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

Abstract

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction in the major groove of DNA containing 5-ethynyluracil (U ) with azides was used for turning off sequence-specific protein-DNA interactions. The concept was first demonstrated on switching off cleavage of short modified DNA by restriction endonuclease BamHI-HF. Finally, DNA template containing U was used for in vitro transcription with E. coli RNA polymerase and the transcription was turned off by CuAAC with 3-azidopropane-1,2-diol or 3-azido-7-hydroxycoumarin.

Switching transcription with bacterial RNA polymerase through photocaging, photorelease and phosphorylation reactions in the major groove of DNA

“…The 311-bp templates for transcription containing the Pveg promoter for transcription with E. coli RNAP were designed similarly as previously reported30,45 and were prepared by PCR using modified dU hm TP , dU NB TP ,37 dC hm TP or dC NB TP 39 instead of the corresponding natural pyrimidine nucleotide (Scheme 1). In all cases, full length amplicons were obtained efficiently and after isolation were used as templates for in vitro transcription experiments.…”

“…The resulted transcripts (RNA) were about 145 nucleobases long. Multiple round in vitro transcription assays were performed essentially as described 29,30. The experiments were carried out in total volume 10 μL with 5 ng of DNA template.…”

“…We also found that DNA templates containing 5-hydroxymethyluracil ( U hm ), a rare natural base whose biological role is yet unknown,25–28 can enhance (up to 3.5 times) transcription depending on the promoter 29. We envisaged that some bioorthogonal chemical reactions in the major groove of DNA could be used to manipulate the bulkiness of the modification and we recently published the first paper on turning OFF transcription through a click reaction of 5-ethynyluracil in the major groove 30. Understanding of how nucleic acids can be modified and subsequently interact with RNAP is still in its infancy.…”

Dynamic changes in DNA populations revealed by split–combine selection

“…12,13 The products of the click reaction were analyzed by nuclease digest followed by liquid chromatography and mass spectrometry. [14][15][16][17] These analyses reveal a near quantitative modication of the DNA (see ESI †). Subsequently, the ve libraries were mixed at equimolar concentrations and subjected to a split-combine click-SELEX procedure (Scheme 1b).…”

Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

“…Alkyne-modified nucleotides have proven to be of high potential and utility for obtaining highly functionalized DNA exploiting DNA pol-mediated DNA synthesis in primer extension ( 14 – 16 ) and PCR reactions ( 14 , 17 ). Interestingly, alkyne-modified nucleotides are even processed during DNA synthesis in living cells ( 18 , 19 ).…”