Alternate Strategies to Induce Dynamically Modulated Transient Transcription Machineries

Emulating native transient transcription machineries modulating temporal gene expression by synthetic circuits is a major challenge in the area of systems chemistry. Three different methods to operate transient transcription machineries and to modulate the gated transcription processes of target RNAs are introduced. One method involves the design of a reaction module consisting of transcription templates being triggered by promoter fuel strands transcribing target RNAs and in parallel generating functional DNAzymes in the transcription templates, modulating the dissipative depletion of the active templates and the transient operation of transcription circuits. The second approach involves the application of a reaction module consisting of two transcription templates being activated by a common fuel promoter strand. While one transcription template triggers the transcription of the target RNA, the second transcription template transcribes the anti-fuel strand, displacing the promoter strand associated with the transcription templates, leading to the depletion of the transcription templates and to the dynamic transient modulation of the transcription process. The third strategy involves the assembly of a reaction module consisting of a reaction template triggered by a fuel promoter strand transcribing the target RNA. The concomitant nickase-stimulated depletion of the promoter strand guides the transient modulation of the transcription process. Via integration of two parallel fuel-triggered transcription templates in the three transcription reaction modules and application of template-specific blocker units, the parallel and gated transiently modulated transcription of two different RNA aptamers is demonstrated. The nickase-stimulated transiently modulated transcription reaction module is applied as a functional circuit guiding the dynamic expression of gated, transiently operating, catalytic DNAzymes.

For the cyclic transient transcription of the MG aptamer, the reaction module was applied twice with the fuel strand Lr: 0.1 μM and 0.5 μM.The dynamic time-dependent fluorescence changes of the MG-aptamer complex were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (30 ℃).
For the measurement of the dynamic transcription of the MG aptamer at variable concentrations of NTPs, three reaction module samples were prepared including the same concentrations of DNA template T1/A1 (0.05 μM), T7 RNAP (1.25 × 10 3 U/mL) and MG (4 μM), and different concentrations of the NTPs (2 mM, 4 mM and 6 mM, respectively).All the three samples were applied with the same concentrations of the fuel strand Lr (0.1 μM) and the time-dependent fluorescence changes were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (30 ℃).
For the cyclic transient transcription of the DFHBI aptamer, the reaction module including the DNA template T2/A2 (0.1 μM) was applied twice with the fuel strand Lr: 0.1 μM and 0.2 μM.The dynamic time-dependent fluorescence changes of the DFHBI-aptamer complex were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 500 nm (30 ℃).
To gate the respective transcription processes, the reaction module was subjected to the blocker (1) (2 μM) or (2) (4 μM), for a time interval of 10 min, and the respective dynamic transcription processes were triggered by Lr, 0.1 μM, followed by monitoring the time-dependent fluorescence changes of the respective RNA aptamer-ligand complexes using a Cary Eclipse Fluorometer (Varian Inc.) at 30 ℃.
For the cyclic transient transcription of the MG aptamer, the reaction module was applied twice with the fuel strand F: 0.033 μM and 0.33 μM.The dynamic time-dependent fluorescence changes of the MG-aptamer complex were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (37 ℃).
For the measurement of the dynamic transcription of the MG aptamer at variable concentrations of NTPs, three reaction module samples were prepared including the same concentrations of DNA templates, T3/A1 (0.05 μM) and T4/A3 (0.05 μM), T7 RNAP (1.875 × 10 3 U/mL) and MG (4 μM), and different concentrations of the NTPs (1 mM, 2 mM and 4 mM, respectively).All the three samples were applied with the same concentrations of the fuel strand F, 0.1 μM and the timedependent fluorescence changes were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (37 ℃).
For the measurement of the dynamic transcription of the MG aptamer at variable concentrations of DNA template T4/A3, three reaction module samples were prepared including the same concentrations of DNA template, T3/A1 (0.05 μM), T7 RNAP (2.5 × 10 3 U/mL) and MG (4 μM), and different concentrations of the DNA template T4/A3 (0.1 μM, 0.05 μM and 0.02 μM, respectively).All the three samples were applied with the same concentrations of the fuel strand F, 0.15 μM and the time-dependent fluorescence changes were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (37 ℃).
All the samples were loaded into the 10% PAGE gel to perform at 80 V for 16 h (8 ℃).
To gate the respective transcription processes, the reaction module was subjected to the blocker (3) (0.2 μM) or (4) (0.3 μM), for a time interval of 10 min, and the respective dynamic transcription processes were triggered by F, 0.03 μM, followed by monitoring the time-dependent fluorescence changes of the respective RNA aptamer-ligand complexes using a Cary Eclipse Fluorometer (Varian Inc.) at 37 ℃.
For the cyclic transient transcription of the MG aptamer, the reaction module was applied twice with the fuel strand L1: 0.02 μM and 0.1 μM.The dynamic time-dependent fluorescence changes of the MG-aptamer complex were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (37 ℃).
For the measurement of the dynamic transcription of the MG aptamer at variable concentrations of NTPs, three reaction module samples were prepared including the same concentrations of DNA template, T6/A4 (0.02 μM), T7 RNAP (1.25 × 10 3 U/mL), Nt.BbvCI (87.5 U/mL), and MG (4 μM), and different concentrations of the NTPs (1.33 mM, 2.66 mM and 4 mM, respectively).All the three samples were applied with the same concentrations of the fuel strand L1, 0.1 μM and the timedependent fluorescence changes were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (37 ℃).
For the measurement of the dynamic transcription of the MG aptamer at variable concentrations of Nt.BbvCI, three reaction module samples were prepared including the same concentrations of DNA template, T6/A4 (0.02 μM), T7 RNAP (1.25 × 10 3 U/mL) and MG (4 μM), and different concentrations of the Nt.BbvCI (166.7 U/mL, 83.3 U/mL and 0 U/mL, respectively).All the three samples were applied with the same concentrations of the fuel strand L1, 0.02 μM and the timedependent fluorescence changes were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 665 nm (37 ℃).
The dynamic time-dependent fluorescence changes of the DFHBI-aptamer complex were recorded with a Cary Eclipse Fluorometer (Varian Inc.) at λem = 500 nm (37 ℃).
To gate the respective transcription processes, the reaction module was subjected to the blocker (5) (0.4 μM) or (6) (1.2 μM), for a time interval of 10 min, and the respective dynamic transcription processes were triggered by L1 (0.25 μM)/L2 (0.45 μM), followed by monitoring the time-dependent fluorescence changes of the respective RNA aptamer-ligand complexes using a Cary Eclipse Fluorometer (Varian Inc.) at 37 ℃.
Samples of 100 μL solutions of the transcription system were collected at different time-intervals and applied with the couplers (P1/Q1, P2/Q2, 1 μM each), the DNAzyme subunits (X, Y, Z and U, 1 μM each) and the substrates (S1 and S2, 2 μM each) and the time-dependent fluorescence changes of the respective fluorophore labeled fragmented substrates were followed (FAM, λex = 495 nm, λem = 518 nm; Cy5, λex = 635 nm, λem = 665 nm) at 25 ℃.To further, quantitatively, evaluate the temporal transcription of the MG-RNA aptamer according to Figure 1(A), we formulated a kinetic model that follows the stepwise reaction associated with the triggered activation of the reaction module and the subsequent transcription of the RNA product and the concomitant DNAzyme driven depletion of the reaction intermediates to recover the parent "rest" reaction module.The scheme of the stepwise reactions comprising the model are displayed in Figure S2.
To quantitatively, computationally simulate the experimental temporal fluorescence changes generated by the MG-RNA aptamer complex, the temporal fluorescence changes shown in Figure 1(B) were translated into temporal concentrations of the MG-RNA aptamer using an appropriate calibration curve, Figure S1. Figure S3, curves (i), (ii), and (iii) depict the experimental temporal concentration changes of the MG-RNA aptamer generated by different concentrations of the fuel strand (dotted curve).Curve (i) was then computationally fitted to the kinetic model displayed in Figure S2.The fitted simulated curve is displayed in Figure S3, curve i', (solid curve).The derived rate constants are presented in Table S1.The rate constants were, then, applied to simulate the experimental curves (ii) and (iii) and the simulated temporal curves are displayed in curves (ii') and (iii') (solid curve).The fit of simulated curves to the experimental results using the rate constants derived for curve (i), suggest that the kinetic model and the set of rate constants presented adequately the kinetics of the transient transcription processes.To further, quantitatively, evaluate the temporal transcription of the MG-RNA aptamer according to Figure 3(A), we formulated a kinetic model that follows the stepwise reaction associated with the triggered activation of the reaction module and the subsequent transcription of the RNA product and the concomitant strand-displacement driven depletion of the reaction intermediates to recover the parent "rest" reaction module.The scheme of the stepwise reactions comprising the model are displayed in Figure S9.

Kinetic equations of the dissipative DNAzyme-triggered operation of the transient transcription machinery shown in
To quantitatively, computationally simulated the experimental temporal fluorescence changes generated by the MG-RNA aptamer complex, the temporal fluorescence changes shown in Figure 3(B) were translated into temporal concentrations of the MG-RNA aptamer using an appropriate calibration curve, Figure S1. Figure S10, curves (i), (ii), and (iii) depict the experimental temporal concentration changes of the MG-RNA aptamer generated by different concentrations of the fuel strand (dotted curve).Curve (i) was then computationally fitted to the kinetic model displayed in Figure S9.The fitted simulated curve is displayed in Figure S10, curve i', (solid curve).The derived rate constants are presented in Table S2.The rate constants were, then, applied to simulate the experimental curves (ii) and (iii) and the simulated temporal curves are displayed in curves (ii') and (iii') (solid curve).The fit of simulated curves to the experimental results using the rate constants derived for curve (i), suggest that the kinetic model and the set of rate constants presented adequately the kinetics of the transient transcription processes.dynamic, temporal features of the transcription machinery constituents: T4/A3, 1 μM; NTPs, 4 mM each) Lane 5-the transcription template T4/A3 prior to the addition of the fuel strand F and T7 RNAP, in the presence of NTPs; Lane 6-After addition of the fuel strand F (1 μM), in the presence of NTPs; Lane 7-the system upon addition of T7 RNAP (1.25 × 10 4 U/mL), in the presence of NTPs, after a time-interval of two minutes; Lane 8-the system in the presence of T7 RNAP, the NTPs, after a time interval of one hour.The formation of the waste product F/F' and the recovery of the inactive T4/A3 template confirm the transient operation of the fueled transcription machinery generating F/F' as waste.The 8% PAGE was performed for 20 h at 80 V (at 8 ℃).

Kinetic equations of the strand-displacement guided the transient transcription machinery shown in
To support the strand displacement process as a guiding principle to modulate the transcription machinery, we engineered a reaction module, Figure S16(A), Where the promoter-triggering strand Fc, forms a stable transcription template T3/Fc+A1, where Fc cannot be displaced by the transcribed product F' that prohibiting the transiently-modulated transcription machinery of T3/A1.Furthermore, as the transient template T4/A3 includes complementary domain to the fuel strand F, it is important to prove that the template T4/A3 stay intact in the presence of F. This is confirmed by the gel electrophoretic experiment presented in Figure S17.This experiment demonstrates that the addition of F to the active template T4+F/A3 does not yield any displaced product.To further, quantitatively, evaluate the temporal transcription of the MG-RNA aptamer according to Figure 5(A), we formulated a kinetic model that follows the stepwise reaction associated with the triggered activation of the reaction module and the subsequent transcription of the RNA product and the concomitant nickase-driven depletion of the reaction intermediates to recover the parent "rest" reaction module.The scheme of the stepwise reactions comprising the model are displayed in Figure S21.
To quantitatively, computationally simulated the experimental temporal fluorescence changes generated by the MG-RNA aptamer complex, the temporal fluorescence changes shown in Figure 5(B) were translated into temporal concentrations of the MG-RNA aptamer using an appropriate calibration curve, Figure S1. Figure S22, curves (i), (ii), and (iii) depict the experimental temporal concentration changes of the MG-RNA aptamer generated by different concentrations of the fuel strand (dotted curve).Curve (i) was then computationally fitted to the kinetic model displayed in Figure S21.The fitted simulated curve is displayed in Figure S22, curve i', (solid curve).The derived rate constants are presented in Table S3.The rate constants were, then, applied to simulate the experimental curves (ii) and (iii) and the simulated temporal curves are displayed in curves (ii') and (iii') (solid curve).The fit of simulated curves to the experimental results using the rate constants derived for curve (i), suggest that the kinetic model and the set of rate constants presented adequately the kinetics of the transient transcription processes.Table S1.Rate constants derived from the computational simulation of the Pb 2+ -DNAzymemodulated transcription dissipative system shown in Figure 1(A).

Kinetic equations of the nickase-stimulated transient operation of the transcription machinery shown in
Table S2.Rate constants derived from the computational simulation of the strand-displacementmodulated transcription dissipative system shown in Figure 3(A).
-dimethyl-4H-imidazol-4-one) were purchased from Sigma-Aldrich.Oligonucleotides were purchased from Integrated DNA Technologies and Sigma-Aldrich.All the sequences of the oligonucleotides were listed below:

Figure S1 .
Figure S1.Calibration curve corresponding to the fluorescence intensities of MG-RNA aptamer complex, MG (4 μM), in the presence of variable concentrations of the RNA aptamer.The curve is fit linearly and the r 2 = 0.9996.

Figure S2 .
Figure S2.Computational simulation of the dissipative DNAzyme-triggered operation of a transient transcription machinery shown in Figure 1.The kinetic scheme of the sub-reactions associated with the time-dependent concentration changes during the dissipative transitions is summarized in the above equations.Knowing the time-dependent concentration changes of the RNA product R1, we computationally simulated the time-dependent concentration changes by using Matlab R2020a.

Figure S3 .
Figure S3.Temporal concentration changes of the MG-RNA aptamer generated by the transient reaction module shown in Figure 1(A) in the presence of variable concentrations of the fuel triggering strand Lr: (i) 0.05 μM; (ii) 0.1 μM; (iii) 0.15 μM.Solid curves (i', ii', and iii') correspond to the computationally simulated kinetic profiles.Dotted curves (i, ii, and iii) represent the experimental data.

Figure S4 .
Figure S4.Temporal fluorescence intensities of the MG-RNA aptamer generated by the DNAzymetriggered transient reaction module shown in Figure 1(A), with the fuel triggering strand Lr 0.1 μM, experiments repeated for 3 times.

Figure S5 .
Figure S5.Control experiment for the temporal fluorescence intensities of the MG-RNA aptamer generated by the DNAzyme-triggered transient reaction module shown in Figure 1(A) in the absence of the trigger Lr.

Figure S6 .
Figure S6.Temporal fluorescence intensities of the MG-RNA aptamer generated by the transient reaction module shown in Figure 1(A) in the presence of variable concentrations of different concentrations of NTPs: (i) 2 mM; (ii) 4 mM; (iii) 6 mM.

Figure
Figure S7(B) curve (i) depicts the time-dependent fluorescence changes proceeding in the system.A non-dissipative, continuous generation of the MG-RNA aptamer is observed.For comparison, Figure S7(B), curve (ii) depicts the temporal fluorescence changes of the MG-RNA aptamer in the transiently-modulated system shown in Figure 1(A).The results indicate that the ribonucleobase in the triggering strand Lr is essential to drive the Pb 2+ -DNAzyme-dependent transiently-modulated transcription of the MG-RNA aptamer.

Figure S7 .
Figure S7.(A) Schematic reaction module for the DNAzyme-triggered operation of a transcription machinery synthesizing the MG-RNA aptamer by using the control fuel strand Lc demonstrating that the dissipative dynamic transcription of the MG-RNA aptamer is dependent on the DNAzyme cleavage of the trigger.(B) Time-dependent fluorescent changes of the MG-RNA aptamer complexes from the transcription machinery by using the different trigger Lc (0.1 μM, curve (i)) and Lr (0.1 μM, curve (ii)), respectively.Inset is the magnified curve (ii).

Figure S8 .
Figure S8.Schematic reaction module for the DNAzyme-triggered operation of a transient transcription machinery synthesizing the DFHBI-RNA aptamer.

Figure S9 .
Figure S9.Computational simulation of the strand-displacement guided transient transcription machinery shown in Figure 3.The kinetic scheme of the sub-reactions associated with the timedependent concentration changes during the dissipative transitions is summarized in the above equations.Knowing the time-dependent concentration changes of the RNA product R2, we computationally simulated the time-dependent concentration changes by using Matlab R2020a.

Figure S10 .
Figure S10.Temporal concentration changes corresponding to the transcribed MG-RNA aptamer following scheme shown in Figure 3(A) in the presence of variable concentrations of fuel F: (i) 0.05 μM; (ii) 0.1 μM; (iii) 0.15 μM.Solid curves (i', ii', and iii') correspond to the computationally simulated kinetic profiles.Dotted curves (i, ii, and iii) represent the experimental data.

Figure S11 .
Figure S11.Temporal fluorescence intensities of the MG-RNA aptamer generated by the stranddisplacement guided transient reaction module shown in Figure 3(A), in the presence of trigger F 0.1 μM, experiments repeated for 3 times.

Figure S12 .
Figure S12.Control experiment for the temporal fluorescence intensities of the MG-RNA aptamer generated by the strand-displacement guided transient reaction module shown in Figure 3(A) in the absence of the trigger F.

Figure S13 .
Figure S13.Temporal fluorescence changes corresponding to the transcribed MG-RNA aptamer shown in Figure 3(A) using the strand displacement principle in the presence of variable concentrations of NTPs: (i) 1 mM; (ii) 2 mM; (iii) 4 mM.

Figure S14 .
Figure S14.Temporal fluorescence changes corresponding to the transcribed MG-RNA aptamer shown in Figure 3(A) using the strand displacement principle in the presence of variable concentrations of the DNA template T4/A3: (i) 0.1 μM; (ii) 0.05 μM; (iii) 0.02 μM.

Figure S15 .
Figure S15.PAGE electrophoretic separation demonstrating the fueled transient operation of the T4/A3 transcription template transcribing the anti-fuel strand F' and the temporal formation of the F/F' waste product: (Lane 1-Lane 4: reference constituents of the transcription machinery) Lane 1the fuel strand F (2 μM, 10 μL); Lane 2-the reference waste product (1 μM, 10 μL); Lane 3-the template T4/A3 (1 μM, 10 μL), Lane 4-the F-modified T4/A3 (1 μM, 10 μL); (Lane 5-Lane 8:dynamic, temporal features of the transcription machinery constituents: T4/A3, 1 μM; NTPs, 4 mM each) Lane 5-the transcription template T4/A3 prior to the addition of the fuel strand F and T7 RNAP, in the presence of NTPs; Lane 6-After addition of the fuel strand F (1 μM), in the presence of NTPs; Lane 7-the system upon addition of T7 RNAP (1.25 × 10 4 U/mL), in the presence of NTPs, after a time-interval of two minutes; Lane 8-the system in the presence of T7 RNAP, the NTPs, after a time interval of one hour.The formation of the waste product F/F' and the recovery of the inactive T4/A3 template confirm the transient operation of the fueled transcription machinery generating F/F' as waste.The 8% PAGE was performed for 20 h at 80 V (at 8 ℃).
Figure S16(B), curve (i) shows the temporal fluorescence changes of the MG-RNA aptamer upon operation of the transcription machinery displayed in Figure S16(A).Continuous, non-dissipative formation of the MG-RNA aptamer is observed.Figure S16(B), curve (ii) shows the temporal fluorescence changes of the MG-RNA aptamer transcribed according to Figure 3(A).The results demonstrate that the strand displacement of the triggering strand F by the strand F' is essential to induce the transiently-modulated transcription apparatus shown in Figure 3(A).

Figure S16 .
Figure S16.(A) Schematic of the dynamic transcription of the MG-RNA aptamer based on the strand-displacement guided transient transcription machinery by using the control trigger Fc that cannot be displaced by the transcribed F'. (B) Time-dependent fluorescent changes of the MG-RNA aptamer complexes based on the strand displacement principle by using the different triggers: Fc (0.1 μM, curve (i)) and F (0.1 μM, curve (ii)), respectively.Inset is the magnified curve (ii).

Figure S18 .
Figure S18.Schematic of the dynamic transcription of the DFHBI-RNA aptamer using the strand displacement principle guided transient transcription machinery.

Figure S19 .
Figure S19.(A) Temporal fluorescence changes corresponding to the transcribed DFHBI-RNA aptamer using the strand displacement principle shown in Figure S18 in the presence of variable concentrations of fuel F: (i) 0.05 μM; (ii) 0.1 μM; (iii) 0.15 μM.(B) Time-dependent catalytic transcription rates corresponding to the transient synthesis of the DFHBI-RNA aptamer in the presence of different concentrations of fuel F: (i) 0.05 μM; (ii) 0.1 μM; (iii) 0.15 μM.

Figure S20 .
Figure S20.(A) Temporal fluorescence changes upon the cyclic operation of the transient reaction module synthesizing the DFHBI-RNA aptamer.The time marked with an arrow indicates the time reactivation of the reaction module by adding the fuel strand F: 0.05 μM and 0.15 μM.(B) Cyclic catalytic rates corresponding to the stepwise operation of the transient transcription machinery.

Figure S21 .
Figure S21.Computational simulation of the nickase-stimulated transient operation of a transcription machinery shown in Figure 5.The kinetic scheme of the sub-reactions associated with the time-dependent concentration changes during the dissipative transitions is summarized in the above equations.Knowing the time-dependent concentration changes of the RNA product R3, we computationally simulated the time-dependent concentration changes by using Matlab R2020a.

Figure S22 .
Figure S22.Temporal concentration changes corresponding to the transcription of the MG-RNA aptamer upon the triggered nickase-stimulated transcription machinery, in the presence of variable concentrations of the trigger L1: (i) 0.1 μM; (ii) 0.2 μM; (iii) 0.3 μM.Solid curves (i', ii', and iii') correspond to the computationally simulated kinetic profiles.Dotted curves (i, ii, and iii) represent the experimental data.

Figure S23 .
Figure S23.Temporal fluorescence intensities of the MG-RNA aptamer generated by the nickasestimulated guided transient reaction module shown in Figure 5(A), in the presence of trigger L1 0.2 μM, experiments repeated for 3 times.

Figure S24 .
Figure S24.Control experiment for the temporal fluorescence intensities of the MG-RNA aptamer generated by the nickase-stimulated transient reaction module shown in Figure 5(A) in the absence of the trigger L1.

Figure S25 .
Figure S25.Time-dependent fluorescence changes of the MG-aptamer complex based on the nickase-stimulated dissipative transcription machinery in the presence of different concentrations of NTPs: (i) 1.33 mM; (ii) 2.66 mM; (iii) 4 mM.

Figure S26 .
Figure S26.Time-dependent fluorescence changes of the MG-aptamer complex based on the nickase-stimulated dissipative transcription machinery in the presence of different concentrations of Nt.BbvCI: (i) 166.7 U/mL; (ii) 83.3 U/mL; (iii) 0 U/mL.

Figure S27 .
Figure S27.Schematic of the transient transcription of the DFHBI RNA aptamer based on the nickase (Nb.BtsI)-stimulated dissipative transcription machinery.

Figure S32 .
Figure S32.Panel I depicts the temporal transcription of the DFHBI-RNA aptamer driven by the strand-displacement-modulated transcription machinery; Panel II, the temporal transcription of the MG-RNA aptamer stimulated by the nickase-modulated transcription machinery.