T7 RNA polymerase activation and improvement of the transcriptional sequencing by polyamines.
We examined the possibility to improve the effectiveness of the in vitro transcription system using T7 RNA polymerase by coexistence with organic bases. The effect of the additives was evaluated by measuring the amount of RNA products in comparison with that of the control system (without additive). We found that four commercial bases and a series of ethylated polyamine analogues newly designed were active enhancers in the following activation order, 1,8-bis(ethylamino)octane > 1,8-octanediamine > 1,5-bis(ethylamino)pentane > cadaverine > 1,8-bis(ethylamino)-4-azaoctane > spermidine 1,18-bis(ethylamino)-5,14-diazaoctadecane > agmatine. It was shown that RNA products were corresponding, only in the presence of active enhancers, to the full length size of the template DNA, and that sequencing signals were enhanced by the presence of active enhancers with high fidelity so that the transcriptional sequencing was further refined to be a highly sensitive sequencing method from a small amount of linear dsDNA. These results suggest that T7 RNA polymerase was activated by the specific binding of the polyamine additive to produce RNA transcripts with fidelity to the template DNA. Therefore, it is expected that the transcriptional sequencing in the presence of active enhancers might be a powerful and sensitive method, in place of the prevalent DNA amplification method, for genomic science projects and clinical and practical gene diagnoses.
Iwata
M
M
Biopolymer Physics Laboratory, The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan. iwatama@postman.riken.go.jp
Izawa
M
M
Sasaki
N
N
Nagumo
Y
Y
Sasabe
H
H
Hayashizaki
Y
Y
D004247:DNA / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011073:Polyamines / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014764:Viral Proteins
D011073:Polyamines; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases