA with a CG inversion site.

Triplex formation against a target duplex DNA has the potential to become a tool for the genome research. However, there is an intrinsic restriction on the duplex DNA sequences capable of forming the triplex DNA. Recently, we demonstrated the selective formation of the stable antiparallel triplexes containing the CG inversion sites using the 2'-deoxy-1-methylpseudocytidine derivative (ΨdC), whose amino group was conjugated with the 2-aminopyridine at its 5-position as an additional hydrogen bonding unit (AP-ΨdC). The 1-N of 2-aminopyridine was supposed to be protonated to form the hydrogen bond with the guanine of the CG inversion site. In this study, to test the effect of the 3-substitution of the 2-aminopyridine unit of AP-ΨdC on the triplex stability, we synthesized the 3-halogenated 2-aminopyridine derivatives of AP-ΨdC. The pKa values 1-N of the 2-aminopyridine unit of AP-ΨdC as the monomer nucleoside were determined to be 6.3 for 3-CH3 (MeAP-ΨdC), 6.1 for 3-H (AP-ΨdC), 4.3 for 3-Cl (ClAP-ΨdC), 4.4 for 3-Br (BrAP-ΨdC), and 4.7 for 3-I (IAP-ΨdC), suggesting that all the halogenated AP-ΨdCs are not protonated under neutral conditions. Interestingly, although the recognition selectivity depends on the sequence context, the TFO having the sequence of the 3'-G-(IAP-ΨdC)-A-5' context showed the selective triplex formation with the CG inversion site. These results suggest that the protonation at the 1-N position plays an important role in the stable and selective triplex formation of AP-ΨdC derivatives in any sequences.