Modified Nucleoside Triphosphates Research Articles

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.

A toolbox for enzymatic modification of nucleic acids with photosensitizers for photodynamic therapy.

Photodynamic therapy (PDT) is an approved cancer treatment modality. Despite its high efficiency, PDT is limited in terms of specificity and by the poor solubility of the rather lipophilic photosensitizers (PSs). In order to alleviate these limitations, PSs can be conjugated to oligonucleotides. However, most conjugation methods often involve complex organic synthesis and result in the appendage of single modifications at the 3'/5' termini of oligonucleotides. Here, we have investigated the possibility of bioconjugating a range of known PSs by polymerase-mediated synthesis. We have prepared a range of modified nucleoside triphosphates by different conjugation methods and investigated the substrate tolerance of these nucleotides for template-dependent and -independent DNA polymerases. This method represents a mild and versatile approach for the conjugation of single or multiple PSs onto oligonucleotides and can be useful to further improve the efficiency of the PDT treatment.

Fluorescent labeling of RNA and DNA on the Hoogsteen edge using sulfinate chemistry.

We have devised a single pot, low-cost method to add azide groups to unmodified nucleic acids without the need for enzymes or chemically modified nucleoside triphosphates. This involves reacting an azide-containing sulfinate salt with the nucleic acid, leading to replacement of C-H bonds on the nucleobase aromatic rings with C-R, where R is the azide-containing linker derived from the original sulfinate salt. With the addition of azide functional groups, the modified nucleic acid can easily be reacted with any alkyne-labeled compound of interest, including fluorescent dyes as shown in this work. This methodology enables the fluorescent labeling of a wide variety of nucleic acids, including natively folded RNAs, under mild conditions with minimal effects upon biochemical function and ribozyme catalysis. To demonstrate this, we show that a pair of labeled complementary ssDNA oligonucleotides (oligos) can hybridize to form dsDNA, even when labeled with multiple fluorophores per oligo. In addition, we also demonstrate that two different group II introns can splice when prelabeled internally with fluorophores, using our method. Broadly, this demonstrates that sulfinate modification of RNA is compatible with ribozyme function and Watson-Crick pairing, while preserving the labile backbone.

Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

Modified nucleoside triphosphates (NTPs) are powerful probes and medicines, but their anionic character impedes membrane permeability. As such, invasive delivery techniques, transport carriers, or prodrug strategies are required for their in vivo use. Here, we present a fluorescent 2'-deoxyribonucleoside triphosphate "TAMRA-dATP" that exhibits surprisingly high bioavailability in vivo. TAMRA-dATP spontaneously forms nanoparticles in Mg+2-containing buffers that are taken into the vesicles of living cells and animals by energy-dependent processes. In cell cultures, photochemical activation with yellow laser light (561 nm) facilitated endosomal escape of TAMRA-dATP, resulting in its metabolic incorporation into DNA in vitro. In contrast, in vivo studies revealed that TAMRA-dATP is extensively trafficked by active pathways into cellular DNA of zebrafish (Danio rerio) and Caenorhabditis elegans where DNA labeling was observed in live animals, even without photochemical release. Metabolic labeling of DNA in whole, living animals can therefore be achieved by simply soaking animals in a buffer containing TAMRA-dATP or a structurally related compound, Cy3-dATP.

Variety of Nucleotide Polymerase Mutants Aiming to Synthesize Modified RNA.

Significant efforts have been made to develop therapeutic RNA aptamers that exploit synthetic RNA to capture target molecules. However, ensuring RNA aptamers are resistant against intrinsic nucleases remains an issue and restricts their use as therapeutics. Introduction of chemical modifications to the 2' sugar moiety of RNA improves their stability effectively and can be achieved by chemical synthesis using modified phosphoramidites; however, this approach is not suitable for preparing long RNA molecules. Although recombinant nucleotide polymerases can transcribe RNA, these polymerases cannot synthesize modified RNA because they do not recognize 2' modified nucleoside triphosphates. In this review, we focus on several polymerase mutants that tolerate substrates containing modifications of the 2' sugar moiety to synthesize RNA, and the problems that must be overcome to prepare chemically modified RNA with high efficacy by in vitro transcription.

Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications.

Modified nucleoside triphosphates (NTPs) are invaluable tools to probe bacterial enzymatic mechanisms, develop novel genetic material, and engineer drugs and proteins with new functionalities. Although the impact of nucleobase alterations has predominantly been studied due to their importance for protein recognition, sugar and phosphate modifications have also been investigated. However, NTPs are cell impermeable due to their negatively charged phosphate tail, a major hurdle to achieving live bacterial studies. Herein, we review the recent advances made to investigate and evolve bacteria and their processes with the use of modified NTPs by exploring alterations in one of the three moieties: the nucleobase, the sugar and the phosphate tail. We also present the innovative methods that have been devised to internalize NTPs into bacteria for in vivo applications.

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.

P(V) Reagents for the Scalable Synthesis of Natural and Modified Nucleoside Triphosphates.

Natural and modified nucleoside triphosphates impact nearly every major aspect of healthcare research from DNA sequencing to drug discovery. However, a scalable synthetic route to these molecules has long been hindered by the need for purification by high performance liquid chromatography (HPLC). Here, we describe a fundamentally different approach that uses a novel P(V) pyrene pyrophosphate reagent to generate derivatives that are purified by silica gel chromatography and converted to the desired compounds on scales vastly exceeding those achievable by HPLC. The power of this approach is demonstrated through the synthesis of a broad range of natural and unnatural nucleoside triphosphates (dNTPs and xNTPs) using protocols that are efficient, inexpensive, and operationally straightforward.

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.

Synthesis and Enzymatic Characterization of Sugar-Modified Nucleoside Triphosphate Analogs.

Chemical modification of nucleic acids can be achieved by the enzymatic polymerization of modified nucleoside triphosphates (dN*TPs). This approach obviates some of the requirements and drawbacks imposed by the more traditional solid-phase synthesis of oligonucleotides. Here, we describe the protocol that is necessary to synthesize dN*TPs and evaluate their substrate acceptance by polymerases for their subsequent use in various applications including selection experiments to identify aptamers. The protocol is exemplified for a sugar-constrained nucleoside analog, 7',5'-bc-TTP.

Aptamer chemistry.

Aptamers are single-stranded DNA or RNA molecules capable of tightly binding to specific targets. These functional nucleic acids are obtained by an in vitro Darwinian evolution method coined SELEX (Systematic Evolution of Ligands by EXponential enrichment). Compared to their proteinaceous counterparts, aptamers offer a number of advantages including a low immunogenicity, a relative ease of large-scale synthesis at affordable costs with little or no batch-to-batch variation, physical stability, and facile chemical modification. These alluring properties have propelled aptamers into the forefront of numerous practical applications such as the development of therapeutic and diagnostic agents as well as the construction of biosensing platforms. However, commercial success of aptamers still proceeds at a weak pace. The main factors responsible for this delay are the susceptibility of aptamers to degradation by nucleases, their rapid renal filtration, suboptimal thermal stability, and the lack of functional group diversity. Here, we describe the different chemical methods available to mitigate these shortcomings. Particularly, we describe the chemical post-SELEX processing of aptamers to include functional groups as well as the inclusion of modified nucleoside triphosphates into the SELEX protocol. These methods will be illustrated with successful examples of chemically modified aptamers used as drug delivery systems, in therapeutic applications, and as biosensing devices.

Transport of Nucleoside Triphosphates into Cells by Artificial Molecular Transporters.

Chemically modified nucleoside triphosphates (NTPs) are widely exploited as unnatural metabolites in chemical biology and medicinal chemistry. Because anionic NTPs do not permeate cell membranes, their corresponding neutral precursors are employed in cell-based assays. These precursors become active metabolites after enzymatic conversion, which often proceeds insufficiently. Here we show that metabolically-active NTPs can be directly transported into eukaryotic cells and bacteria by the action of designed synthetic nucleoside triphosphate transporters (SNTTs). The transporter is composed of a receptor, which forms a non-covalent complex with a triphosphate anion, and a cell-penetrating agent, which translocates the complex across the plasma membrane. NTP is then released from the complex in the intracellular milieu and accumulates in nuclei and nucleoli in high concentration. The transport of NTPs proceeds rapidly (seconds to minutes) and selectively even in the presence of other organic anions. We demonstrate that this operationally simple and efficient means of transport of fluorescently labelled NTPs into cells can be used for metabolic labeling of DNA in live cells.

DUTPs conjugated with zwitterionic Cy3 or Cy5 fluorophore analogues are effective substrates for DNA amplification and labelling by Taq polymerase.

To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3− and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3′ ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.

Modified nucleoside triphosphates exist in mammals.

DNA and RNA contain diverse chemical modifications that exert important influences in a variety of cellular processes. In addition to enzyme-mediated modifications of DNA and RNA, previous in vitro studies showed that pre-modified nucleoside triphosphates (NTPs) can be incorporated into DNA and RNA during replication and transcription. Herein, we established a chemical labeling method in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis for the determination of endogenous NTPs in the mammalian cells and tissues. We synthesized 8-(diazomethyl)quinoline (8-DMQ) that could efficiently react with the phosphate group under mild condition to label NTPs. The developed method allowed sensitive detection of NTPs, with the detection limits improved by 56-137 folds. The results showed that 12 types of endogenous modified NTPs were distinctly determined in the mammalian cells and tissues. In addition, the majority of these modified NTPs exhibited significantly decreased contents in human hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues. Taken together, our study revealed the widespread existence of various modified NTPs in eukaryotes.

Trifluoroacetophenone-Linked Nucleotides and DNA for Studying of DNA-Protein Interactions by 19F NMR Spectroscopy.

A series of 7-[4-(trifluoroacetyl)phenyl]-7-deazaadenine and -7-deazaguanine as well as 5-substituted uracil and cytosine 2'-deoxyribonucleosides and mono- and triphosphates were synthesized through aqueous Suzuki-Miyaura crosscoupling of halogenated nucleosides or nucleotides with 4-(trifluoroacetyl)phenylboronic acid. The modified nucleoside triphosphates were good substrates for DNA polymerases applicable in primer extension or PCR synthesis of modified oligonucleotides or DNA. Attempted cross-linking with a serine-containing protein did not proceed, however the trifluoroacetophenone group was a sensitive probe for the study of DNA-protein interactions by 19F NMR.

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis.

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis can be characterized using an in vitro DNA synthesis assay followed by polyacrylamide gel electrophoresis. The goal of this assay is to quantify synthesis of both natural DNA and modified DNA (M-DNA). These approaches are particularly useful for resolving oligonucleotides with single nucleotide resolution, enabling observation of individual steps during enzymatic oligonucleotide synthesis. These methods have been applied to the evaluation of an array of biochemical and biophysical properties such as the measurement of steady-state rate constants of individual steps of DNA synthesis, the error rate of DNA synthesis, and DNA binding affinity. By using modified components including, but not limited to, modified nucleoside triphosphates (NTP), M-DNA, and/or mutant DNA polymerases, the relative utility of substrate-DNA polymerase pairs can be effectively evaluated. Here, we detail the assay itself, including the changes that must be made to accommodate nontraditional primer DNA labeling strategies such as near-infrared fluorescently labeled DNA. Additionally, we have detailed crucial technical steps for acrylamide gel pouring and running, which can often be technically challenging.

Chemically Modified, α-Amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) Receptor RNA Aptamers Designed for in Vivo Use.

Glutamate ion channels have three subtypes, that is, α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors. Excessive activity of these receptor subtypes either individually or collectively is involved in various neurological disorders. RNA aptamers as antagonists of these receptors are potential therapeutics. For developing aptamer therapeutics, the RNA aptamers must be chemically modified to become ribonuclease-resistant or stable in biological fluids. Using systematic evolution of ligands by exponential enrichment (SELEX) and a chemically modified library, prepared enzymatically (i.e., the library contains RNAs with 2'-fluoro modified nucleoside triphosphates or ATPs, CTPs and UTPs, but regular GTPs), we have isolated an aptamer. The short aptamer (69 nucleotides) FN1040s selectively inhibits the GluA1 and GluA2Qflip AMPA receptor subunits, whereas the full-length aptamer (101 nucleotides) FN1040 additionally inhibits GluK1, but not GluK2, kainate receptor, and GluN1a/2A and GluN1a/2B, the two major native NMDA receptors. The two aptamers show similar potency (2-4 μM) and are stable with a half-life of at least 2 days in serum-containing medium or cerebrospinal fluid. Therefore, these two aptamers are amenable for in vivo use.

New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments

5-Ethynyl-2′-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.

Systematic study of constraints imposed by modified nucleoside triphosphates with protein-like side chains for use in in vitro selection.

Successful selection of modified DNAzymes depends on the potential for modified nucleoside triphosphates (dNTPs) to replace their unmodified counterparts in enzyme catalyzed primer extension reactions and, once incorporated, to serve as template bases for information transfer prior to PCR amplification. To date, the most densely modified DNAzymes have been selected from three modified dNTPs: 8-histaminyl-deoxyadenosine (dAimTP), 5-guanidinoallyl-deoxyuridine (dUgaTP), and 5-aminoallyl-deoxycytidine (dCaaTP) to provide several RNA-cleaving DNAzymes with greatly enhanced rate constants compared to unmodified counterparts. Here we report biophysical and enzymatic properties of these three modified nucleosides in the context of specific oligonucleotide sequences to understand how these three modified nucleobases function in combinatorial selection. The base-pairing abilities of oligonucleotides bearing one or three modified nucleosides were investigated by thermal denaturation studies and as templates for enzymatic polymerization with both modified and unmodified dNTPs. While we address certain shortcomings in the use of modified dNTPs, we also provide key evidence of faithful incorporation and enzymatic read-out, which strongly supports their continued use in in vitro selection.

Rolling Circle Amplification with Chemically Modified Nucleoside Triphosphates.

Modified nucleoside triphosphates (dN*TPs) represent facile and versatile precursors for the introduction of chemical diversity into nucleic acids. While dN*TPs have been utilized in a plethora of practical applications, very little attention has been devoted to the assessment of their compatibility with isothermal amplification strategies. In this context, rolling circle amplification (RCA) is a wide-spread enzymatic replication method in which small single-stranded DNA (ssDNA) circles serve as templates in primer extension reactions yielding very long, ssDNA products. RCA is a pivotal tool for the generation of biosensor and diagnostic devices and is currently evaluated for its usefulness to create novel drug delivery systems. This unit describes the experimental procedures for the synthesis of modified RCA products using dN*TPs bearing chemical alterations at any possible location of the nucleosidic scaffold. Two ligation methods are presented for the generation of the DNA nanocircles that serve as templates for RCA, followed by a description of the RCA method itself and an assessment of the nuclease resistance of the ensuing products. © 2016 by John Wiley & Sons, Inc.