RESUMEN
2-Selenouridine ((Se)U) is one of the naturally occurring modifications of Se-tRNAs ((Se)U-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the (Se)U-RNA via 2-selenouridine triphosphate ((Se)UTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized (Se)UTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of (Se)U-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed (Se)U-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of (Se)U-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.
Asunto(s)
Compuestos de Organoselenio/síntesis química , ARN/genética , Transcripción Genética , Uridina Trifosfato/análogos & derivados , Anticodón , Catálisis , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Compuestos de Organoselenio/química , ARN/química , ARN Catalítico/química , ARN Catalítico/genética , ARN Mensajero/química , ARN Mensajero/genética , Uridina Trifosfato/síntesis química , Uridina Trifosfato/química , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
5'-Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2'-deoxyribo- and ribonucleoside 5'-triphosphates (dNTPs and NTPs) from any natural or modified base. This one-pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non-bridging oxygen atom of the α-phosphate. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5'-triphosphates, as well as analysis and confirmation of natural and sulfur- or selenium-modified nucleic acids, are described in this protocol unit.
Asunto(s)
Química Orgánica/métodos , Nucleósidos/química , Nucleósidos/síntesis química , Fenómenos Químicos Orgánicos , Oxidación-Reducción , Fosfatos/química , Polifosfatos/química , Compuestos de Selenio/químicaRESUMEN
Phosphoroselenoate RNA (PSe-RNA) is nuclease resistant and has great potentials in X-ray crystal structure and function studies of noncoding RNAs and protein-RNA interactions. In order to conveniently synthesize PSe-RNA via transcription, we have developed a one-pot synthetic method for the nucleoside 5'-(α-P-seleno)-triphosphates (NTPαSe) analogs without protecting any functionality of the ribonucleosides. The NTPαSe diastereomers have been purified, fully characterized, and incorporated into RNAs by T7 RNA polymerase. The transcribed RNAs are diastereomerically pure, and the Se-derivatized ribozymes are generally active. Furthermore, we have established an affinity purification strategy by using immobilized boronate to conveniently purify NTPαSe analogs. Though the affinity-purified NTPαSe analogs are diastereomeric mixtures, they can be directly used in transcription without a significant impact on the transcription efficiency. Moreover, we found that the PSe-nucleotide is stable during polyacrylamide gel purification, indicating that the PSe-RNAs can be purified straightforwardly for crystal structural and functional studies.
Asunto(s)
Compuestos de Organoselenio/síntesis química , Fosfatos/síntesis química , ARN/genética , Transcripción Genética , Estructura Molecular , ARN/químicaRESUMEN
By differentiating the functional groups on nucleosides, we have designed and developed a one-pot synthesis of deoxyribonucleoside 5'-triphosphates without any protection on the nucleosides. A facile synthesis is achieved by generating an in situ phosphitylating reagent that reacts selectively with the 5'-hydroxyl groups of the unprotected nucleosides. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. This novel approach is straightforward and cost-effective for triphosphate synthesis.
Asunto(s)
ADN/síntesis química , Nucleósidos/química , Fosfatos/síntesis química , Polimerizacion , Estructura MolecularRESUMEN
By generating a selective phosphitylating reagent in situ, nucleoside 5'-triphosphates can be conveniently synthesized in one pot. This novel strategy without nucleoside protection has been developed to largely simplify synthesis of the nucleoside triphosphates. This demonstrated principle can be applied to the 5'-triphosphate synthesis of both native and modified nucleosides.
Asunto(s)
Nucleótidos/química , Nucleótidos/síntesis química , Polifosfatos/química , ARN/genética , Transcripción Genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleótidos/metabolismo , ARN/biosíntesisRESUMEN
BACKGROUND: To prevent DNA damage caused by the ultraviolet (UV) radiation of sunlight, sunscreens are commonly used to protect human skin. Current analysis of sunscreens' effectiveness is done through complicated procedures, including human exposure. OBJECTIVE: We sought to design a simple system using thymidine-thymidine (TT) dinucleotides to analyze the effectiveness of sunscreens. METHODS: We can directly analyze sunscreen effectiveness and the formation of TT photolesions simply by using UV spectrophotometry and high-performance liquid chromatography (HPLC). Efficient sunscreen has protective effects against UV irradiation damage. RESULTS: We have developed a simple method using TT dinucleotide, UV, and HPLC for the analysis of sunscreen effectiveness. Our research indicates that the analytical results from UV are consistent with those of HPLC, which is used to monitor the formation of the TT photolesions. Moreover, both UV and HPLC analyses indicate that TT dinucleotides are better protected against UV damage, using the sunscreens with higher UVB sun protection factor (SPF) value, and that sunscreens with higher SPF lead to reduced photolesion formation. Our UV and HPLC analyses confirm the SPF grading of commercial sunscreens. LIMITATIONS: In this experiment, only sunscreens were tested. The experiment, therefore, does not apply to other commercial products, such as cosmetic materials that claim UV protection as a secondary benefit. CONCLUSION: In conclusion, we have established a simple strategy to analyze the effectiveness of sunscreens and the quality of these potential cancer-preventive products.
Asunto(s)
Cromatografía Líquida de Alta Presión , Neoplasias Cutáneas/prevención & control , Protectores Solares/química , Rayos Ultravioleta , Daño del ADN/efectos de la radiación , Femenino , Humanos , Masculino , Ensayo de Materiales , Valores de Referencia , Sensibilidad y Especificidad , Piel/efectos de la radiación , Espectrofotometría/métodos , Protectores Solares/uso terapéuticoRESUMEN
Selenium (Se) can provide unique biochemical and biological functions, and properties to macromolecules, including protein and RNA. Although Se has not yet been found in DNA, identification of the presence of Se in natural tRNAs has led to discovery of the naturally occurring 2-selenouridine and 5-[(methylamino)methyl]-2-selenouridine (mnm(5)se(2)U). The Se-atoms at C(2) of the modified uridines are introduced by 2-selenouridine synthase via displacement of the S-atoms in the corresponding 2-thiouridine nucleotides of the tRNAs, and selenophosphate is used as the Se donor. The research indicated that mnm(5)se(2)U is located at the first or wobble position of the anticodons in several bacterial tRNAs, including tRNA(Lys), tRNA(Glu), and tRNA(Gln). The 2-seleno functionality on this modified nucleotide probably improves the translation accuracy and/or efficiency. These observations in vivo suggest that the presence of Se can provide natural RNAs with useful properties to better function and survival. To further investigate the biochemical and structural properties of Se-derivatized nucleic acids (SeNA), we have pioneered chemical and enzymatic synthesis of Se-derivatized nucleic acids, and introduced Se into both RNA and DNA at a variety of positions by atom-specific replacement of oxygen. This review outlines the recent advancements in chemical and biochemical syntheses, and studies of SeNAs, and their potential applications in structural and functional investigation of nucleic acids and their protein complexes.
Asunto(s)
Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Selenio/metabolismo , Ácidos Nucleicos/química , Células Procariotas/metabolismo , Proteínas/química , Selenio/químicaRESUMEN
The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.