Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

Abstract

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli. Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. coli enzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.

Turning Off Transcription with Bacterial RNA Polymerase through CuAAC Click Reactions of DNA Containing 5‐Ethynyluracil

“…play criticalr oles in regulation of gene expression through activation or inhibition by enhancing or preventing binding of transcription factors (TFs) and RNA polymerases (RNAP). Following severals cattered examples of prior works on transcription of non-natural modified DNA, [3] we have reported [4] as ystematic studyo ft ranscription of DNA templates modified in the major groove by bacterial RNA polymerases and found that mostb ulkierc hemical modificationsc ompletely inhibitedt ranscription, whereas the templatesc ontaining some smallm odifications( e.g.,7 -deazaadenine, 7-deazaguanine or 5-ethynyluracil) still supporteds ignificant transcription. [2] While the portfolio of natural epigenetic modifications is limited, there is ac hallenging opportunity to introduce new non-natural modifications to expand the epigenetic landscapea nd offer new tools for regulation of gene expression.…”

“…[1] Enzymatic DNA methylation and demethylation is used duringt he differentiation of cells to switch on and off certain genes. [4] More recently,w eh avef ound that DNA containing 5-hydroxymethyluracil can even give enhanced (up to 3.5 times) transcription dependingo nt he promoter. Following severals cattered examples of prior works on transcription of non-natural modified DNA, [3] we have reported [4] as ystematic studyo ft ranscription of DNA templates modified in the major groove by bacterial RNA polymerases and found that mostb ulkierc hemical modificationsc ompletely inhibitedt ranscription, whereas the templatesc ontaining some smallm odifications( e.g.,7 -deazaadenine, 7-deazaguanine or 5-ethynyluracil) still supporteds ignificant transcription.…”

“…[9] The corresponding 5-ethynyluracil (U E )d eoxyribonucleoside (dU E )i su sed for metabolic labeling [10,11] and its triphosphate (dU E TP)i sagood substrate for DNA polymerases [11,12] to produce DNA containing this base, whichistoleratedby most restriction endonucleases (REs) [12] that can still cleavet he U E -modified DNA and the DNA containing this base is still ap roficient template in bacterial transcription. [4] The organic azide partner for the CuAACc lick reaction should be waters oluble and reasonably bulky.T herefore, we selected an easily available 3-azidopropane-1,2-diol (APD)a nd tried the click reactiono nn ucleoside dU E and on triphosphate dU E TP (Scheme 1). In both cases, the reactions proceeded smoothly to give the corresponding dihydroxypropyltriazoles dU DHT and dU DHT TP (in the latter case in low yield due to partialh ydrolysis of the triphosphatea nd difficult isolation).…”

“…[4] We used a3 39-mer template with Pveg promoter from Bacillus subtilis which is also well-recognizedb yt he E. coli RNAP holoenzyme containing the primary sigma factor, s 70 . We performed an in vitro transcriptions tudy on modified DNA templates with Escherichia coli RNA polymerase (RNAP) as reported previously.…”

“…[4] We used a3 39-mer template with Pveg promoter from Bacillus subtilis which is also well-recognizedb yt he E. coli RNAP holoenzyme containing the primary sigma factor, s 70 . In accord with the previous work, [4] the U E -containing DNA template (DNA E )g ave approximately 43 %t ranscription compared to naturalD NA (Scheme 3a,b), whereas the bulkiert riazolecontaining DNA DHT template abolished transcription (Figure 2). Though the dU DHT TP was as lightly worse substrate for the KOD XL polymerase, we were still able to obtaint he modified amplicons in both cases ( Figure S5 in the Supporting Information).…”