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Enzymatic Incorporation of Emissive Pyrimidine Ribonucleotides
Author(s) -
Srivatsan Seergazhi G.,
Tor Yitzhak
Publication year - 2009
Publication title -
chemistry – an asian journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.18
H-Index - 106
eISSN - 1861-471X
pISSN - 1861-4728
DOI - 10.1002/asia.200800370
Subject(s) - pyrimidine , uracil , oligonucleotide , polymerase , nucleotide , rna polymerase , t7 rna polymerase , rna , uridine , nucleoside , chemistry , biochemistry , ribonucleoside , enzyme , microbiology and biotechnology , biology , dna , gene , escherichia coli , bacteriophage
Making an emission : T7 RNA polymerase incorporates a series of thiophene‐modified uridine triphosphate (UTP) analogues to generate emissive RNA transcripts. Labeling experiments suggested that the enzyme frequently pauses at the incorporation position and, when incorporation does take place, T7 RNA polymerase fails to elongate the modified oligonucleotides and yields aborted transcripts. TPs=triphosphates.The enzymatic incorporation of a series of emissive pyrimidine analogues into RNA oligonucleotides is explored. T7 RNA polymerase is challenged with accepting three non‐natural, yet related, triphosphates as substrates and incorporating them into diverse RNA transcripts. The three ribonucloside triphosphates differ only in the modification of their uracil nucleus and include a thieno[3,2‐d]pyrimidine nucleoside, a thieno[3,4‐d]pyrimidine derivative, and a uridine containing a thiophene ring conjugated at its 5‐position. All thiophene‐containing uridine triphosphates (UTPs) get incorporated into RNA oligonucleotides at positions that are remote to the promoter, although the yields of the transcripts vary compared with the transcript obtained with only native triphosphates. Among the three derivatives, the 5‐modified UTP is found to be the most “polymerase‐friendly” and is well accommodated by T7 RNA polymerase. Although the fused thiophene analogues cannot be incorporated next to the promoter region, the 5‐modified non‐natural UTP gets incorporated near the promoter (albeit in relatively low yields) and even in multiple copies. Labeling experiments shed light on the mediocre incorporation of the fused analogues, suggesting the enzyme frequently pauses at the incorporation position. When incorporation does take place, the enzyme fails to elongate the modified oligonucleotide and yields aborted transcripts. Taken together, these results highlight the versatility and robustness, as well as the scope and limitation, of T7 RNA polymerase in accepting and incorporating reporter nucleotides into modified RNA transcripts.