Nucleobase Selectivity Research Articles

Probing the nucleobase selectivity of RNA polymerases with dual-coding substrates

Formycin A (FOR) and Pyrazofurin A (PYR) are nucleoside analogues with antiviral and antitumor properties. They are known to interfere with nucleic acid metabolism, but their direct effect on transcription is less understood. We explored how RNA polymerases (RNAPs) from bacteria, mitochondria, and viruses utilize FOR, PYR, and oxidized purine nucleotides. All tested polymerases incorporated FOR in place of adenine and PYR in place of uridine. FOR also exhibited surprising dual-coding behavior, functioning as a cytosine substitute, particularly for viral RNAP. In contrast, 8-oxoadenine and 8-oxoguanine were incorporated in place of uridine in addition to their canonical Watson-Crick codings. Our data suggest that the interconversion of canonical anti- and alternative syn-conformers underlies dual-coding abilities of FOR and oxidized purines. Structurally distinct RNAPs displayed varying abilities to utilize syn-conformers during transcription. By examining base pairings that led to substrate incorporation and the entire spectrum of geometrically compatible pairings, we have gained new insights into the nucleobase selection processes employed by structurally diverse RNAPs. These insights may pave the way for advancements in antiviral therapies.

Simple and Easy Synthesis of γ-Amido-dNTPs in Water and Their Polymerase Reaction Properties.

γ-Amido-modified 2'-deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs) are becoming increasingly important as biological tools. We herein describe the simple and easy synthesis of γ-amido-dNTPs and -NTPs from commercially available corresponding dNTPs and NTPs in a one-pot reaction using water-soluble carbodiimide and ammonia solution. We examined the effects of synthesized γ-amido-dNTPs on the DNA polymerase reaction. The results obtained showed the incorporation of these derivatives into the DNA primer while maintaining nucleobase selectivity; however, their incorporation efficiency by DNA polymerase was lower than that of dNTP. This is the first study to demonstrate the successful synthesis of four sets of γ-amido-dNTPs and clarify their properties.

Designer Receptors for Nucleotide‐Resolution Analysis of Genomic 5‐Methylcytosine by Cellular Imaging

We report programmable receptors for the imaging‐based analysis of 5‐methylcytosine (5mC) in user‐defined DNA sequences of single cells. Using fluorescent transcription‐activator‐like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co‐stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.

Designer Receptors for Nucleotide‐Resolution Analysis of Genomic 5‐Methylcytosine by Cellular Imaging

AbstractWe report programmable receptors for the imaging‐based analysis of 5‐methylcytosine (5mC) in user‐defined DNA sequences of single cells. Using fluorescent transcription‐activator‐like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co‐stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.

Photosensitised biphotonic chemistry of pyrimidine derivatives.

Photosensitised biphotonic irradiation of DNA has been rarely addressed, probably due to the difficulties in the experimental design. This is associated with the selection of nucleobases and sensitisers with appropriate absorption spectra and photochemical reactivity, in combination with a laser source emitting intense UVA light of the adequate wavelength. The present paper presents a new strategy involving absorption of a first UVA photon by an adequate sensitiser followed by triplet energy transfer to a pyrimidine (Pyr) derivative and absorption of a second UVA photon by the resulting Pyr triplet excited state. The feasibility of the proposed strategy has been demonstrated using two model reactions: (i) the Norrish-Yang photocyclisation of a tert-butyluracil and (ii) the photohydration of its uracil analogue, lacking the tert-butyl substituent.

Crystal structure and substrate binding mode of ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3)

Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3) is a membrane-bound glycoprotein that regulates extracellular levels of nucleotides. NPP3 is known to contribute to the immune response on basophils by hydrolyzing ATP and to regulate the glycosyltransferase activity in Neuro2a cells. Here, we report on crystal structures of the nuclease and phosphodiesterase domains of rat NPP3 in complex with different substrates, products and substrate analogs giving insight into details of the catalytic mechanism. Complex structures with a phosphate ion, the product AMP and the substrate analog AMPNPP provide a consistent picture of the coordination of the substrate in which one zinc ion activates the threonine nucleophile whereas the other zinc ion binds the phosphate group. Co-crystal structures with the dinucleotide substrates Ap4A and UDPGlcNAc reveal a binding pocket for the larger leaving groups of these substrates. The crystal structures as well as mutational and kinetic analysis demonstrate that the larger leaving groups interact only weakly with the enzyme such that the substrate affinity is dominated by the interactions of the first nucleoside group. For this moiety, the nucleobase is stacked between Y290 and F207 and polar interactions with the protein are only formed via water molecules thus explaining the limited nucleobase selectivity.

Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal Transcription-Activator-Like Effector Repeats.

5-Methylcytosine (5mC) and its oxidized derivatives are regulatory elements of mammalian genomes involved in development and disease. These nucleobases do not selectively modulate Watson-Crick pairing, preventing their programmable targeting and analysis by traditional hybridization probes. Transcription-activator-like effectors (TALEs) can be engineered for use as programmable probes with epigenetic nucleobase selectivity. However, only partial selectivities for oxidized 5mC have been achieved so far, preventing unambiguous target binding. We overcome this limitation by destroying and re-inducing nucleobase selectivity in TALEs via protein engineering and chemoselective nucleobase blocking. We engineer cavities in TALE repeats and identify a cavity that accommodates all eight human DNA nucleobases. We then introduce substituents with varying size, flexibility, and branching degree at each oxidized 5mC. Depending on the nucleobase, substituents with distinct properties effectively block TALE-binding and induce full nucleobase selectivity in the universal repeat. Successful transfer to affinity enrichment in a human genome background indicates that this approach enables the fully selective detection of each oxidized 5mC in complex DNA by programmable probes.

Topological Line Defects Around Graphene Nanopores for DNA Sequencing

Topological line defects in graphene represent an ideal way to produce highly controlled structures with reduced dimensionality that can be used in electronic devices. In this work, we propose using extended line defects in graphene to improve nucleobase selectivity in nanopore-based DNA sequencing devices. We use a combination of quantum mechanics/molecular mechanics and nonequilibrium Green’s function methods to investigate the conductance modulation, fully accounting for solvent effects. By sampling over a large number of different orientations generated from molecular dynamics simulations, we theoretically demonstrate that distinguishing between the four nucleobases using line defects in a graphene-based electronic device appears possible. The changes in conductance are associated with transport across specific molecular states near the Fermi level and their coupling to the pore. Through the application of a specifically tuned gate voltage, such a device would be able to discriminate the four types of...

Isolation of Human Genomic DNA Sequences with Expanded Nucleobase Selectivity.

We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases.

Mammalian Nucleotidyl Cyclases and Their Nucleotide Binding Sites.

Mammalian membranous and soluble adenylyl cyclases (mAC, sAC) and soluble guanylyl cyclases (sGC) generate cAMP and cGMP from ATP and GTP, respectively, as substrates. mACs (nine human isoenzymes), sAC, and sGC differ in their overall structures owing to specific membrane-spanning and regulatory domains but consist of two similarly folded catalytic domains C1 and C2 with high structure-based homology between the cyclase species. Comparison of available crystal structures - VC1:IIC2 (a construct of domains C1a from dog mAC5 and C2a from rat mAC2), human sAC and sGC, mostly in complex with substrates, substrate analogs, inhibitors, metal ions, and/or modulators - reveals that especially the nucleotide binding sites are closely related. An evolutionarily well-conserved catalytic mechanism is based on common binding modes, interactions, and structural transformations, including the participation of two metal ions in catalysis. Nucleobase selectivity relies on only few mutations. Since in all cases the nucleoside moiety is embedded in a relatively spacious cavity, mACs, sAC, and sGC are rather promiscuous and bind nearly all purine and pyrimidine nucleotides, including CTP and UTP, and many of their derivatives as inhibitors with often high affinity. By contrast, substrate specificity of mammalian adenylyl and guanylyl cyclases is high due to selective dynamic rearrangements during turnover.

Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase

Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phosphoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.

Adaptive Ligand Binding by the Purine Riboswitch in the Recognition of Guanine and Adenine Analogs

Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity.

Intrinsic lifetimes of the excited state of DNA and RNA bases.

The lifetimes of the excited state of free nucleobases were measured in the gas phase for the first time. They are, respectively, 1.0 and 0.8 ps for the purine bases adenine (shown above) and guanine and 3.2, 2.4, and 6.4 ps for the pyrimidine bases cytosine, uracil, and thymine at 267 nm. The longer lifetimes of the pyrimidine bases may be associated with their higher propensity toward photodegradation, especially in the case of thymine. The ultrashort lifetime of nucleobases conventionally known in solution was found to be an intrinsic molecular property due to extremely facile internal conversion, and therefore the lifetime should be largely independent of the medium at this energy, that is, whether in vacuo, in solution, or in vivo. The evolutionary selection of nucleobases as the durable carriers of genetic information is suggested to be due to their inherent immunity from photochemical reactions.

Heavy metal mutagenicity: insights from bioinorganic model chemistry

The mutagenicity of metal species may be the result of a direct interaction with the target molecule DNA. Possible scenarios leading to nucleobase mispairing are discussed, and selected examples are presented. They include changes in nucleobase selectivity as a consequence of alterations in acid–base properties of nucleobase atoms and groups involved in complementary H bond formation, guanine deprotonation, and stabilization of rare nucleobase tautomers by metal ions. Oxidative nucleobase damage brought about by metal species will not be considered.