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1.
Science ; 372(6541): 516-520, 2021 04 30.
Article in English | MEDLINE | ID: mdl-33926955

ABSTRACT

Cells have two purine pathways that synthesize adenine and guanine ribonucleotides from phosphoribose via inosylate. A chemical hybrid between adenine and guanine, 2-aminoadenine (Z), replaces adenine in the DNA of the cyanobacterial virus S-2L. We show that S-2L and Vibrio phage PhiVC8 encode a third purine pathway catalyzed by PurZ, a distant paralog of succinoadenylate synthase (PurA), the enzyme condensing aspartate and inosylate in the adenine pathway. PurZ condenses aspartate with deoxyguanylate into dSMP (N6-succino-2-amino-2'-deoxyadenylate), which undergoes defumarylation and phosphorylation to give dZTP (2-amino-2'-deoxyadenosine-5'-triphosphate), a substrate for the phage DNA polymerase. Crystallography and phylogenetics analyses indicate a close relationship between phage PurZ and archaeal PurA enzymes. Our work elucidates the biocatalytic innovation that remodeled a DNA building block beyond canonical molecular biology.


Subject(s)
2-Aminopurine/analogs & derivatives , Adenylosuccinate Synthase/chemistry , Bacteriophages/chemistry , Bacteriophages/enzymology , Biosynthetic Pathways , DNA, Viral/chemistry , Viral Nonstructural Proteins/chemistry , 2-Aminopurine/chemistry , 2-Aminopurine/metabolism , Adenylosuccinate Synthase/classification , Adenylosuccinate Synthase/genetics , Bacteriophages/genetics , Crystallography, X-Ray , DNA, Viral/genetics , Genome, Viral , Phylogeny , Viral Nonstructural Proteins/classification , Viral Nonstructural Proteins/genetics
2.
ACS Chem Biol ; 15(11): 2872-2884, 2020 11 20.
Article in English | MEDLINE | ID: mdl-33090769

ABSTRACT

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long-standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However, the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches, and very little is known about polymerase-mediated synthesis. Herein, we report the selective and high yielding enzymatic construction of a silver-mediated base pair (dImC-AgI-dPurP) as well as a two-step protocol for the synthesis of DNA duplexes containing such an artificial metal base pair. Guided by DFT calculations, we also shed light into the mechanism of formation of this artificial base pair as well as into the structural and energetic preferences. The enzymatic synthesis of the dImC-AgI-dPurP artificial metal base pair provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet.


Subject(s)
DNA/chemistry , Purine Nucleosides/chemistry , Silver/chemistry , Base Pairing , Biocatalysis , DNA/genetics , Density Functional Theory , Genetic Code , Models, Molecular , Polyphosphates/chemistry , Purine Nucleosides/genetics
3.
Chembiochem ; 21(23): 3398-3409, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32673442

ABSTRACT

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.


Subject(s)
Coordination Complexes/metabolism , Copper/metabolism , DNA-Directed DNA Polymerase/metabolism , Imidazoles/metabolism , Coordination Complexes/chemical synthesis , Coordination Complexes/chemistry , Copper/chemistry , DNA-Directed DNA Polymerase/chemistry , Imidazoles/chemistry , Molecular Structure
4.
Proc Natl Acad Sci U S A ; 117(29): 16790-16798, 2020 07 21.
Article in English | MEDLINE | ID: mdl-32631977

ABSTRACT

Nucleic acid aptamers selected through systematic evolution of ligands by exponential enrichment (SELEX) fold into exquisite globular structures in complex with protein targets with diverse translational applications. Varying the chemistry of nucleotides allows evolution of nonnatural nucleic acids, but the extent to which exotic chemistries can be integrated into a SELEX selection to evolve nonnatural macromolecular binding interfaces is unclear. Here, we report the identification of a cubane-modified aptamer (cubamer) against the malaria biomarker Plasmodium vivax lactate dehydrogenase (PvLDH). The crystal structure of the complex reveals an unprecedented binding mechanism involving a multicubane cluster within a hydrophobic pocket. The binding interaction is further stabilized through hydrogen bonding via cubyl hydrogens, previously unobserved in macromolecular binding interfaces. This binding mechanism allows discriminatory recognition of P. vivax over Plasmodium falciparum lactate dehydrogenase, thereby distinguishing these highly conserved malaria biomarkers for diagnostic applications. Together, our data demonstrate that SELEX can be used to evolve exotic nucleic acids bearing chemical functional groups which enable remarkable binding mechanisms which have never been observed in biology. Extending to other exotic chemistries will open a myriad of possibilities for functional nucleic acids.


Subject(s)
Aptamers, Nucleotide/chemistry , L-Lactate Dehydrogenase/chemistry , Malaria/diagnosis , Protozoan Proteins/chemistry , Biomarkers/blood , Biomarkers/chemistry , Humans , Hydrogen Bonding , L-Lactate Dehydrogenase/blood , Malaria/blood , Molecular Diagnostic Techniques/methods , Molecular Dynamics Simulation , Plasmodium vivax/enzymology , Protein Binding
5.
Bioorg Med Chem ; 28(11): 115487, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32284226

ABSTRACT

The incorporation of nucleotides equipped with C-glycosidic aromatic nucleobases into DNA and RNA is an alluring strategy for a number of practical applications including fluorescent labelling of oligonucleotides, expansion of the genetic alphabet for the generation of aptamers and semi-synthetic organisms, or the modulation of excess electron transfer within DNA. However, the generation of C-nucleoside containing oligonucleotides relies mainly on solid-phase synthesis which is quite labor intensive and restricted to short sequences. Here, we explore the possibility of constructing biphenyl-modified DNA sequences using enzymatic synthesis. The presence of multiple biphenyl-units or biphenyl residues modified with electron donors and acceptors permits the incorporation of a single dBphMP nucleotide. Moreover, templates with multiple abasic sites enable the incorporation of up to two dBphMP nucleotides, while TdT-mediated tailing reactions produce single-stranded DNA oligonucleotides with four biphenyl residues appended at the 3'-end.


Subject(s)
Biphenyl Compounds/metabolism , DNA-Directed DNA Polymerase/metabolism , DNA/metabolism , Oligonucleotides/biosynthesis , Biphenyl Compounds/chemistry , DNA/chemistry , Humans , Molecular Structure , Oligonucleotides/chemistry
6.
Org Biomol Chem ; 17(35): 8083-8087, 2019 09 21.
Article in English | MEDLINE | ID: mdl-31460550

ABSTRACT

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.


Subject(s)
Aptamers, Nucleotide/chemistry , Arabinose/analogs & derivatives , Uridine Triphosphate/chemistry , Arabinose/chemistry , Binding Sites , Carbohydrate Conformation , DNA/chemistry , DNA/genetics
7.
Chembiochem ; 20(24): 3032-3040, 2019 12 13.
Article in English | MEDLINE | ID: mdl-31216100

ABSTRACT

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.


Subject(s)
Base Pairing , Chemical Phenomena , Metals/chemistry , Nucleotides/chemistry , Nucleotides/metabolism , Sulfhydryl Compounds/chemistry , Base Sequence , DNA Nucleotidylexotransferase/metabolism , Hydrogen-Ion Concentration , Nucleotides/genetics
8.
Chimia (Aarau) ; 73(6): 368-373, 2019 May 29.
Article in English | MEDLINE | ID: mdl-31118118

ABSTRACT

Advances in the chemical synthesis of RNA have opened new possibilities to address current questions in RNA biology. Access to site-specifically modified oligoribonucleotides is often a pre-requisite for RNA chemical-biology projects. Driven by the enormous research efforts for development of oligonucleotide therapeutics, a wide range of chemical modifications have been developed to modulate the intrinsic properties of nucleic acids in order to fit their use as therapeutics or research tools. The RNA synthesis platform, supported by the NCCR RNA & Disease, aims to provide access to a large variety of chemically modified nucleic acids. In this review, we describe some of the recent projects that involved work of the platform and highlight how RNA chemistry supports new discoveries in RNA biology.


Subject(s)
RNA/genetics , Biology , Oligonucleotides
9.
J Inorg Biochem ; 191: 154-163, 2019 02.
Article in English | MEDLINE | ID: mdl-30529723

ABSTRACT

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.


Subject(s)
Base Pairing , DNA-Directed DNA Polymerase/metabolism , Imidazoles/chemistry , Metals/chemistry , Nucleotides/chemistry , Coordination Complexes/chemistry , Ligands
10.
Molecules ; 23(7)2018 Jul 23.
Article in English | MEDLINE | ID: mdl-30041480

ABSTRACT

Rolling circle amplification (RCA) is a robust way to generate DNA constructs, which are promising materials for biomedical applications including drug delivery because of their high biocompatibility. To be employed as a drug delivery platform, however, the DNA materials produced by RCA need to be shaped into nanoparticles that display both high cellular uptake efficiency and nuclease resistance. Here, we showed that the DNA nanoparticles (DNPs) can be prepared with RCA and modified nucleotides that have side-chains appended on the nucleobase are capable of interacting with the DNA strands of the resulting RCA products. The incorporation of the modified nucleotides improved cellular uptake efficiency and nuclease resistance of the DNPs. We also demonstrated that these DNPs could be employed as carriers for the delivery of a photosensitizer into cancer cells to achieve photodynamic therapy upon irradiation at both the in vitro and in vivo levels.


Subject(s)
DNA , Nanoparticles , Nucleotides , Photosensitizing Agents/administration & dosage , Photosensitizing Agents/chemistry , Animals , Cell Line , DNA/chemistry , Disease Models, Animal , Drug Delivery Systems , Humans , Light , Mice , Nanoparticles/chemistry , Neoplasms/pathology , Neoplasms/therapy , Nucleotides/chemistry , Particle Size , Photochemotherapy , Xenograft Model Antitumor Assays
11.
Adv Drug Deliv Rev ; 134: 3-21, 2018 09.
Article in English | MEDLINE | ID: mdl-29626546

ABSTRACT

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.


Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Humans , Ligands
12.
Int J Mol Sci ; 18(11)2017 Nov 16.
Article in English | MEDLINE | ID: mdl-29144411

ABSTRACT

Recent progresses in organic chemistry and molecular biology have allowed the emergence of numerous new applications of nucleic acids that markedly deviate from their natural functions. Particularly, DNA and RNA molecules-coined aptamers-can be brought to bind to specific targets with high affinity and selectivity. While aptamers are mainly applied as biosensors, diagnostic agents, tools in proteomics and biotechnology, and as targeted therapeutics, these chemical antibodies slowly begin to be used in other fields. Herein, we review recent progress on the use of aptamers in the construction of smart DNA origami objects and MRI and PET imaging agents. We also describe advances in the use of aptamers in the field of neurosciences (with a particular emphasis on the treatment of neurodegenerative diseases) and as drug delivery systems. Lastly, the use of chemical modifications, modified nucleoside triphosphate particularly, to enhance the binding and stability of aptamers is highlighted.


Subject(s)
Aptamers, Nucleotide/chemistry , Drug Delivery Systems/methods , Nanotechnology/methods , Neuroprotective Agents/administration & dosage , SELEX Aptamer Technique/methods , Animals , Aptamers, Nucleotide/pharmacokinetics , Biosensing Techniques/methods , Humans
13.
Chem Commun (Camb) ; 53(97): 13031-13034, 2017 Dec 05.
Article in English | MEDLINE | ID: mdl-29164188

ABSTRACT

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.


Subject(s)
DNA Nucleotidylexotransferase/chemistry , Enzymes, Immobilized/chemistry , Immobilized Nucleic Acids/chemistry , DNA Nucleotidylexotransferase/metabolism , Enzymes, Immobilized/metabolism , Immobilized Nucleic Acids/metabolism
14.
Org Biomol Chem ; 15(20): 4449-4455, 2017 May 23.
Article in English | MEDLINE | ID: mdl-28485736

ABSTRACT

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.


Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA/chemistry , Imidazoles/chemistry , Nucleotides/chemistry , DNA/metabolism , DNA-Directed DNA Polymerase/metabolism , Imidazoles/metabolism , Nucleotides/metabolism
15.
Bioorg Med Chem Lett ; 27(4): 897-900, 2017 02 15.
Article in English | MEDLINE | ID: mdl-28089700

ABSTRACT

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.


Subject(s)
Deoxyuracil Nucleotides/chemistry , Polyphosphates/chemistry , DNA/chemistry , DNA/metabolism , DNA-Directed DNA Polymerase/metabolism , Deoxyuracil Nucleotides/chemical synthesis , Polymerase Chain Reaction , SELEX Aptamer Technique
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