With the application of nucleic acids in the treatment of diseases, human attention to nucleic acids in medicine is gradually increasing. In order to obtain a large amount of nucleic acids in a short time for use in the pharmaceutical field, the in vitro synthesis of nucleic acids is particularly important. The in vitro Synthesis of nucleic acids is mainly divided into biological enzymatic synthesis and chemical solid-phase synthesis, and the two synthetic pathways have their own advantages and disadvantages. Biological enzymatic synthesis can quickly synthesize long-chain nucleic acids with a certain fidelity, but it needs to rely on different polymerases for non-natural nucleic acids (modified nucleic acids). Chemical solid-phase synthesis can achieve the synthesis of non-natural nucleic acids by adding different phosphoramidite monomers, but it is limited by phosphoramidite monomer synthesis (cumbersome synthesis path) and nucleic acid length (only shorter nucleic acid chains can be synthesized). Therefore, here we will introduce several examples of studying the in vitro synthesis of modified RNA and single-stranded DNA (ssDNA).
The enzymatic method has the following advantages for producing Nucleotides:
Recently, the article "Template-independent enzymatic synthesis of RNA oligonucleotides" published in Nature biotechnology reported a platform for enzymatic synthesis of RNA based on water phase, which can write natural and modified RNA oligonucleotides one base at a time without the need for a template sequence.
The entire synthesis platform requires three components for enzymatic extension: 3′-blocked reversible terminator nucleotides, enzymes capable of their robust and indiscriminate incorporation, and an initiator oligonucleotide. The reversible terminator group prevents uncontrolled extension of the enzyme and limits the extension sequence to a single sequence. The sequence and length of the initial oligonucleotide can be different, and it can be replaced with some functional modifications at the carrier site, such as a fluorescent group. The typical cycle of enzymatic synthesis begins with step 1, where the initial oligonucleotide primer is extended in the presence of reversible terminator nucleoside triphosphate (RT-NTP) and enzyme to form an extension product of N+1*, where N and the asterisk represent the length of the initial oligonucleotide primer and the reversible terminator blocking group, respectively. Then, step 2 is performed to remove the reversible terminator blocking group to obtain an N+1 extension product without a protecting group. Then, RT-NTP is added to proceed to the next synthesis cycle. Finally, the oligonucleotide is separated from the solid support after the complete sequence is obtained.
Fig. 1 Overview of the synthesis process of template-independent enzymatic RNA oligonucleotides. (Wiegand, et al., 2024)
Allyl ether was selected as the reversible terminator group, and RT-NTPs with reversible terminator groups of A, U, G and C were synthesized. The ability of these four RT-NTPs to perform single-cycle enzymatic RNA synthesis was investigated. The results showed that the initial primer oligonucleotide disappeared, the N+1 extension product appeared successfully, and the allyl ether group could be successfully removed.
According to the same method, five controlled enzymatic synthesis cycles were attempted using a Cy5-labeled primer, and the deprotected extension product was successfully obtained. At the same time, after introducing the modification into the substrate, it was found that this method could extend 2’-F and 2’-OMe modified RT-NTPs, but the reversible blocking group needed to be replaced for PS modification.
In summary, the authors developed a controllable non-template-dependent enzymatic RNA Oligonucleotide Synthesis method. By simulating solid-phase synthesis, it can get rid of the dependence on templates in RNA enzymatic synthesis, and it can not only be used for the synthesis of natural RNA sequences, but also for the synthesis of commonly used modified RNA sequences in ASO.
Different from the idea of the previous article, the article "Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases" published in Nature Communications also reported a strategy for synthesizing base-modified RNA by enzymatic methods. The main idea of this article is to engineer thermophilic DNA polymerases SFM4-3 and TGK to introduce single or multiple modified nucleotides into RNA.
In the past, the commonly used method for obtaining modified RNA long chains was through in vitro transcription (IVT), but this method requires the initial sequence to contain GGG trinucleotides for some incompatible complex Modifications and is prone to premature transcription termination for complex structured RNA. Some studies have shown that SFM4-3 derived from Thermococcus aquaticus (Taq) A-family polymerase or TGK polymerase derived from Thermococcus gorgonarius B-family DNA polymerase (Tgo) can synthesize 2'-modified DNA or xenonucleic acids (XNA). Based on this idea, this paper uses engineered DNA polymerases (SFM4-3 and TGK) to synthesize base-modified RNA under different conditions.
Fig. 2 Comparison of currently used methods with the presented engineered DNA polymerase-based route to base-modified RNA. (Brunderová, et al., 2024)
The authors synthesized different modification groups: click groups (ethynyl, E), hydrophobic groups (pentyn-1-yl, Pent and phenyl, Ph), chloroacetamide (CA) and formylthienyl (FT), as well as mBdp, Cy5 and Cy3 containing different tags. The extension of different polymerase variants was tested for the insertion of a single modified nucleotide at a single site and four sites, and TGK and SFM4-3 were able to smoothly extend to full length. For different modified nucleotides, the authors divided them into three groups: rAPhTP, rUPhTP, rCPhTP and rGTP; rAETP, rUBioTP, rCPhTP and rGTP; rAPhTP, rUBioTP, rCmBdpTP and rGTP. For these three groups, TGK can obtain full-length products with high yields, while T7 RNAP only obtains partial full-length products in the case of the second group of substrates.
Because the extension efficiency of TGK is better than that of SFM4-3, TGK was used for subsequent studies. In order to remove the DNA primer, rATP, rUTP, rCCy5TP and rGTP monomers were used as substrates, and ssDNA containing a single dU modification was used as a primer for primer extension. The extension product was then treated with uracil-DNA glycosylase (UDG) to form a base-deficient site, and DNase eventually degraded the DNA template. In order to obtain multi-modified RNA with fluorescent modifications at the end, the authors introduced a fluorescently modified dT at the downstream adjacent position of the primer dU site. Based on this, hypermodified RNAs with fluorescent labels at the 5' end (rAETP, rCPhTP, rUBioTP and rGPentTP) were obtained.
Based on this, the authors can achieve site-specific RNA labeling of two different sites in one pot, and designed mRNA encoding nanoluciferase with an internal ribosome entry site, which can emit light after successful translation.
In summary, the authors developed an engineered thermostable DNA polymerase for modified RNA synthesis, which is compatible with various modified rNXTPs. This method can achieve traceless removal of primers and 5'-end labeling. Unlike the first article, which does not rely on the template, this article designed a clever method to achieve degradation of the DNA template and introduce a label at the 5'-end.
ssDNA is usually synthesized by chemical solid phase synthesis, but solid phase synthesis has the disadvantages of high cost and high organic waste generation. Enzymatic synthesis of ssDNA has the advantages of simple synthesis steps and low cost, and it is more convenient to synthesize unconventional ssDNA than solid phase synthesis. However, it is very difficult to separate ssDNA from complementary chains of the same length in enzymatic synthesis. The article "Robust Enzymatic Production of DNA G-Quadruplex, Aptamer, DNAzyme, and Other Oligonucleotides: Applications for NMR" reported in JACS proposed an enzymatic synthesis method called "palindrome-nicking-dependent amplification" (PaNDA), which can synthesize a large amount of ssDNA simply and stably. Under non-denaturing conditions, ssDNA products can be easily separated by anion exchange chromatography.
PaNDA consists of three components: Bst 2.0 DNA polymerase with strand displacement function; Nt.BstNBI nicking Enzyme; input DNA, where the input DNA contains a template sequence and a palindromic primer sequence. The polymerase extends the DNA primer chain and produces double-stranded DNA (dsDNA), and the Nt.BstNBI nicking enzyme recognizes the palindromic sequence in the template chain and generates a nick between the 4th and 5th bases at the 3' end of the recognition sequence. This nick allows the DNA polymerase to perform the same extension process again, displacing the previous ssDNA product. Therefore, ssDNA gradually accumulates through the above cycle and is finally separated by anion exchange chromatography.
Fig. 3 Enzymatic synthesis of ssDNA by PaNDA. (Wang, et al., 2024)
The PaNDA method can add radiolabeled dNTPs to the substrate to achieve radiolabeling of ssDNA, and can characterize sequences containing G-quadruplexes by NMR. The authors verified the 22 nt telomeric repeat DNA G-quadruplex, and this method can be used to characterize whether the DNA aptamer contains G-quadruplexes. Finally, this method can also perform rapid isotope labeling of DNAzyme.
In summary, the PaNDA strategy developed by the authors can prepare ssDNA simply and quickly, and can also perform isotope labeling of ssDNA. Due to the application of NMR in the characterization of nucleic acid structure, PaNDA can be combined with NMR to achieve faster characterization.
Enzymatic synthesis and solid-phase synthesis are the mainstream methods for in vitro synthesis of nucleic acids, and both have their advantages and disadvantages. The methods in the above three articles try to improve the shortcomings of enzymatic synthesis, such as the dependence on DNA templates in RNA synthesis, the difficulty in synthesizing sequences containing modified nucleotides, and the difficulty in separating the template chain in ssDNA. All three methods have certain application value, and the engineered polymerase mentioned in the second article should be the main direction for solving the synthesis of modified RNA. Enzymatic synthesis requires more efficient polymerases to achieve in vitro synthesis of natural nucleotides and modified nucleotides, which makes the work of engineered polymerases particularly important.
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