Selenium-atom-modified thymidine enhances the specificity and sensitivity of DNA polymerization and detection.

Nucleobase mismatches can jeopardize DNA polymerization specificity, causing mutations and errors in DNA replication and detection. Herein we report the first synthesis of novel 2-Se-thymidine triphosphate (SeTTP), describe the single-selenium atom-specific modification strategy (SAM) against T/G mismatches, and demonstrate SAM-assisted polymerization and detection with much higher specificity and sensitivity. SAM can effectively suppress the formation of non-specific products in DNA polymerization and detection. Thus, SAM enhances the specificity of DNA synthesis by approximately 10 000 fold, and in turn, it allows the detection of clinical COVID-19 viral RNA in low copy numbers (single-digit copies), while the conventional RT-qPCR does not.

High-quality RT-PCR with chemically modified RNA controls.

In detecting infectious diseases, such as coronavirus 2019 (COVID-19), real-time reverse-transcription polymerase chain reaction (RT-PCR) is one of the most important technologies for RNA detection and disease diagnosis. To achieve high quality assurance, appropriate positive and negative controls are critical for disease detection using RT-PCR kits. In this study, we have found that commercial kits often adopt DNAs instead of RNAs as the positive controls, which can't report the kit problems in reverse transcription, thereby increasing risk of the false negative results when testing patient samples. To face the challenge, we have proposed and developed the chemically modified RNAs, such as phosphoroselenaote and phosphorothioate RNAs (Se-RNA and S-RNA), as the controls. We have found that while demonstrating the high thermostability, biostability, chemostability and exclusivity (or specificity), both Se-RNA and S-RNA can be fine templates for reverse transcription, indicating their potentials as both positive and negative controls for RT-PCR kits.

Synthesis of Selenium-Triphosphates (dNTPαSe) for More Specific DNA Polymerization.

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.

Temperature-Induced Replacement of Phosphate Proton with Metal Ion Captured in Neutron Structures of A-DNA.

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.