T7 transcription kit for synthesis of nuclease-resistant RNA. Contains a mix of NTPs with 2’‑fluoro CTP and 2′-fluoro UTP. The T7 RNA polymerase is optimized towards incorporation of 2’‑fluoro modified nucleotides. Incorporation of 2′-fluoro-pyrimidines protects oligonucleotides from digestion by extracellular nucleases and prolongs oligonucleotide half-life in the nuclease-rich environment. The efficiency of T7 assays strongly depends on balanced adjustment and high quality of all reaction components. The kit incorporates carefully optimized reagents and conditions that improves the efficiency of RNA synthesis.
2′-fluoro pyrimidine RNAs are frequently applied in SELEX for aptamers synthesis as well as for development of nuclease-resistant small interfering RNAs (siRNA) with silencing ability. 2′-fluoro modifications offer better secondary structure formation capabilities as compared to 2′-amino modifications, and display higher compatibility with downstream enzymatic manipulations as compared to 2′-O-methyl modified RNA. Half-life of 2′- fluoro modified aptamers depends on their precise sequence and secondary structure, as well as on the composition and the nuclease load of the application environment. Typical half-life values in human or animal sera range between 30 minutes to several hours. In contrast, entirely non-modified RNAs are quickly degraded under these conditions and half-live is usually too short for being reliably measured.
2′-fluoro pyrimidine RNA is compatible with reverse transcription reaction with NG dART Reverse Transcriptase (Cat. No. E0801).
NTP mix consists of 2’-deoxy-2’-fluorocytidine 5’-triphosphate, 2’-deoxy-2’-fluorouridine 5’-triphosphate as well as non-modified ATP and GTP, respectively. The NTPs mix does not contain any non-modified CTP or UTP.