RESUMO
2'-Deoxynucleoside 5'-(alpha-P-seleno)-triphosphates (dNTPαSe) have been conveniently synthesized using a protection-free, one-pot strategy. One of two diastereomers of each dNTPαSe can be efficiently recognized by DNA polymerases, while the other is neither a substrate nor an inhibitor. Furthermore, this Se-atom modification can significantly inhibit non-specific DNA polymerization caused by mis-priming. Se-DNAs amplified with dNTPαSe via polymerase chain reaction have sequences identical to the corresponding native DNA. In conclusion, a simple strategy for more specific DNA polymerization has been established by replacing native dNTPs with dNTPαSe.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/síntese química , Polifosfatos/química , Selênio/química , Humanos , Polimerização , Especificidade por SubstratoRESUMO
Nucleic acids can fold into well-defined 3D structures that help determine their function. Knowing precise nucleic acid structures can also be used for the design of nucleic acid-based therapeutics. However, locations of hydrogen atoms, which are key players of nucleic acid function, are normally not determined with X-ray crystallography. Accurate determination of hydrogen atom positions can provide indispensable information on protonation states, hydrogen bonding, and water architecture in nucleic acids. Here, we used neutron crystallography in combination with X-ray diffraction to obtain joint X-ray/neutron structures at both room and cryo temperatures of a self-complementary A-DNA oligonucleotide d[GTGG(CSe)CAC]2 containing 2'-SeCH3 modification on Cyt5 (CSe) at pH 5.6. We directly observed protonation of a backbone phosphate oxygen of Ade7 at room temperature. The proton is replaced with hydrated Mg2+ upon cooling the crystal to 100 K, indicating that metal binding is favored at low temperature, whereas proton binding is dominant at room temperature.