Universal Nucleobase Research Articles

Biocatalytic Nucleobase Diversification of 4'-Thionucleosides and Application of Derived 5-Ethynyl-4'-thiouridine for RNA Synthesis Detection.

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Biocatalytic Nucleobase Diversification of 4′‐Thionucleosides and Application of Derived 5‐Ethynyl‐4′‐thiouridine for RNA Synthesis Detection

AbstractNucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4′‐thiouridine in a one‐pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4′‐thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5‐iodo‐4′‐thiouridine on multi‐milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5‐ethynyl‐4′‐thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

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.

4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.

Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2′-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA–protein interactions.

Using NMR and molecular dynamics to link structure and dynamics effects of the universal base 8-aza, 7-deaza, N8 linked adenosine analog.

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N8-(2′deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.

7-Aminobutynyl-8-Aza-7-Deazahypoxanthine as a Quasi-Universal Nucleobase

Several 7-(hydroxy, amino, methylureido, and guanidino)alkynyl-substituted 8-aza-7-deaza- hypoxanthine analogues were investigated as potential universal nucleobases. 7-Aminobutynyl-8-aza-7-deazahypoxanthine was found to be the most promising quasi-universal nucleobase with improved hybridization and polymerase chain reaction (PCR) enhancing properties as compared to commonly used hypoxanthine (the nucleobase of inosine). It demonstrated improved ambiguity for pairing with A, T, and C bases and its base pairing properties can be summarized as follows: X:C∼X:A∼X:T > X:G. The improvement in PCR performance directly correlated with primer's Tm. Primers containing multiple 7-aminobutynyl-8-aza-7-deazahypoxanthines were successfully used without noticeable inhibition of Taq polymerase activity provided the modifications are positioned more than two bases away from the 3′ end.

A computational proposal for the experimentally observed discriminatory behavior of hypoxanthine, a weak universal nucleobase

A computational model composed of six nucleobases was used to investigate why hypoxanthine does not yield duplexes of equal stability when paired opposite each of the natural DNA nucleobases. The magnitudes of all nearest-neighbor interactions in a DNA helix were calculated, including hydrogen-bonding, intra- and interstrand stacking interactions, as well as 1-3 intrastrand stacking interactions. Although the stacking interactions in DNA relevant arrangements are significant and account for at least one third of the total stabilization energy in our nucleobase complexes, the trends in the magnitude of the stacking interactions cannot explain the relative experimental melting temperatures previously reported in the literature. Furthermore, although the total hydrogen-bonding interactions explain why hypoxanthine preferentially pairs with cytosine, the experimental trend for the remaining nucleobases (A, T, G) is not explained. In fact, the calculated pairing preference of hypoxanthine matches that determined experimentally only when the sum of all types of nearest-neighbor interactions is considered. This finding highlights a strong correlation between the relative magnitude of the total nucleobase-nucleobase interactions and measured melting temperatures for DNA strands containing hypoxanthine despite the potential role of other factors (including hydration, temperature, sugar-phosphate backbone). By considering a large range of sequence combinations, we reveal that the binding preference of hypoxanthine is strongly dependent on the nucleobase sequence, which may explain the varied ability of hypoxanthine to universally bind to the natural bases. As a result, we propose that future work should closely examine the interplay between the dominant nucleobase-nucleobase interactions and the overall strand stability to fully understand how sequence context affects the universal binding properties of modified bases and to aid the design of new molecules with ambiguous pairing properties.

Replication of a Universal Nucleobase Provides Unique Insight into the Role of Entropy during DNA Polymerization and Pyrophosphorolysis

Most models accounting for the efficiency and fidelity of DNA polymerization invoke the use of either hydrogen bonding contacts or complementarity of shape and size between the formed base pair. This report evaluates these mechanisms by quantifying the ability of a high-fidelity DNA polymerase to replicate 5-nitroindole, a purine mimetic devoid of classic hydrogen bonding capabilities. 5-NITP acts as a universal nucleotide since it is incorporated opposite any of the four natural nucleobases with nearly equal efficiencies. Surprising, the polymerization reaction is not reciprocal as natural nucleotides are poorly incorporated opposite 5-nitroindole in the template strand. Incorporation opposite 5-nitroindole is more efficient using natural nucleotides containing various modifications that increase their base stacking potential. However, 5-substituted indolyl nucleotides that contain pi-electron and/or hydrophobic groups are incorporated opposite the non-natural nucleobase with the highest catalytic efficiencies. The collective data set indicate that replication of a non-natural nucleobase is driven by a combination of the hydrophobic nature and pi-electron surface area of the incoming nucleotide. In this mechanism, the overall hydrophobicity of the incoming nucleobase overcomes the lack of hydrogen bonding groups that are generally required for optimal DNA polymerization. However, the lack of hydrogen bonds between base pairs prevents primer extension. This final aspect is manifest by the appearance of unusually high pyrophosphorolysis activity by the T4 DNA polymerase that is only observed with the non-natural nucleobase in the template. These results highlight the importance of hydrogen bonding interactions during primer extension and pyrophosphorolysis.

Binding affinity of a peptide nucleic acid containing pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone as a nucleobase for oligonucleotides

In order to examine the potentiality as a universal nucleobase of pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone (PPT) incorporated in a peptide-nucleic acid (PNA), we synthesized a PNA with PPT, that is H-Gly-CCT(PPT)TCC-Lys-NH2, and investigated the duplex-formation ability of this PNA for the oligodeoxyribonucleotide, (5')GGAXAGG(3') (X = A, G, C or T) on the basis of the Tmvalue of a 1:1 mixture of the PNA and the oligonucleotide. As a result, we found that H-Gly-CCT(PPT)TCC-Lys-NH2 has good binding affinity for all the tested oligonucleotides, i.e., (5')GGAAAGG(3'), (5')GGAGAGG(3'), (5')GGACAGG(3') and (5')GGATAGG(3') and forms stable duplexes with these nucleotides. Thus, it was revealed that PPT serves as an excellent universal base in PNA to discriminate all natural nucleobases.

A computational characterization of the hydrogen-bonding and stacking interactions of hypoxanthine

The hydrogen-bonding and stacking interactions of hypoxanthine, a potential universal nucleobase, were calculated using a variety of methodologies (CCSD(T), MP2, B3LYP, PWB6K, AMBER). All methods predict that the hydrogen-bonding interaction in the hypoxanthine-cytosine pair is approximately 25 kJ mol(-1) stronger than that in the other dimers. Although the calculations support suggestions from experiments that hypoxanthine preferentially binds with cytosine, the trend in the calculated hydrogen-bond strengths for the remaining natural nucleobases do not show a strong correlation with the experimentally predicted binding preferences. However, our calculations suggest that the stacking interactions of hypoxanthine are similar in magnitude to the hydrogen-bonding interactions at all levels of theory (with the exception of B3LYP, which incorrectly predicts stacked dimers to be unstable). Therefore, stacking interactions should also be considered when analyzing the stability of DNA helices containing hypoxanthine and the use of larger models that account for both hydrogen-bonding and stacking within DNA duplexes will likely result in better agreement with experimental observations. For the majority of the dimers, PWB6K and AMBER provide reasonable binding strengths at reduced computational costs, and therefore will be useful techniques for considering larger models.

Nitrosubstituted aromatic molecules as universal nucleobases: Computational analysis of stacking interactions

MP2 stacking interactions between nitrosubstituted rings (3-nitropyrrole, 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole), the corresponding unsubstituted rings and natural nucleobases were investigated. Although the enhancement in stacking provided by the nitro group decreases with an increase in the size of the aromatic ring, the largest stacking interactions were found for 5-nitroindole dimers. Nevertheless, the stacking interactions of nitrosubstituted rings cannot compensate for the loss of hydrogen-bonding interactions upon incorporation into DNA. The calculated stacking energies help explain experimentally observed behaviors of these molecules, and suggest that ring composition and size, in addition to external substituents, should be considered when designing novel universal nucleobases.

Characterization of the binding interactions between a unique class of modified purines and the natural nucleobases

Density-functional theory was used to study the hydrogen-bonding properties of a series of modified purines that contain unusual glycosylation positions. These unique molecules have been previously studied experimentally for their ability to behave as universal nucleobases, which must bind with an equal affinity to all natural DNA bases. The thermodynamics and kinetics for rotation about the glycosidic bond are considered since two hydrogen-bonding faces must be readily available for these molecules to bind with all natural nucleobases. The binding energies between modified and natural bases are also calculated. The present study provides support for the universal properties of some modified nucleobases and offers potential explanations for why other modifications do not bind indiscriminately.

Synthesis of 2'-aminoalkyl-substituted fluorinated nucleobases and their influence on the kinetic properties of hammerhead ribozymes.

Hammerhead ribozymes are ribonucleic acids that catalyse the hydrolytic cleavage of RNA. They interfere with gene expression in a highly specific manner and recognize the mRNA target through Watson-Crick base pairing. To overcome the problem of point mutations (Watson-Crick "mismatches") occurring in viral genomes, we developed 2'-aminoethyl-substituted fluorinated nucleosides, which are universal nucleobases. The highly efficient synthetic pathway, which features a direct phthaloylamination of a primary alcohol under Mitsunobu conditions, leads to modified phosphoroamidites. The 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-D-ribofuranose nucleoside analogue does not differentiate between the four natural nucleosides and leads to a RNA duplex that is as stable as the unmodified parent duplex. Upon incorporation into a ribozyme, the analogue's catalytic activity is equal for all four possible substrates, and the cleavage rates for the modified ribozymes are significantly higher (up to a factor of 13) than for the natural Watson-Crick "mismatch" base pairs. In agreement with the thermodynamic data obtained by measurement of the T(m) values of the RNA 12-mers, the cleavage rates for the 2'-substituted fluorinated benzimidazole derivative 4 are slightly higher than for the corresponding fluorinated benzene derivative 3.

The Effect of Universal Fluorinated Nucleobases on the Catalytic Activity of Ribozymes

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2 , containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The Km value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM−1, the corresponding kcat is 2.91 · 10−3 min−1. A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2′-(β-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.

Striving to Understand the Properties of Universal Nucleobases: A Computational Study of Azole Carboxamides

Density functional theory (DFT) is used to study the properties of a series of azole carboxamides in attempts to better understand why these molecules do not have an equal affinity for all natural DNA (RNA) nucleobases, which is an important criterion for universal bases. The thermodynamics and kinetics for bond rotations that afford four azole carboxamide conformers, which each bind to a different natural (DNA) base, are studied. It is concluded that a particular conformer of some azole carboxamides is favorably stabilized; therefore, these molecules will likely preferentially bind to a particular natural base. The geometries and binding energies are calculated for complexes formed between azole carboxamides and natural bases. Our calculations indicate that some complexes are highly distorted and therefore likely reduce the stability of duplexes. Our calculations also indicate that azole carboxamides bind to natural bases with varying affinities. Furthermore, the azole carboxamide binding interactions are generally significantly less than those in the corresponding natural base pair, with the exception of the thymine (or uracil) azole carboxamide complexes. Our calculations provide insight into interactions between azole carboxamides and the natural bases and allow suggestions to be made regarding why these compounds do not function as universal nucleobases.

Development of a Universal Nucleobase and Modified Nucleobases for Expanding the Genetic Code

This unit presents protocols for the synthesis and characterization of nucleosides with unnatural bases in order to develop bases for the expansion of the genetic alphabet or for nonselective pairing opposite natural bases. Protocols describe the design, synthesis, and characterization of unnatural base pairs involving 1-beta-D-2-deoxyribosyl-N- and -C-nucleosides. Determination of the thermodynamic and kinetic parameters of unnatural nucleosides is accomplished by incorporation into oligonucleotides and subsequent evaluation as described herein.

ChemInform Abstract: N‐Pent‐4‐enoyl (PNT) Group as a Universal Nucleobase Protector: Applications in the Rapid and Facile Synthesis of Oligonucleotides, Analogues, and Conjugates.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

N-pent-4-enoyl ( PNT) group as a universal nucleobase protector: Applications in the rapid and facile synthesis of oligonucleotides, analogs, and conjugates

Rapid synthesis of phosphoric diester and phosphorothioate oligonucleotides in 1 micromol to 1 millimol scale has been achieved using PNT nucleoside phosphoramidites. Furthermore, facile synthesis of mixed backbone oligonucleotides (MBOs) as antisense agents can be carried out using PNT nucleoside phosphoramidites in conjunction with PNT nucleoside H-phosphonates and PNT nucleoside phosphonamidites. The versatility of the PNT group is further demonstrated by its use in the preparation of bioreversible oligonucleotide conjugates.