Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Enzymatic Construction of Artificial Base Pairs: The Effect of Metal Shielding.

Th formation of metal base pairs is a versatile method for the introduction of metal cations into nucleic acids that has been used in numerous applications including the construction of metal nanowires, development of energy, charge-transfer devices and expansion of the genetic alphabet. As an alternative, enzymatic construction of metal base pairs is an alluring strategy that grants access to longer sequences and offers the possibility of using such unnatural base pairs (UBPs) in SELEX experiments for the identification of functional nucleic acids. This method remains rather underexplored, and a better understanding of the key parameters in the design of efficient nucleotides is required. We have investigated the effect of methylation of the imidazole nucleoside (dImnMe TP) on the efficiency of the enzymatic construction of metal base pairs. The presence of methyl substituents on dImTP facilitates the polymerase-driven formation of dIm4Me -AgI -dIm and dIm2Me TP-CrIII -dIm base pairs. Steric factors rather than the basicity of the imidazole nucleobase appear to govern the enzymatic formation of such metal base pairs. We also demonstrate the compatibility of other metal cations rarely considered in the construction of artificial metal bases by enzymatic DNA synthesis under both primer extension reaction and PCR conditions. These findings open up new directions for the design of nucleotide analogues for the development of metal base pairs.

Chemical methods for the modification of RNA.

RNA is often considered as being the vector for the transmission of genetic information from DNA to the protein synthesis machinery. However, besides translation RNA participates in a broad variety of fundamental biological roles such as gene expression and regulation, protein synthesis, and even catalysis of chemical reactions. This variety of function combined with intricate three-dimensional structures and the discovery of over 100 chemical modifications in natural RNAs require chemical methods for the modification of RNAs in order to investigate their mechanism, location, and exact biological roles. In addition, numerous RNA-based tools such as ribozymes, aptamers, or therapeutic oligonucleotides require the presence of additional chemical functionalities to strengthen the nucleosidic backbone against degradation or enhance the desired catalytic or binding properties. Herein, the two main methods for the chemical modification of RNA are presented: solid-phase synthesis using phosphoramidite precursors and the enzymatic polymerization of nucleoside triphosphates. The different synthetic and biochemical steps required for each method are carefully described and recent examples of practical applications based on these two methods are discussed.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids.

The terminal deoxynucleotidyl transferase (TdT) belongs to the X family of DNA polymerases. This unusual polymerase catalyzes the template-independent addition of random nucleotides on 3'-overhangs during V(D)J recombination. The biological function and intrinsic biochemical properties of the TdT have spurred the development of numerous oligonucleotide-based tools and methods, especially if combined with modified nucleoside triphosphates. Herein, we summarize the different applications stemming from the incorporation of modified nucleotides by the TdT. The structural, mechanistic, and biochemical properties of this polymerase are also discussed.

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa -Perturbed Nucleotides.

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pKa -perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pKa perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.

Compatibility of 5-ethynyl-2'F-ANA UTP with in vitro selection for the generation of base-modified, nuclease resistant aptamers.

A modified nucleoside triphosphate bearing two modifications based on a 2'-deoxy-2'-fluoro-arabinofuranose sugar and a uracil nucleobase equipped with a C5-ethynyl moiety (5-ethynyl-2'F-ANA UTP) was synthesized. This nucleotide analog could enzymatically be incorporated into DNA oligonucleotides by primer extension and reverse transcribed to unmodified DNA. This nucleotide could be used in SELEX for the identification of high binding affinity and nuclease resistant aptamers.

On the enzymatic incorporation of an imidazole nucleotide into DNA.

The expansion of the genetic alphabet with an additional, artificial base pair is of high relevance for numerous applications in synthetic biology. The enzymatic construction of metal base pairs is an alluring strategy that would ensure orthogonality to canonical nucleic acids. So far, very little is known on the enzymatic fabrication of metal base pairs. Here, we report on the synthesis and the enzymatic incorporation of an imidazole nucleotide into DNA. The imidazole nucleotide dIm is known to form highly stable dIm-Ag+-dIm artificial base pairs that cause minimal structural perturbation of DNA duplexes and was considered to be an ideal candidate for the enzymatic construction of metal base pairs. We demonstrate that dImTP is incorporated with high efficiency and selectivity opposite a templating dIm nucleotide by the Kf exo-. The presence of Mn2+, and to a smaller extent Ag+, enhances the efficiency of this polymerization reaction, however, without being strictly required. In addition, multiple incorporation events could be observed, albeit with modest efficiency. We demonstrate that the dIm-Mn+-dIm cannot be constructed by DNA polymerases and suggest that parameters other than stability of a metal base pair and its impact on the structure of DNA duplexes govern the enzymatic formation of artificial metal base pairs.

Synthesis of C8-N-Arylamine-Modified 2'-Deoxyguanosine-5'-Triphosphates and Their Effects on Primer Extension by DNA Polymerases.

C8-N-arylamine adducts of 2'-deoxyguanosine (2'-dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8-N-acetyl-N-arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8-N-acetyl-N-arylamine-2'-deoxyguanosine-5'-triphosphates and C8-NH-arylamine-2'-deoxyguanosine-5'-triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8-dG adducts were studied in primer-extension assays by using Klenow fragment exo(-) of Escherichia coli DNA polymerase I and human DNA polymerase ß. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N-acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8-N-arylamine-2'-deoxyguanosine-5'-triphosphates in chemical carcinogenesis.

Efficient Automated Solid-Phase Synthesis of DNA and RNA 5'-Triphosphates.

A fast, high-yielding and reliable method for the synthesis of DNA- and RNA 5'-triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite was coupled to the 5'-end. Oxidation of the formed 5'-phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5'-cycloSal-oligonucleotides. Reaction of the support-bonded 5'-cycloSal-oligonucleotide with pyrophosphate yielded the corresponding 5'-triphosphates. The 5'-triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.

Towards the enzymatic formation of artificial metal base pairs with a carboxy-imidazole-modified nucleotide.

The identification of synthetic nucleotides that sustain the formation of orthogonal, unnatural base pairs is an important goal in synthetic biology. Such artificial synthons have been used for the generation of semi-synthetic organisms as well as functional nucleic acids with enhanced binding properties. The enzymatic formation of artificial metal-base pairs is a vastly underexplored and alluring alternative to existing systems. Here, we report the synthesis and biochemical characterization of 1­(2-deoxy­ß­d­ribofuranosyl) imidazole­4­carboxylate nucleoside triphosphate (dImCTP) which is equipped with a carboxylic acid moiety on the imidazole moiety in order to increase the coordination environment to [2 + 2] and [2 + 1]. A clear metal dependence was observed for the single incorporation of the modified nucleotide into DNA by the DNA polymerase from Thermus aquaticus (Taq). The presence of AgI in primer extension reactions conducted with combinations of 1­(2­deoxy­ß­d­ribofuranosyl) imidazole nucleoside triphosphate (dImTP) and dImCTP supported the unusual [2 + 1] coordination pattern. The efficiency of the tailing reactions mediated by the terminal deoxynucleotidyl transferase (TdT) was markedly improved when using dImCTP instead of dImTP. Even though products with multiple modified nucleotides were not observed, the appendage of additional metal binding ligands on the imidazole nucleobase appears to be a valid approach to improve the biochemical properties of modified triphosphates in the context of an expansion of the genetic alphabet with metal base pairs.

Facile immobilization of DNA using an enzymatic his-tag mimic.

Methods for immobilization of DNA on solid supports are in high demand. Herein, we present a generally applicable enzymatic method for the immobilization of DNA without any prior chemical derivatization. This strategy relies on the homopolymerization of the modified triphosphate dImTP by the TdT. The resulting enzymatic his-tag mimic ensures binding of DNA on Ni-NTA agarose. The usefulness of this method is highlighted by the immobilization of functional nucleic acids without impairing their specific activities.

Solid-Phase Synthesis of DNA and RNA 5'-O-Triphosphates Using cycloSal Chemistry.

A chemical method for the synthesis of short strands of DNA and RNA 5'-O-triphosphates is described that makes use of the conventional coupling and oxidizing reagents that are readily available on standard DNA/RNA synthesizers. After automated solid-phase synthesis of oligonucleotides and 5'-O-detritylation, a novel cycloSal-phosphoramidite, 5-chloro-saligenyl-N,N-diisopropylphosphoramidite, was coupled to the 5'-hydroxyl group and the product oxidized. The resulting support-bonded 5'-cycloSal-oligonucleotide was reacted with pyrophosphate to yield 5'-O-triphosphorylated DNA/RNA oligonucleotides after cleavage from the support in high purity and excellent yields. The reaction sequence was adapted to be used completely on a standard automated oligonucleotide synthesizer. © 2016 by John Wiley & Sons, Inc.