Reversible Control of Gene Expression by Guest-Modified Adenosines in a Cell-Free System via Host–Guest Interaction

Gene expression technology has become an indispensable tool for elucidating biological processes and developing biotechnology. Cell-free gene expression (CFE) systems offer a fundamental platform for gene expression-based technology, in which the reversible and programmable control of transcription can expand its use in synthetic biology and medicine. This study shows that CFE can be controlled via the host–guest interaction of cucurbit[7]uril (CB[7]) with N6-guest-modified adenosines. These adenosine derivatives were conveniently incorporated into the DNA strand using a post-synthetic approach and formed a selective and stable base pair with complementary thymidine in DNA. Meanwhile, alternate addition of CB[7] and the exchanging guest molecule induced the reversible formation of a duplex structure through the formation and dissociation of a bulky complex on DNA. The kinetics of the reversibility was fine-tuned by changing the size of the modified guest moieties. When incorporated into a specific region of the T7 promoter sequence, the guest-modified adenosines enabled tight and reversible control of in vitro transcription and protein expression in the CFE system. This study marks the first utility of the host–guest interaction for gene expression control in the CFE system, opening new avenues for developing DNA-based technology, particularly for precise gene therapy and DNA nanotechnology.


Materials and instrumentation
All the chemicals and solvents were purchased from commercial suppliers (FUJIFILM Wako Pure Chemical, the Tokyo Chemical Industry, Kanto Chemical, Nacalai Tesque, Strem Chemicals, BLDpharm, Glen Research, and Sigma-Aldrich) and used without further purification.The reactions were conducted under an argon atmosphere in oven-dried glassware unless otherwise specified.The NMR spectra were recorded with Bruker AVANCE III 400, Bruker AVANCE III 500 or Bruker AVANCE III 600 spectrometer.
Stopped-flow fluorescence measurements were performed using RSP-2000 equipped with a temperaturecontrolled reaction chamber (Unisoku) connected with a heat bath circulator.Light was collected from 75 W xenon arc lamp housing equipped with a MD200 monochromator (Unisoku) to the reaction chamber through a bundled optical fiber.PCR was performed using T100 Thermal Cycler (Bio-Rad).The gel images were obtained and quantified by ChemiDoc MP Imaging System (Bio-Rad).

Post-synthetic conversion of I Pu into the modified adenosines in ODNs
The ODN-bound CPGs (0.1 μmol) were placed in a 1.5 mL screw-capped eppendorf tube.A solution of amine (compound 2-6, 30-40% ethylamine in ethanol, or ethylenediamine; 5-20 μL) in MeOH (400 μL) was added, and the mixture was kept overnight at 50 °C in a shaker (Note: the reaction can also proceed efficiently at room temperature).After cooling to room temperature, 28% NH4OH (400 μL) was added to the mixture, and the shaking was continued for another 2 h at room temperature.The reaction mixture was concentrated using a centrifugal evaporator.The residue was diluted with distilled H2O and washed twice with EtOAc.The aqueous layer was collected, filtered through a filter unit (DISMIC-13HP, ADVANTEC) and evaporated under reduced pressure.The crude ODN was purified by reverse-phase HPLC using a Nacalai Tesque COSMOSIL 5C18-MS-II column (4.6ID × 250 mm) with 0.1 M triethylammonium acetate buffer at pH 7.0 (buffer A) and CH3CN (buffer B).A linear gradient of 5 to 25% of buffer B over 20 min was applied at the flow rate of 3 mL/min except for ODN1 (X = A AD ) and ODN5-8 (X = A AD ) in which a linear gradient of 10 to 35% for over 20 min and 10 to 60% for over 20 min was utilized.The column oven was set to 50 °C, and the peaks were detected at 254 nm.The appropriate fraction was collected and lyophilized.The purified ODN was dissolved in distilled H2O and ion-exchanged using Strong Acidic Cation Exchange Resin No.6 (Na + -form, FUJIFILM Wako Pure Chemical).The eluent was desalted by passing through Nap-10 column (Cytiva) and lyophilized.The purity and structural integrity of each synthesized ODN were confirmed by RP-HPLC and MALDI-TOF MS analyses, respectively.

Enzymatic digestion of the ODNs incorporating guest-modified nucleosides
To a solution of ODN (2 nmol) in 1× Nucleoside Digestion Mix Reaction Buffer (New England Biolabs) was added Nucleoside Digestion Mix (4 μL, New England Biolabs), and the reaction mixture (40 μL in total) was incubated at 37 °C for 4 h.The digested mixture was analyzed by reverse-phase HPLC using a Nacalai Tesque COSMOSIL 5C18-MS-II column (4.6ID × 250 mm) with 50 mM ammonium formate (buffer A) and CH3CN (buffer B).A linear gradient of 5 to 15% over 10 min followed by 15 to 40% over 20 min of buffer B was applied at the flow rate of 1 ml/min at 35°C, and the peaks were detected at 254 nm.For A ADcontaining ODNs, a linear gradient of 5 to 15% over 10 min followed by 15 to 100% over 20 min of buffer B was applied.The appropriate fraction was collected and analyzed by ESI-TOF MS measurement.

UV melting temperature measurement of duplex DNA
A solution containing 2 μM of each ODN, 10 mM sodium phosphate buffer (pH 7.0), and 150 mM NaCl was heated at 90 °C and gradually cooled down to room temperature for annealing.UV melting curves were recorded with a quartz cell with a 1 cm path length at temperatures between 10 and 80 °C with a ramping and a scanning rate of 1.0 °C/min at 260 nm.The melting curves from alternating addition of CB[7] and Ad Eda were recorded after incubating the ODN solution with 4 μM of CB[7] for 30 min and 10 μM of Ad Eda for 30 h at 37 °C.Each Tm value is presented as an average of three measurements.The absorbance was normalized using the following equation: Normalized Abs260 = {Abs260(t °C)-Abs260(10 °C)} / {Abs260(80 °C) -Abs260(10 °C)}.

Time course monitoring of the fluorescence upon the addition of CB[7]
A solution of duplex DNA was prepared by annealing ODN3 (0.2 μM) and ODN4 (0.3 μM) in 10 mM sodium phosphate buffer (pH 7.0) and 150 mM NaCl as described above.The buffered duplex solution (ODN3-ODN4) and CB[7] solution (2 μM in 10 mM sodium phosphate buffer and 150 mM NaCl at pH 7.0) were set in each chamber.After the thermal equilibration at 37 °C, an equal volume (∼60 μL) of each solution was mixed in the reaction chamber at 37 °C, and the reaction progress was followed by monitoring the increase in the fluorescence (λex = 495 nm, λem > 525 nm).Relative fluorescence intensity was defined as the ratio of the fluorescence intensity with respect to that of the end of the measurement.

Time course monitoring of the fluorescence upon the addition of Ad Eda
A solution of the duplex DNA (prepared as described above) in 10 mM sodium phosphate buffer (pH 7.0), 150 mM NaCl and CB[7] (2 μM) was incubated at at 37 °C for 30 min.The duplex-CB[7] buffer solution and Ad Eda buffer solution (4 μM in 10 mM sodium phosphate buffer (pH 7.0) and 150 mM NaCl) were set in each chamber.After the thermal equilibration at 37 °C, an equal volume (∼60 μL) of each solution was mixed in the reaction chamber at 37 °C, and the reaction progress was followed by monitoring the decrease in the fluorescence with λex = 495 nm and λem > 525 nm.In case of Am2 dA, the time-course change of the fluorescence monitored under the same conditions except that the measurement was performed on a spectrofluorometer FP-8300 (JASCO; λex = 495 nm and λem = 520 nm) due to its inherently slow reaction rate.Relative fluorescence intensity was defined as the ratio of the fluorescence intensity with respect to that in the beginning of the measurement.

Curve fitting for calculating apparent rate constant (i) Reaction rate for duplex dissociation process
Assuming that the dissociation of CB[7] from the guest-modified adenosines are considerably slow under the given conditions, the duplex dissociation reaction induced by CB[7] binding to the guest-modified DNA can be approximated as a second-order reaction, and the rate equation at a given time "t" for DNA + CB → DNA • CB (complex of DNA and CB[7]) can be described as follows: [DNA]0, [CB]0: initial concentrations of DNA and CB[7], kin: the rate constant for duplex dissociation In the case [CB7] ≫ [DNA], the reaction can be approximated as pseudo-first order reaction as follows: By defining the constant as the fluorescence intensity at the end of the reaction as "b" and the fluorescence intensity at the start of the reaction as "a + b", the fitting equation can be expressed as in equation (3).

𝒚 = 𝒂 𝐞𝐱𝐩(−𝒌 𝐢𝐧 [𝐂𝐁] 𝟎 𝒕) + 𝒃 (𝟑) (ii) Reaction rate for duplex re-hybridization process
The duplex re-formation reaction by the addition of the exchange guest is the rate-determining reaction for the dissociation process of the CB[7]-guest complex.Therefore, this reaction can be approximated as a first-order reaction of the CB[7]-guest complex.The first-order reaction equation at a certain time "t" is expressed as in equation ( 4).
[] = [ • ]  ( − (−  )) () Defining the fluorescence intensity at the end of the reaction as "b" and the fluorescence intensity at the beginning of the reaction as "a + b" as constants, the curve fitting equation is expressed as in equation ( 5).

Preparation of SQ-DNAs by polymerase extension reaction
A solution of ODN5 (4 μM) and the 100 mer template ODN6 (2 μM) in 1× ThermoPol Reaction Buffer (New England Biolabs) was mixed with dNTP (400 μM each, Toyobo) and Deep Vent (exo -) polymerase (0.04 U/μL, New England BioLabs) on ice.The reaction mixture was heated at 95 °C for 20 s followed by the incubation at 50 °C for 30 min.An aliquot (2 μL) of the reaction mixture was mixed with a loading buffer (18 μL, 2.5× TBE buffer and 50% glycerol) and analyzed on 20% polyacrylamide native gel at 200 V.The bands were stained with SYBR Gold Nucleic Acid Gel Stain (Invitrogen) and visualized using ChemiDoc MP Imaging System with a SYBR Gold filter.The extended duplex DNA (SQ-DNA) was purified by QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's protocol.

In vitro transcription of SQ-DNA and fluorescnce measurement of transcribed Squash aptamer
A solution of 1 μM SQ-DNA, 40 mM Tris-HCl buffer (pH 8.0), 5 mM dithiothreitol, 20 mM MgCl2, 2 mM spermidine and rNTP (2 mM each) was mixed with T7 RNA Polymerase ver 2.0 (10 U/μL, Takara Bio), and the reaction mixture was incubated at 37 °C.Part of the reaction mixture (2 μL) was taken at each time point (0, 30, 60, 90, 120 min) and mixed with a solution (98 μL) of 5 μM DFHBI-1T, 40 mM HEPES-KOH (pH 7.4), 100 mM KCl and 0.5 mM MgCl2.Fluorescence spectra were measured on a FP-6500 spectrofluorometer with λex = 451 nm and λem = 503 nm at 25 °C.The transcription reactions in the presence of CB[7] and Ad Eda were performed in the same manner except that the reaction mixture was incubated with CB[7] (8 μM) at 37 °C for 30 min prior to the addition of T7 RNA polymerase.Ad Eda (10 μM) was added immediately before the addition of T7 RNA polymerase.For the iterative control of transcription activity, CB[7] and Ad Eda were added to the reaction mixture at the indicated timing.

Single-nucleotide insertion against the guest-modified adenosines
To a solution of the FAM-labelled ODN7 (0.1 μM), ODN8 (0.15 μM) and Phusion DNA polymerase (0.02 U/μL, New England BioLabs) in 1× Phusion HF Buffer (New England Biolabs) was added each dNTP (50 μM) on ice, and the reaction mixture (10 μL in total) was incubated at 37 °C for 5 min.An aliquot (4 μL) of the reaction mixture was mixed with a loading buffer (8 μL, 95% formamide containing 20 mM EDTA) and heated at 95 °C for 5 min.The products were analyzed on 20% polyacrylamide denaturing gel containing 7 M urea at 300 V.The gel images were obtained using ChemiDoc MP Imaging System with a Alexa 488 filter.

Full-length extension using the template ODN containing the guest-modified adenosines
A solution of the FAM-labelled ODN7 (30 nM) and ODN8 (45 nM) in 1× Phusion HF Buffer was mixed with dNTP (400 μM each) and Phusion DNA polymerase (0.02 U/μL) on ice, and the reaction mixture (40 μL in total) was incubated at 55 °C for 30 min.An aliquot (12 μL) of the reaction mixture was mixed with a loading buffer (12 μL, 95% formamide containing 20 mM EDTA) and heated at 95 °C for 5 min.The products were analyzed on 20% polyacrylamide denaturing gel as described above.

Preparation of the modified DHFR gene by PCR
A solution (20 μL) of DHFR-DNA [2] (10 ng, GeneFrontier), ODN8 (0.5 μM, Fwd-primer), ODN9 (0.5 μM, Rev-primer), dNTP (400 μM each) and Phusion DNA Polymerase (0.02 U/μL) in 1× Phusion HF Buffer was prepared on ice.The PCR was performed using a T100 Thermal Cycler (Bio-Rad) with the following cycle: 95 °C for 10 s, 56 °C for 20 s, 70 °C for 30 s (20 cycles).The reaction mixture was diluted 200-fold with distilled H2O and subjected to another round (20 cycles) of PCR under the same conditions as described above.The reaction mixture was mixed with 10× Loading Buffer (5 μL, Takara Bio) and analyzed by 1.5% agarose gel electrophoresis at 100 V.The bands were stained with SYBR Gold Nucleic Acid Gel Stain and visualized using ChemiDoc MP Imaging System with a SYBR Gold filter.Alternatively, the PCR products were visualized by UV shadowing for gel purification.The appropriate band was excised from the agarose gel, and the amplified DNA was isolated using NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL) according to the manufacturer's protocol.

In vitro transcription of DHFR-DNA
A solution of DHFR-DNA (10 ng), 40 mM Tris-HCl buffer (pH 8.0), 5 mM dithiothreitol, 20 mM MgCl2, 2 mM spermidine, RNase Inhibitor (1.75 U/μL, Nacalai Tesque) and rNTP (2 mM each, New England Biolabs) was mixed with T7 RNA Polymerase ver 2.0 (10 U/μL), and the reaction mixture (20 μL in total) was incubated at 37 °C for 2 h.The reaction was quenched with 10× Loading Buffer (2 μL, Takara Bio), and the products were analyzed by 1.5% agarose gel elctrophoresis at 100 V.The gel was run with Low Range ssRNA Ladder (New England Biolabs).The bands were stained with SYBR Gold Nucleic Acid Gel Stain and visualized using ChemiDoc MP Imaging System with a SYBR Gold filter.The transcription reactions in the presence of CB[7] and Ad Eda were performed in the same manner except that the reaction mixture was incubated with CB[7] (80 μM) at 37 °C for 30 min prior to the addition of T7 RNA polymerase.Ad Eda (100 μM) was added immediately before the addition of T7 RNA polymerase.
After the indicated reaction time, RNase A Solution (1 μL, Promega) was added, and the reaction mixture was incubated at 37 °C for additional 15 min to digest the unreacted FluoroTect

Analytical data of the ODNs used in this study
[M-H] - Found (m/z)

4 .
Figure S3.UV melting curves and Tm values of the DNA duplexes containing A, Am2 dA, Am3 dA, and Am4 dA at position X and each canonical nucleoside at position Y.Each Tm value is average of three measurements and provided with standard errors (S.E.).

Figure S4 .
Figure S4.(a) Structures of Et dA and Eda dA for investigating the recognition mode of Am2 dA-T pair.(b) UV melting curves and Tm values of the DNA duplexes containing Et dA and AEt dA at position X and each canonical nucleoside at position Y.Each Tm value is average of three measurements and provided with standard errors (S.E.).(c) Comparison of the Tm values of the DNA duplexes (ODN1/ODN2) containing A-T, Et dA-T, AEt dA-T, and Am2 dA-T pairs at position X-Y.

Figure S7 .
Figure S7.The time course change of fluorescence obtained from the DNA duplex containing Am2 dA in the presence of different concentration of CB[7].The signal change was monitored by stopped-flow measurement (λex = 495 nm and λem > 525 nm).The kinetic parameters were determined by a non-linear least squares regression analysis of the respective curve.

Figure S8 .
Figure S8.The time course change of fluorescence obtained by using different concentrations of the DNA duplex containing Am2 dA. 1 μM CB[7] was added and the signal change was monitored by stopped-flow measurement (λex = 495 nm and λem > 525 nm).The kinetic parameters were determined by a non-linear least squares regression analysis of the respective curve.

Figure S9 .
Figure S9.The time course change of fluorescence obtained from the guest exchange reaction of the DNA containing Am2 dA complexed with CB[7].(a) The guest exchange reaction in the presence of different concentrations of Ad Eda .(b) The guest exchange reaction using different exchanging guest (2 μM).The signal change was monitored by fluoresncece measurement (λex = 495 nm and λem = 520 nm).The kinetic parameters were determined by a non-linear least squares regression analysis of the respective curve.

7.
Figure S10.UV melting curves and summary of Tm values of the DNA duplexes containing Nad dA and Bic dA at position X and each canonical nucleoside at position Y.

Figure S11 .
Figure S11.UV melting curves and summary of Tm values of the DNA duplexes containing Nad dA-T and Bic dA-T pairs upon alternating treatment with CB[7] and Ad Eda .

Figure S12 .
Figure S12.The time course change of fluorescence obtained by using different concentration of DNA duplex containing Nad dA and Bic dA. 1 μM CB[7] was added and the signal change was monitored by stopped-flow measurement (λex = 495 nm and λem > 525 nm).The kinetic parameters were determined by a non-linear least squares regression analysis of the respective curve.

Figure S13 .
Figure S13.The time course change of fluorescence obtained from the DNA duplex containing Nad dA and Bic dA in the presence of different concentration of CB[7].The signal change was monitored by stoppedflow measurement (λex = 495 nm and λem > 525 nm).The kinetic parameters were determined by a nonlinear least squares regression analysis of the respective curve.

Figure S14 .Figure S15 .
Figure S14.Base pairing properties of A AD nucleoside reported by Xiao et al.(a) UV melting curves and summary of the Tm values of the DNA duplexes containing A AD at position X and each canonical nucleoside at position Y.(b) UV melting curves of the DNA duplexes containing A AD -T pair upon alternating treatment with CB[7] and Ad Eda .(c) Comparison of the base pairing properties of A, Am2 dA and A AD in DNA duplexes.

Figure S19 .
Figure S19.Fluorescence spectra obtained from the transcription reaction of SQ-DNA-8 (X = Nad dA).The reactions were performed in the absence and after alternating addition of CB[7] (8 μM) and Ad Eda (10 μM).The fluorescence was measured using aliquots of the transcription mixture sampled at different time points (0, 30, 60, 90, 120 min) in the presence of DFHBI-1T.

Figure S25 .
Figure S25.Transcription monitoring of SQ-DNA-8 (X = A AD ).The reactions were performed in the absence and after alternating addition of CB[7] (8 μM) and Ad Eda (10 μM).(a) Fluorescence spectra of each reaction mixture sampled at different time points (0, 30, 60, 90, 120 min).(b) A summary of the relative transcription efficiency.A AD induced transcription inhibition regardless of the host-guest interaction.
TMGreenLys tRNA.The digest was mixed with Sample Buffer Solution without 2-ME (2x) for SDS-PAGE (11 μL, Nacalai Tesque) and heated at 95 °C for 3 min.The sample was analyzed by SDS-PAGE using SuperSep Ace, 12.5%, 17 well (FUJIFILM Wako Pure Chemical) at 300 V.The gel image was obtained using ChemiDoc MP Imaging System with an Alexa 488 filter.The reactions in the presence of CB[7] and Ad Eda were performed in the same manner except that a mixture of DNA, CB[7] (8 or 80 μM) and Solution I was incubated at 37 °C for 30 min before the addition of the other reagents.Ad Eda (10 or 100 μM) was added immediately before the addition of Solution II, Solution III, FluoroTect GreenLys tRNA and RNase inhibitor.

Table S1 .
Sequences and MADLI-TOF MS data of the ODNs used in this study.