Switching transcription with bacterial RNA polymerase through photocaging, photorelease and phosphorylation reactions in the major groove of DNA† †Electronic supplementary information (ESI) available: Contains detailed experimental procedures, characterization data and additional figures and gels. See DOI: 10.1039/c9sc00205g

Biomimetic switching of in vitro transcription was developed by photochemical deprotection of photocaged 5hmU or 5hmC in template DNA (ON) and by enzymatic phosphorylation (OFF).


Preparation of plasmid with 311-mer region containing specific promotor region
The 1177 plasmid containing the Pveg promoter 3 was used to clone a 99 bp DNA sequence upstream of the promoter. The sequence was inserted to facilitate subsequent PCR reaction with modified nucleotides. First, two complementary strands of comercially available 99-mer oligos Insert_99_EcoRI (100 uL of 0.1 mM) and Insert_99_EcoRI_REV (100 uL of 0.1 mM) were annealed under the following conditions: 95°C 5 min; gradient from 95°C to 25°C (change in S4 temperature every 2s). To check annealed DNA, 2% agarose gel stained by GelRed was run.
( Figure S1A) The prepared 99-mer dsDNA was then cleaved with EcoRI restriction endonuclease to create sticky ends and ligated into the 1177 plasmid into the EcoRI site at the upstream edge of the promoter. Such prepared plasmid was transformed into E.coli DH5 and colonies with inserts were identified and verified by sequencing, yielding the final construct LK2130.

Enzymatic synthesis of template (Temp Pveg2 )
The 311-mer (Temp Pveg2 ) template was prepared by PCR with forward (Prim FOR ) and reverse (Prim REV ) NON-labelled primers from plasmids containing specific promoter regions cloned in p770 between EcoRI and HindIII sites. 3,4 One PCR reaction mixture (20 μL) contained Taq DNA polymerase for ThermoPol buffer (New England Biolabs; 5000 U/mL; 1.2 μL) with ThermoPol buffer (2 μL), natural dNTPs (4 mM; 1.125 μL), primers (20 μM; 3 μL; Prim FOR and 20 μM; 3 μL; Prim REV ) and appropriate plasmid template (42 ng). The reaction was performed in total volume 160 μL (8x20 μL). Forty PCR cycles were run in the thermal cycler under the following conditions: preheating for 3 minutes at 94°C, denaturation for 1 minute at 94°C, annealing for 1 minute at 68°C, extension for 1.5 minutes at 75°C, followed by final extension step of 5 minutes at 75°C. 160 μL of PCR reaction mixture was combined into one Eppendorf vial and PCR product was purified using Agencourt AMPure XP magnetic particles. In the last step of purification, the product was eluted with 200 μL of MilliQ water at the final concentration of 132 ng/μL of Temp Pveg2 . After purification, 180 ng of DNA was loaded on a control 1.3% agarose gel stained with GelRed (Biotium) and analyzed in 0.5xTBE buffer. The sequence of template was confirmed by DNA sequencing. (Figure S1B

Synthesis of modified DNA templates
To prepare modified templates for transcription, PCR reactions were performed in a total final volume of 20 μL. Natural DNAs (positive controls on agarose gels), which were used as a ( Figure S2) Moreover for the purpose of transcription, 32 P-labelled DNA was determined throught 7% PAGE gels exposed to Fuji MS phosphor storage screens. (Figure S9; Figure S11 Figure S2A).

Study of cleavage of modified DNA with AluI
Approx. 100 ng of either natural, hm-modified or NB-modified DNAs were incubated at 37 °C with addition of CutSmart Buffer (1 µL) and restriction enzyme AluI (1.8 µL) during 1 hour. The products of cleavage were then monitored with 1.3% agarose gel stained with GelRed. (Scheme S1, Figure S3)

Study of cleavage of modified DNA with RsaI
Approx. 100 ng of either natural, hm-modified or NB-modified DNAs were incubated at 37 °C with addition of CutSmart Buffer (1 µL) and restriction enzyme RsaI (1.4 µL) for 1 hour.
In the case of samples, which were synthesized in the presence of dC X TPs, 1.8 µL of RsaI enzyme was used for a cleavage DNA ( Figure S3). The products of cleavage were then monitored with 1.3% agarose gel stained with GelRed. (Figure S3) Despite the fact that the short 40-mer hm-modified DNA (synthesized in the presence of dC hm TP) was easily cleaved with RsaI 2 , hydroxymethyl-modification in long DNA coming from dC hm TP was not tolerated with restriction enzymes -AluI or RsaI. Because of that, the kinetic study of deprotection was firstly performed just with modified DNA synthesized in the presence of modified dU X TP (X=NB or hm).  analyzed on a 5% PAA gel without urea and subsequently dried and exposed to Fuji MS phosphor storage screens and scanned with a Molecular Imager FX (BIORAD). The results

Study of transcription for hm-and NB-modified DNA templates
were analyzed with Quantity One program (BIORAD).

Multiple round transcriptions
Multiple round in vitro transcription assays were performed essentially as described 4  for 10 min at 37°C and started with NTPs. The reactions were allowed to proceed for 10 min at 37°C. Subsequently, the reactions were stopped by the addition of 10 μl of formamide stop solution (95% formamide, 20 mM EDTA, pH 8.0). 10 μL of each sample was loaded onto 7% polyacrylamide gels. The gels were dried and exposed to Fuji MS phosphor storage screens and scanned with a Molecular Imager FX (BIORAD) and analysed with Quantity One program (BIORAD).

Quantification of relative transcriptions
Firstly, the transcript signals (background was subtracted) were normalized to DNA template signals. Subsequently, signals of transcriptions of non-modified and modified DNA templates were normalized to the signal of natural DNA (T + or C + ), which was set as 100 % (T + or C + -S11 transcription of nonmodified DNA diluted in water). At least three independent experiments were performed.  (Figure S4) Then, the irradiated DNA was used as a template for an in vitro transcription assay (Figure S5).

Transcription data for kinetic study of deprotection of NB-modified DNA irradiated by UV lamps either 355 nm (1 mW) or 400 nm (25 mW)
Multiple-round in vitro transcriptions were carried out under the general conditions (see

Kinetic study of deprotection of U NB -modified DNA irradiated by UV lamp 400 nm (3 W)
The purified 32P-labelled NB-modified DNA was diluted to the final concentration of approx. The degree of deprotection of NB-modified DNA was controlled with cleavage by restriction endonuclease -RsaI (1.4 ul for 100 ng of DNA). (Figure S9) Then, the irradiated DNA was used as a template for an in vitro transcription assay. (Figure S10, Figure 1  µL of (50 mM) DTT in 1.5 mL Eppendorf tubes at room temperature. The sides of the Eppendorf tubes were covered with alu-foil and samples were irradiatiated from the top of the opened tube. The degree of deprotection of NB-modified DNA was not controlled with cleavage by restriction endonuclease, because even small hydroxymethyl modification is not tolerated with restriction enzymes -RsaI and AluI (see chapter 3.) (Figure S11) Then, the irradiated DNA was used as a template for an in vitro transcription assay (Figure S12).   Figure S13). Therefore, the RE cleavage could not be reliably used for determination of the outcome of phosphorylation and we directly proceeded to the transcription study.

Sequencing of PCR products:
The correct sequence of all PCR products was confirmed with LIGHTRUN tube Sequencing service (GATC Biotech AG, Germany) using standard Sanger sequencing. In the case of C NB modified dsDNA, C NB modified DNA was used as a template for their rePCR with natural dNTPs according to the protocol reported in the sections 2.3. of the SI, and the resulting product was sent for sequencing.
The red underlined part shows exact match of sequenced DNA with expected sequence in the presence of reverse primer. Blue underlined part shows exact match of sequenced DNA with expected sequence in the presence of forward primer. Double, violet underlined part was correctly sequenced in the presence of either forward or reverse primer.