Enzymatic RNA Synthesis Research Articles

Template-Independent Enzymatic RNA Synthesis.

A route to prepare ribonucleoside triphosphates featuring a 3'-aminoxy (3'-O-NH 2 ) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3'-O-NH 2 group within the extended primer regenerates the 3'-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Polθ. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.

Advances in evolved T7 RNA polymerases for expanding the frontiers of enzymatic nucleic acid synthesis.

In vitro RNA synthesis technologies are crucial in developing therapeutic RNA drugs, such as mRNA vaccines and RNA interference (RNAi) therapies. Enzymatic RNA synthesis, recognized for its sustainability and efficiency, enables the production of extensive RNA sequences under mild conditions. Among the enzymes utilized, T7 RNA polymerase is distinguished by its exceptional catalytic efficiency, enabling the precise and rapid transcription of RNA from DNA templates by recognizing the specific T7 promoter sequence. With the advancement in clinical applications of RNA-based drugs, there is an increasing demand for the synthesis of chemically modified RNAs that are stable and resistant to nuclease degradation. To this end, researchers have applied directed evolution to broaden the enzyme's substrate scope, enhancing its compatibility with non-canonical substrates and reducing the formation of by-products. This review summarizes the progress in engineering T7 RNA polymerase for these purposes and explores prospective developments in the field.

Metals in nanomotion: probing the role of extracellular vesicles in intercellular metal transfer.

Metals in living organisms and environments are essential for key biological functions such as enzymatic activity, and DNA and RNA synthesis. This means that disruption of metal ion homeostasis and exchange between cells can lead to diseases. EVs are believed to play an essential role in transporting metals between cells, but the mechanism of metal packaging and exchange remains to be elucidated. Here, we established the elemental composition of EVs at the nanoscale and single-vesicle level and showed that the metal content depends on the cell type and culture microenvironment. We also demonstrated that EVs participate in the exchange of metal elements between cells. Specifically, we used two classes of EVs derived from papaya fermented fluid (PaEVs), and decidual mesenchymal stem/stromal cells (DEVs). To show that EVs transfer metal elements to cells, we treated human osteoblast-like cells (MG63) and bone marrow mesenchymal stem cells (BMMSCs) with both classes of EVs. We found that both classes of EVs contained various metal elements, such as Ca, P, Mg, Fe, Na, Zn, and K, originating from their parent cells, but their relative concentrations did not mirror the ones found in the parent cells. Single-particle analysis of P, Ca, and Fe in DEVs and PaEVs revealed varying element masses. Assuming spherical geometry, the mean mass of P was converted to a mean size of 62 nm in DEVs and 24 nm in PaEVs, while the mean sizes of Ca and Fe in DEVs were smaller, converting to 20 nm and 30 nm respectively. When EVs interacted with BMMSCs and MG63, DEVs increased Ca, P, and Fe concentrations in BMMSCs and increased Fe concentration in MG63, while PaEVs increased Ca concentrations in BMMSCs and had no effect on MG63. The EV cargo, including proteins, nucleic acids, and lipids, differs from their origin in composition, and this variation extends to the element composition of EVs in our study. This fundamental understanding of EV-mediated metal exchange between cells could offer a new way of assessing EV functionality by measuring their elemental composition. Additionally, it will contribute novel insights into the mechanisms underlying EV production and their biological activity.

Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.

Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA-based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and yields of synthetic RNA. Here, we engineer human DNA polymerase theta (Polθ) variants and demonstrate their ability to synthesize long (95-200 nt) RNA oligonucleotides with canonical ribonucleotides and ribonucleotide analogs commonly used for stabilizing RNA for therapeutic and genome engineering applications. In contrast to natural promoter-dependent RNA polymerases, Polθ variants synthesize RNA by initiating from DNA or RNA primers, which enables the production of RNA without short abortive byproducts. Remarkably, Polθ variants show the lower capacity to misincorporate ribonucleotides compared to T7 RNA polymerase. Automation of this enzymatic RNA synthesis technology can potentially increase yields while reducing costs of synthetic RNA oligonucleotide production.

Modified Nucleotides for Chemical and Enzymatic Synthesis of Therapeutic RNA.

In recent years, RNA has emerged as a medium with a broad spectrum of therapeutic potential, however, for years, a group of short RNA fragments was studied and considered therapeutic molecules. In nature, RNA plays both functions, with coding and non-coding potential. For RNA, like any other therapeutic, to be used clinically, certain barriers must be crossed. Among them, there are biocompatibility, relatively low toxicity, bioavailability, increased stability, target efficiency and low off-target effects. In the case of RNA, most of these obstacles can be overcome by incorporating modified nucleotides into its structure. This may be achieved by both, in vitro and in vivo biosynthetic methods, as well as chemical synthesis. Some advantages and disadvantages of each approach are summarized here. The wide range of nucleotide analogues has been tested for their utility as monomers for RNA synthesis. Many of them have been successfully implemented, and a lot of pre-clinical and clinical studies involving modified RNA have been carried out. Some of these medications have already been introduced into clinics. After the huge success of RNA-based vaccines that were introduced into widespread use in 2020, and the introduction to the market of some RNA-based drugs, RNA therapeutics containing modified nucleotides appear to be the future of medicine.

One-step enzymatic modification of RNA 3' termini using polymerase θ.

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3′ terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3′ termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30–50 2′-deoxyribonucleotides to the 3′ terminus of RNA molecules of various lengths and sequences, and extends RNA 3′ termini with an assortment of 2′-deoxy and 2′,3′-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3′ terminal modification of RNA and potentially enzymatic synthesis of RNA.

Polynucleotide phosphorylase: Not merely an RNase but a pivotal post-transcriptional regulator.

Almost 60 years ago, Severo Ochoa was awarded the Nobel Prize in Physiology or Medicine for his discovery of the enzymatic synthesis of RNA by polynucleotide phosphorylase (PNPase). Although this discovery provided an important tool for deciphering the genetic code, subsequent work revealed that the predominant function of PNPase in bacteria and eukaryotes is catalyzing the reverse reaction, i.e., the release of ribonucleotides from RNA. PNPase has a crucial role in RNA metabolism in bacteria and eukaryotes mainly through its roles in processing and degrading RNAs, but additional functions in RNA metabolism have recently been reported for this enzyme. Here, we discuss these established and noncanonical functions for PNPase and the possibility that the major impact of PNPase on cell physiology is through its unorthodox roles.

Synthesis and Engineering of Circular RNAs.

Circular RNAs (circRNAs) have been discovered in all kingdoms of life. They are produced from introns as well as from exons. However, strongest interest is in circRNAs that are transcribed and spliced from exons of protein and noncoding genes in eukaryotic cells including humans. Therefore, synthesis and engineering of circRNAs as models for structure and function studies are strongly required. In vitro, methods for RNA synthesis and circularization are available. Chemical synthesis allows for preparation of RNAs incorporating nonnatural nucleotides in small RNA segments, whereas enzymatic synthesis is advantageous for production of long RNAs, however, without the possibility for site-specific modification. Strategies for chemical and enzymatic RNA synthesis may be combined to obtain long modified linear RNA strands for subsequent circularization. Here, we describe two alternative protocols for synthesis and circularization in dependence on downstream applications and template structure.

ChemInform Abstract: Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages.

Information-bearing nucleic acids display universal 3'-5' linkages, but regioisomeric 2'-5' linkages occur sporadically in non-enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site-specific 2'-5' linkages by an engineered polymerase using 3'-deoxy- or 3'-O-methyl-NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3'-5' linked DNA with good fidelity. This enables a fast and simple method for structural mutagenesis by the position-selective incorporation of 2'-5' linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild-type base sequence as we demonstrate for the 10-23 RNA endonuclease DNAzyme.

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages

Information‐bearing nucleic acids display universal 3′‐5′ linkages, but regioisomeric 2′‐5′ linkages occur sporadically in non‐enzymatic RNA synthesis and may have aided prebiotic RNA replication. Herein we report on the enzymatic synthesis of both DNA and RNA with site‐specific 2′‐5′ linkages by an engineered polymerase using 3′‐deoxy‐ or 3′‐O‐methyl‐NTPs as substrates. We also report the reverse transcription of the resulting modified nucleic acids back to 3′‐5′ linked DNA with good fidelity. This enables a fast and simple method for “structural mutagenesis” by the position‐selective incorporation of 2′‐5′ linkages, whereby nucleic acid structure and function may be probed through local distortion by regioisomeric linkages while maintaining the wild‐type base sequence as we demonstrate for the 10–23 RNA endonuclease DNAzyme.

A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.

Universal Aptamer-Based Real-Time Monitoring of Enzymatic RNA Synthesis

In vitro transcription is an essential laboratory technique for enzymatic RNA synthesis. Unfortunately, no methods exist for analyzing quality and quantity of the synthesized RNA while the transcription proceeds. Here we describe a simple, robust, and universal system for monitoring and quantifying the synthesis of any RNA in real time without interference from abortive transcription byproducts. The distinguishing feature is a universal fluorescence module (UFM), consisting of the eGFP-like Spinach aptamer and a highly active hammerhead ribozyme, which is appended to the RNA of interest (ROI). In the transcription mixture, the primary transcript is cleaved rapidly behind the ROI, thereby releasing always the same UFM, independent of the ROI sequence, polymerase, or promoter used. The UFM binds to the target of the Spinach aptamer, the fluorogenic dye DFHBI, and thereby induces a strong fluorescence signal. This design allows real-time quantification, standardization, parallelization, and high-throughput screening.

Use of a novel 5'-regioselective phosphitylating reagent for one-pot synthesis of nucleoside 5'-triphosphates from unprotected nucleosides.

5'-Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2'-deoxyribo- and ribonucleoside 5'-triphosphates (dNTPs and NTPs) from any natural or modified base. This one-pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non-bridging oxygen atom of the α-phosphate. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5'-triphosphates, as well as analysis and confirmation of natural and sulfur- or selenium-modified nucleic acids, are described in this protocol unit.

The synthesis of 2′-methylseleno adenosine and guanosine 5′-triphosphates

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2′-SeCH3-uridine and 2′-SeCH3-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2′-methylseleno-2′-deoxyadenosine and -guanosine 5′-triphosphates (2′-SeCH3-ATP and 2′-SeCH3-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

BackgroundManufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.ResultsSequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse.ConclusionsThe feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

Efficient Enzymatic Synthesis of Phosphoroselenoate RNA by Using Adenosine 5′‐(α‐P‐Seleno)triphosphate

Se the way to RNA: Adenosine 5′-(α-P-seleno)triphosphate (ATPαSe) has been synthesized and efficiently and diastereoselectively incorporated into RNA through enzymatic synthesis (see scheme). The digestion of the phosphoroselenoate RNA with phosphodiesterase is slower than that of the nonmodified RNA. This enzymatic synthesis can be generally applied in the preparation of multimilligram quantities of selenium-derivatized RNAs for X-ray crystallography.

Efficient Enzymatic Synthesis of Phosphoroselenoate RNA by Using Adenosine 5′‐(α‐P‐Seleno)triphosphate

Selenylierte RNA: Adenosin-5′-(α-P-seleno)triphosphat (ATPαSe) wurde synthetisiert und anschließend enzymatisch effizient und diastereoselektiv in RNA eingebaut (siehe Schema). Die Phosphoroselenoat-RNA wird von Phosphodiesterase langsamer verdaut als nichtmodifizierte RNA. Mit dieser enzymatischen Synthese gelingt allgemein die Erzeugung von Selen-derivatisierten RNAs im Multimilligramm-Maßstab.

INCORPORATION OF RIBONUCLEOSIDE 5′-(α-P-BORANO)TRIPHOSPHATES INTO A 20-MER RNA BY T7 RNA POLYMERASE

The enzymatic synthesis of short boranophosphate RNA was studied by comparing the yield and pattern of abortive products of in vitro transcription at steady-state conditions with that of normal RNA. Boranophosphate short RNA can be readily synthesized by T7 RNA polymerase.

Combination of chemical and enzymatic RNA synthesis.

The potential of standard in vitro transcription reactions can be dramatically expanded, if chemically synthesized low-mol-wt compounds are used as building blocks in combination with standard nucleotide 5' triphosphates (NTPs). Short oligonucleotides that terminate in guanosine effectively compete with guanosine 5' triphosphate (GTP) as starter building blocks, and they are incorporated at the 5'-end of transcripts. Applications include production of RNAs with "unfriendly 5'-ends" (they do not begin with G), variations of the 5'-sequence are possible with the same DNA template, site-specific insertion of nucleotide modifications, and addition of 5'-labels, such as fluorescein for detection or biotin for capture. Clearly, chemically synthesized, modified NTPs are inserted at internal sites. The combination with phosphorothioate linkages for detection has been developed into a powerful high-throughput method to study site-specific interference of modifications with RNA function.

Reading, writing, and modulating genetic information with boranophosphate mimics of nucleotides, DNA, and RNA.

The P-boranophosphates are efficient and near perfect mimics of natural nucleic acids in permitting reading and writing of genetic information with high yield and accuracy. Substitution of a borane (-BH3) group for oxygen in the phosphate ester bond creates an isoelectronic and isosteric mimic of natural nucleotide phosphate esters found in mononucleotides, i.e., AMP and ATP, and in RNA and DNA polynucleotides. Compared to natural nucleic acids, the boranophosphate RNA and DNA analogs demonstrate increased lipophilicity and resistance to endo- and exonucleases, yet they retain negative charge and similar spatial geometry. Borane groups can readily be introduced into the NTP and dNTP nucleic acid monomer precursors to produce alpha-P-borano nucleoside triphosphate analogs (e.g., NTPalphaB and dNTPalphaB). The NTPalphaB and dNTPalphaB are, in fact, good to excellent substrates for RNA and DNA polymerases, respectively, and allow ready enzymatic synthesis of RNA and DNA with P-boranophosphate linkages. Further, boranophosphate polymer products are good templates for replication, transcription, and gene expression; boronated RNA products are also suitable for reverse transcription to cDNA. Fully substituted boranophosphate DNA can activate the RNase H cleavage of RNA in RNA:DNA hybrids. Moreover, certain dideoxy-NTPalphaB analogs appear to be better substrates for viral reverse transcriptases than the regular ddNTPs, and may offer promising prodrug alternatives in antiviral therapy. These properties make boranophosphates promising candidates for diagnostics; aptamer selection; gene therapy; and antiviral, antisense, and RNAi therapeutics. The boranophosphates constitute a versatile family of phosphate mimics for processing genetic information and modulating gene function.