Synthesis Of Oligoribonucleotides Research Articles

Lipophilic Conjugates for Carrier-Free Delivery of RNA Importable into Human Mitochondria.

Defects in human mitochondrial genome can cause a wide range of clinical disorders that still do not have efficient therapies. The natural pathway of small noncoding RNA import can be exploited to address therapeutic RNAsinto themitochondria. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and "in solution" chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.

Solid‐Phase Synthesis of RNA 5′‐Azides and Their Application for Labeling, Ligation, and Cyclization Via Click Chemistry

RNAs with 5' functional groups have been gaining interest as molecular probes and reporter molecules. Copper-catalyzed azide-alkyne cycloaddition is one of the most straightforward methods to access such molecules; however, RNA functionalization with azide group has been posing a synthetic challenge. This article describes a simple and efficient protocol for azide functionalization of oligoribonucleotides 5'-end in solid-phase. An azide moiety is attached directly to the C5'-end in two steps: (i) -OH to -I conversion using methyltriphenoxyphosphonium iodide, and (ii) -I to -N3 substitution using sodium azide. The reactivity of the resulting compounds is exemplified by fluorescent labeling using both copper(I)-catalyzed (CuAAC) and strain-promoted (SPAAC) azide-alkyne cycloaddition reactions, ligation of two RNA fragments, and cyclization of short bifunctionalized oligonucleotides. The protocol makes use of oligoribonucleotides synthesized by standard phosphoramidite approach on solid support, using commercially available 2'-O-PivOM-protected monomers. Such a protection strategy eliminates the interference between the iodination reagent and silyl protecting groups (TBDMS, TOM) commonly used in RNA synthesis by phosphoramidite approach. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Solid-phase synthesis of oligoribonucleotide 5'-azides Basic Protocol 2: CuAAC labeling of oligoribonucleotide 5'-azides in solution Alternate Protocol 1: CuAAC labeling of oligoribonucleotide 5'-azides on solid support Basic Protocol 3: SPAAC labeling of oligoribonucleotide 5'-azides Basic Protocol 4: CuAAC ligation of oligoribonucleotide 5'-azides Basic Protocol 5: CuAAC cyclization of oligoribonucleotide 5'-azides Support Protocol: HPLC Purification.

Chemical Synthesis of Oligoribonucleotide (ASL of tRNALysT. brucei) Containing a Recently Discovered Cyclic Form of 2‐Methylthio‐N6‐threonylcarbamoyladenosine (ms2ct6A)

AbstractInvited for the cover of this issue is Elzbieta Sochacka and co‐workers at Lodz University of Technology, Poland. The image depicted the manual chemical synthesis of tRNA fragment containing hypermodified cyclic 2‐methylthio‐N6‐threonylcarbamoyladenosine (ms2ct6A). Read the full text of the article at 10.1002/chem.201902411.

Front Cover: Chemical Synthesis of Oligoribonucleotide (ASL of tRNALysT. brucei) Containing a Recently Discovered Cyclic Form of 2‐Methylthio‐N6‐threonylcarbamoyladenosine (ms2ct6A) (Chem. Eur. J. 58/2019)

Front Cover: Chemical Synthesis of Oligoribonucleotide (ASL of tRNA^Lys<i>T. brucei</i>) Containing a Recently Discovered Cyclic Form of 2‐Methylthio‐<i>N</i>⁶‐threonylcarbamoyladenosine (ms²ct⁶A) (Chem. Eur. J. 58/2019)

Chemical Synthesis of Oligoribonucleotide (ASL of tRNALys T. brucei) Containing a Recently Discovered Cyclic Form of 2-Methylthio-N6 -threonylcarbamoyladenosine (ms2 ct6 A).

The synthesis of the protected form of 2-methylthio-N6 -threonylcarbamoyl adenosine (ms2 t6 A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms2 t6 A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms2 t6 A-oligonucleotide (the anticodon stem and loop fragment of tRNALys from T. brucei). Finally, stereochemically secure ms2 t6 A→ms2 ct6 A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms2 ct6 A unit. The applied post-synthetic approach provides two sequentially homologous ms2 t6 A- and ms2 ct6 A-oligonucleotides that are suitable for further comparative structure-activity relationship studies.

Synthesis of 5'-Thio-3'-O-ribonucleoside Phosphoramidites.

The chemical synthesis of phosphoramidite derivatives of all four 5'-deoxy-5'-thioribonucleosides is described. These phosphoramidites contained trityl (A, G, C, and U), dimethoxytrityl (A and G), or tert-butyldisulfanyl (G) as the 5'-S-protecting group. The application of several of these phosphoramidites for solid-phase synthesis of oligoribonucleotides containing a 2'-O-photocaged 5'-S-phosphorothiolate linkage or 5'-thiol-labeled RNAs is also further investigated.

Plant organellar DNA primase-helicase synthesizes RNA primers for organellar DNA polymerases using a unique recognition sequence.

DNA primases recognize single-stranded DNA (ssDNA) sequences to synthesize RNA primers during lagging-strand replication. Arabidopsis thaliana encodes an ortholog of the DNA primase-helicase from bacteriophage T7, dubbed AtTwinkle, that localizes in chloroplasts and mitochondria. Herein, we report that AtTwinkle synthesizes RNA primers from a 5′-(G/C)GGA-3′ template sequence. Within this sequence, the underlined nucleotides are cryptic, meaning that they are essential for template recognition but are not instructional during RNA synthesis. Thus, in contrast to all primases characterized to date, the sequence recognized by AtTwinkle requires two nucleotides (5′-GA-3′) as a cryptic element. The divergent zinc finger binding domain (ZBD) of the primase module of AtTwinkle may be responsible for template sequence recognition. During oligoribonucleotide synthesis, AtTwinkle shows a strong preference for rCTP as its initial ribonucleotide and a moderate preference for rGMP or rCMP incorporation during elongation. RNA products synthetized by AtTwinkle are efficiently used as primers for plant organellar DNA polymerases. In sum, our data strongly suggest that AtTwinkle primes organellar DNA polymerases during lagging strand synthesis in plant mitochondria and chloroplast following a primase-mediated mechanism. This mechanism contrasts to lagging-strand DNA replication in metazoan mitochondria, in which transcripts synthesized by mitochondrial RNA polymerase prime mitochondrial DNA polymerase γ.

Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: Lipophilic conjugates with cleavable bonds

Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo. In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells.

Solution phase synthesis of short oligoribonucleotides on a precipitative tetrapodal support.

An effective method for the synthesis of short oligoribonucleotides in solution has been elaborated. Novel 2'-O-(2-cyanoethyl)-5'-O-(1-methoxy-1-methylethyl) protected ribonucleoside 3'-phosphoramidites have been prepared and their usefulness as building blocks in RNA synthesis on a soluble support has been demonstrated. As a proof of concept, a pentameric oligoribonucleotide, 3'-UUGCA-5', has been prepared on a precipitative tetrapodal tetrakis(4-azidomethylphenyl)pentaerythritol support. The 3'-terminal nucleoside was coupled to the support as a 3'-O-(4-pentynoyl) derivative by Cu(I) promoted 1,3-dipolar cycloaddition. Couplings were carried out with 1.5 equiv of the building block. In each coupling cycle, the small molecular reagents and byproducts were removed by two quantitative precipitations from MeOH, one after oxidation and the second after the 5'-deprotection. After completion of the chain assembly, treatment with triethylamine, ammonia and TBAF released the pentamer in high yields.

Draft Genome Sequence of Bacillus pumilus 7P, Isolated from the Soil of the Tatarstan Republic, Russia.

Here, we present a draft genome sequence of Bacillus pumilus strain 7P. This strain was isolated from soil as an extracellular RNase-producing microorganism. The RNase of B. pumilus 7P is considered to be a potential antiviral and therapeutic antitumor agent, and it might be appropriate for agriculture and academic synthesis of oligoribonucleotides.

ChemInform Abstract: Protective Groups in the Chemical Synthesis of Oligoribonucleotides

AbstractReview: 130 refs.

Protective groups in the chemical synthesis of oligoribonucleotides

The review is devoted to the chemical synthesis of oligoribonucleotides and the protecting groups used. In particular the existent methods of blocking 2'-OH function in nucleotide monomers for the RNA synthesis are discussed in detail.

Automated Solid-Phase Synthesis of RNA Oligonucleotides Containing a Nonbridging Phosphorodithioate Linkage via Phosphorothioamidites

This work describes a general method for the synthesis of oligoribonucleotides containing a site-specific nonbridging phosphorodithioate linkage via automated solid-phase synthesis using 5'-O-DMTr-2'-O-TBS-ribonucleoside 3'-N,N-dimethyl-S-(2,4-dichlorobenzyl) phosphorothioamidites (2a-2d). The 3'-phosphorothioamidites (2a-2d) can be conveniently prepared in good yields (86-99%) via a one-pot reaction from the corresponding 5'-O-DMTr-2'-O-TBS-ribonucleosides (1a-1d).

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.

Stereocontrolled Solid-Phase Synthesis of Phosphorothioate Oligoribonucleotides Using 2′-O-(2-Cyanoethoxymethyl)-nucleoside 3′-O-Oxazaphospholidine Monomers

A method for the synthesis of P-stereodefined phosphorothioate oligoribonucleotides (PS-ORNs) was developed. PS-ORNs of mixed sequence (up to 12mers) were successfully synthesized by this method with sufficient coupling efficiency (94-99%) and diastereoselectivity (≥98:2). The coupling efficiency was greatly improved by the use of 2-cyanoethoxymethyl (CEM) groups in place of the conventional TBS groups for the 2'-O-protection of nucleoside 3'-O-oxazaphospholidine monomers. The resultant diastereopure PS-ORNs allowed us to clearly demonstrate that an ORN containing an all-(Rp)-PS-backbone stabilizes its duplex with the complementary ORN, whereas its all-(Sp)-counterpart has a destabilizing effect.

New Insights into the Mechanism of Initial Transcription

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

ChemInform Abstract: Synthesis, Purification, and Characterization of Oligoribonucleotides That Act as Agonists of TLR7 and/or TLR8

AbstractReview: 18 refs.

The Roles of Tryptophans in Primer Synthesis by the DNA Primase of Bacteriophage T7

DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.

Improved Method for the Solid-Phase Synthesis of Oligoribonucleotide 5′-Triphosphates

We have developed an improved solid-phase method for the synthesis of 5'-triphosphates (5'-TPs) of oligoribonucleotides. The method is based on the use of salicyl phosphorochloridite as the phosphitylating reagent and the improvement is characterized by the use of the highly reactive pyrophosphorylating reagent tris(tetra-n-butylammonium) hydrogen pyrophosphate instead of the conventional tri-n-butylammonium salt for the nucleophilic substitution reaction to form the cyclic ester intermediate. The improved method can be used to generate oligoribonucleotide 5'-TPs efficiently and reproducibly.

Synthesis of Oligoribonucleotides Containing 2′-O-Methoxymethyl Group by the Phosphotriester Method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 ′-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 ′-О-methoxymethyl-containing oligoribonucleotides were examined.