Buffer Formulations

(1) Binding Buffer: PBS pH 7.4 (used in hybridization)

(2) 5x Transcription Buffer (used in T7 transcription)

(3) 10x Reaction in Cas system (used in Cas reaction)

Procedure

1. Calculate the required mass of solid solutes using the formula: Mass (mg) = Concentration (mM) × Volume (mL) × Molecular Weight (g/mol)
2. Rinse reagent bottles with Type I water. Weigh the solids using a balance, transfer them to the bottles, and add Type I water up to the target volume mark. Cap the bottles and label them with the solution names.
3. Place the prepared solutions in an ultrasonic cleaner and sonicate for approximately 3 minutes to accelerate solute dissolution.
4. Adjust the pH of the buffer solutions:
   • Remove the electrode from the KCl solution and rinse the pH meter electrode with Type I water and dry it
   • Calibrate the pH meter using standard buffer solutions (pH 4.00, 6.98, and 8.18)
   • First measure the buffer's pH and adjust it by adding NaOH or HCl dropwise until pH reaches standard.
   • Rinse the electrode with Type I water between measurements
   • After completion, rinse the electrode and store it in KCl solution
5. Filter the solutions for homogeneity and to remove any undissolved solids:
   • Rinse the filtration apparatus with Type I water
   • Set up the vacuum filtration system using 45 μm filter paper
   • Secure the collection flask and funnel with clamps to prevent air leaks
   • Turn on the vacuum pump to begin filtration
   • After filtration, disconnect the air tube before turning off the pump
   • Dismantle the filtration setup and transfer the filtrate to reagent bottles
   • Rinse the filtration apparatus with Type I water between different solutions

Strand Hybridization Protocol for Polyacrylamide Gel Electrophoresis

Scope of Application: The experiments in the following table were conducted according to this protocol. The terms "LBO" and "SRO" mentioned hereafter are collective designations for these sequences.

Objective

For both the T7 and Cas systems, we initially hybridized the LBO and SRO strands at a 1:1 ratio and verified the hybridization efficiency via PAGE electrophoresis.

Procedure

1. Prepare the hybridization strands according to the table below.

   Group Reactants	LBO1	SRO1	LBO:SRO 1:1
   10μM LBO1(μL)	1	0	1
   10μM SRO1(μL)	0	1	1
   PBS(μL)	9	9	8
   Overall system	10 μL

2. Place the prepared hybridization strands in boiling water and allow them to cool naturally to room temperature to facilitate hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

Strand Displacement Protocol 1 for Polyacrylamide Gel Electrophoresis

Scope of Application: The experiments in the following table were conducted according to this protocol.

Objective

Using polyacrylamide gel electrophoresis for preliminary qualitative study on whether strand displacement reaction can occur after adding antibiotics.

Procedure

1. Prepare the hybridization strands according to the table below.

   Group Reactants	1	2	3	4	5	6	7	8
   10μM LBO(μL)	1	0	1	1	1	1	1	1
   10μM SRO(μL)	0	1	1	1	1	1	1	1
   PBS(μL)	9	9	8	7	7	7	7	7

2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds. Then place the prepared hybridization stands in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization.
3. After annealing and hybridization, add antibiotic solution to each tube according to the table below to initiate the strand displacement reaction.

   Reactants	1	2	3	4	5	6	7	8
   VAN(μL)	0	0	0	1	1	1	1	1
   Overall(μL)	10

   Note: The concentrations of added vancomycin were 50 μM, 100 μM, 200 μM, 500 μM, and 1 mM respectively for group 4-8, and are diluted ten times in the reaction system, and the final concentrations are respectively 5μM, 10μM, 20μM, 50μM, 100μM.

4. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

Strand Displacement Protocol 2 for Polyacrylamide Gel Electrophoresis

Scope of Application: The experiments in the following table were conducted according to this protocol.

Improvements

We added the antibiotic solution during the preparation of the hybridization chains, rather than adding it after annealing as previously done, to ensure thorough occurrence of the strand displacement reaction.

Procedure

1. Prepare the hybridization chains according to the table below.

   Group Reactants	1	2	3	4	5	6	7	8
   10μM LBO (μL)	1	0	1	1	1	1	1	1
   10μM SRO (μL)	0	1	1	1	1	1	1	1
   PBS (μL)	9	9	8	7	7	7	7	7
   VAN (μL)	0	0	0	1(50μM VAN)	1(100μM VAN)	1(200μM VAN)	1(500μM VAN)	1(1mM VAN)
   Overall system	10 μL

   Note: The concentrations of added vancomycin were 50 μM, 100 μM, 200 μM, 500 μM, and 1 mM respectively, and are diluted ten times in the reaction system, and the final concentrations are respectively 5μM, 10μM, 20μM, 50μM, 100μM.

2. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

Strand Proportional Hybridization Protocol for Polyacrylamide Gel Electrophoresis

Scope of Application: The experiments in the following table were conducted according to this protocol.

Objective

We found that for the LBO and SRO strands of the T7 system, the hybridization efficiency between LBO and SRO is not sufficiently high. When hybridized at a 1:1 ratio of LBO:SRO-T7, a significant amount of free SRO remains in the system, leading to signal leakage. Therefore, it is necessary to identify an appropriate SRO:LBO ratio that keeps the background signal within an acceptable range. (In contrast, the LBO and SRO of the Cas system hybridize at a ratio close to 1:1. So during the strand displacement experiments, the ratio of LBO to SRO was not optimized through screening at this stage.)

We first conducted a preliminary study on the hybridization of LBO and SRO strands using polyacrylamide gel electrophoresis to find a suitable ratio.

Procedure

1. Take 8 centrifuge tubes (1.5ml each) and label them as LBO, SRO, LBO:SRO=1:1, LBO:SRO=1:1.5, LBO:SRO=1:2, LBO:SRO=1:2.5, LBO:SRO=1:3, LBO:SRO=1:3.5.
2. Prepare the hybridization strands according to the table below.

   Group Reactants	LBO1	SRO1	LBO:SRO 1:1	LBO:SRO 1:1.5	LBO:SRO 1:2	LBO:SRO 1:2.5	LBO:SRO 1:3	LBO:SRO 1:3.5
   10μM LBO(μL)	1	0	1	1.5	2	2.5	3	3.5
   10μM SRO(μL)	0	1	1	1	1	1	1	1
   PBS(μL)	9	9	8	7.5	7	6.5	6	5.5
   Overall system	10 μL

3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used as a sample for polyacrylamide gel electrophoresis.

Fluorescence Quantification Protocols

Strand Proportional Hybridization Protocol for Fluorescence Quantification (used in T7 system)

Scope of Application: The experiments in the following table were conducted according to this protocol.

Objective

In the absence of antibiotics, hybridize SRO with LBO and measure the fluorescence of the system. The fluorescence value reflects the amount of free SRO in the system, which may activate downstream amplification pathways and generate background signals. Therefore, it is necessary to identify an appropriate SRO:LBO ratio that keeps the background signal within an acceptable range.

Procedure

1. Different SRO:LBO ratios were tested to evaluate the background fluorescence levels under each condition, enabling the selection of an optimal SRO:LBO ratio for sensor construction.

   Reagent	1:1	1:2	1:3	1:5	1:7	1:9	Positive control
   10μM LBO(μL)	0	0	0	0	0	0	0
   10μM SRO(μL)	0	0	0	0	0	0	0
   10μM LBO-BHQ(μL)	3	6	9	15	21	27	0
   10μM SRO-FAM(μL)	3	3	3	3	3	3	3
   PBS(μL)	24	21	18	12	6	0	27
   Overall(μL)	30	30	30	30	30	30	30

2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for constructing the strand displacement reaction system.

Strand Displacement Protocol for Fluorescence Quantification (used in T7 system)

Objective

Based on the ratio hybridization experiments, we determined that a 5:1 ratio of LBO:SRO is optimal in the T7 system. Therefore, this ratio was selected for quantitative fluorescence analysis to assess whether the system could produce antibiotic concentration-dependent fluorescence changes after the strand displacement reaction was initiated by adding antibiotics.

Procedure

1. Prior to the strand displacement reaction, we first prepare the required hybridization chains in a unified manner to ensure that all experimental groups utilize the same hybridization chains when constructing the reaction system. Prepare the hybridization chains according to the table below.

   Reagent	Experimental group	Positive control	Negative control
   10μM LBO(μL)	0	0	15
   10μM SRO(μL)	0	0	3
   10μM LBO-BHQ(μL)	100	0	0
   10μM SRO-FAM(μL)	20	3	0
   PBS(μL)	80	27	12
   Overall(μL)	200	30	30

2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for construct the strand displacement reaction system.
4. Set up the reaction system as follows:

   Groups	1	2	3	4	5	6	7	8
   VAN (μL)	0	0	0	5	5	5	5	5
   Hybridized strands (μL)	5	5	5	5	5	5	5	5
   PBS(μL)	45	45	45	40	40	40	40	40
   Types of hybridized strands(μL)	LBO+SRO-FAM	LBO+SRO	LBO-BHQ + SRO-FAM

   Microplate reader parameters:

   • Temperature: 37°C
   • Excitation wavelength: 490 nm
   • Emission wavelength: 520 nm
   • Time: 30 min

Strand Displacement Protocol for Fluorescence Quantification (used in Cas system)

Scope of Application: The experiments in the following table were conducted according to this protocol.

Objective

In the Cas system, the LBO and SRO strands can hybridize at a ratio close to 1:1. Consequently, this ratio was selected for quantitative fluorescence analysis to determine whether the system exhibits antibiotic concentration-dependent fluorescence changes following the strand displacement reaction triggered by antibiotic addition.

Procedure

1. Prior to the strand displacement reaction, we first prepare the required hybridization chains in a unified manner to ensure that all experimental groups utilize the same hybridization chains when constructing the reaction system. Prepare the hybridization chains according to the table below.

   Reagent	Experimental group	Positive control
   10mM LBO-BHQ(μL)	200	0
   10mM SRO-FAM(μL)	40	3
   PBS(μL)	160	27
   Overall(μL)	400	30

2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.
3. Place the prepared hybridization chains in boiling water and allow them to cool naturally to room temperature to facilitate DNA hybridization. The annealed solution can then be used for construct the strand displacement reaction system.
4. Construct strand displacement reaction system (Each group repeated 5 times)

   Reagents	No RNA						100 nM RNA						Positive control
   	0	1	2	3	4	5	0	1	2	3	4	5
   1 μM crRNA (μL)	0	0	0	0	0	0	5	5	5	5	5	5	0
   VAN (μL)	0	5	5	5	5	5	0	5	5	5	5	5	0
   Strands (μL)	5	5	5	5	5	5	5	5	5	5	5	5	5
   PBS (μL)	45	40	40	40	40	40	40	35	35	35	35	35	45
   Types of hybridized strands	LBO-BHQ + SRO-FAM												SRO-FAM

   Note: The concentrations of added vancomycin in group 1-5 were 50 μM, 100 μM, 200 μM, 500 μM, and 1 mM respectively, and are diluted ten times in the reaction system, and the final concentrations are respectively 5μM, 10μM, 20μM, 50μM, 100μM.

5. Microplate reader parameters:
   • Temperature: 37°C
   • Excitation wavelength: 490 nm
   • Emission wavelength: 520 nm
   • Time: 30 min

Improvement

In subsequent experiments, to further reduce signal leakage, we slightly increased the LBO:SRO ratio to 1.3:1 and conducted the experiments using the same method as described in the protocol.

Polyacrylamide Gel Electrophoresis (used in T7 and Cas system)

Procedure

1. Gel preparation

1. Assemble the gel cassette and check for leaks.
2. Prepare the gel according to the formulation in the table below.
3. After preparation, vortex to mix thoroughly. Add the gel into the electrophoresis tank using a pipette, insert a comb, and avoid generating bubbles during the process.
4. Wait 30-45 minutes for the gel to solidify. Add 1x TBE as the electrophoresis buffer until the outer liquid level is half the height of the inner liquid level. Then, remove the comb.
5. Add 10x Loading Buffer to the annealed sample at a 1/10 volume ratio, mix well, and centrifuge.
6. Add 10 μL of sample to each well of the solidified gel, and load an equal amount of marker into the wells at both ends.

2. Running the gel

1. Load the DNA samples into the wells of the gel.
2. Apply electricity at 120V with a maximum current of 600mA, and run the electrophoresis for about 30 minutes.

3. Gel Staining and Observation

1. Prepare the developing solution.
2. Cut the gel and place it into a gel staining box. Add approximately 25 ml of developing solution, and incubate protected from light on a shaker for 15 minutes.
3. Develop and observe the gel bands using a gel imaging system.

Transcription Protocol for SSS-M Aptamer Sensor (Before Annealing-Condition Change)

Objective

To evaluate the transcriptional response of the SSS-S single-stranded aptamer sensor under different vancomycin (VAN) concentrations.

Materials & Reagents

• 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
• T7 Template DNA strand (10 µM stock)
• SSS-M aptamer DNA (10 µM stock; annealed to 1 µM before use)
• NTP mix (25 mM)
• DFHBSI (1 mM stock)
• Pyrophosphatase (PPase) 0.001 U/µL
• T7 RNA polymerase (50 µM)
• Vancomycin (VAN) (1 mM stock; also prepare 50 µM and 200 µM working solutions)
• DEPC-treated RNase-free water

Procedure

1. Annealing SSS-M DNA
   • Objective: Generate a 1 µM annealed stock of SSS-S aptamer DNA for direct use in transcription reactions.
   • Mix 10.0 µL of 10 µM SSS-M DNA with 90.0 µL DEPC water to obtain 1 µM SSS-S DNA (100 µL total).
   • Annealing program:
     • Heat at 95 °C for 5 min
     • Slowly cool to room temperature (e.g., 0.1 °C/s)
     • Hold on ice until use
   • For this experiment, 30 µL of 1 µM SSS-M is required for the transcription system. Preparing 100 µL provides extra volume to account for pipetting losses.
2. Transcription Master Mix (510 µL)

   Mix in a 1.5 mL centrifuge tube (without VAN and T7 polymerase):

   Component	Final conc.	Volume (µL)
   5× Transcription buffer	1×	120.0
   T7 Template strand (10 µM)	1 µM	60.0
   Annealed SSS-M (1 µM)	50 nM	30.0
   NTP mix (25 mM)	2 mM	48.0
   DFHBSI (1 mM)	1 µM	0.6
   Pyrophosphatase (0.001 U/µL)	0.00001 U	6.0
   DEPC water	---	245.4
   Total	---	510.0

   This master mix volume is sufficient for 12 wells at 42.5 µL/well.

3. Plate Setup

   Distribute 42.5 µL of transcription master mix per well. Then add VAN and T7 polymerase to reach a final volume of 50 µL/well.

   Group	Transcription Mix (µL)	VAN (µL)	VAN conc. (final)	T7 polymerase (µL, 50 µM)	DFHBSI only (µL)
   Group 0	42.5	5.0 (PBS)	0 µM	2.5	0
   Group 1	42.5	5.0 (50 µM)	50 µM	2.5	0
   Group 2	42.5	5.0 (200 µM)	200 µM	2.5	0
   Group 3	42.5	5.0 (1 mM)	1 mM	2.5	0
   Negative Ctrl	0	0	---	0	50 (1 µM DFHBSI)

4. Anneal SSS-M DNA as described in Step 1.
5. Prepare transcription master mix (510 µL) in a 1.5 mL centrifuge tube (Step 2).
6. Distribute 42.5 µL of the master mix into each designated well of a 384-well plate.
7. Quickly add VAN (5 µL/well):
   • Group 0: PBS 5 µL
   • Group 1: 50 µM VAN 5 µL
   • Group 2: 200 µM VAN 5 µL
   • Group 3: 1 mM VAN 5 µL
   • Negative control: none
8. Immediately add T7 RNA polymerase (2.5 µL/well, 50 µM) to all wells except negative control. Seal the plate with film, gently tap or briefly centrifuge to collect liquid at the bottom. Incubate at 37 °C for 10 hours. Record fluorescence every 1 minutes at Ex/Em = 506/560 nm.

Controls

• Group 0 (PBS) serves as negative VAN control.
• Negative control wells (50 µL DFHBSI without transcription components or T7) measure background fluorescence.

Transcription Protocol for SSS-M Aptamer Sensor: Template Concentration Gradient

Purpose

Evaluate how template DNA concentration (≈1 µM / 100 nM / 10 nM final) affects the transcriptional response of the SSS-M sensor under 0 µM vs 1 mM vancomycin (VAN) conditions.

Materials & Reagents

• 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
• SSS-M aptamer DNA (10 µM stock → anneal to 1 µM before use)
• T7 Template DNA stocks for per-well addition: 10 µM, 1 µM, 100 nM
• NTP mix (25 mM)
• DFHBSI (1 mM stock)
• Pyrophosphatase (PPase) 0.001 U/µL
• T7 RNA polymerase (50 µM)
• Vancomycin (VAN) (1 mM stock; PBS for 0 µM)
• DEPC-treated RNase-free water

Procedure

1. Annealing SSS-M DNA (to 1 µM)
   • Mix 10.0 µL of 10 µM SSS-M with 90.0 µL DEPC water → 1 µM SSS-S (100 µL total).
   • Anneal: 95 °C for 5 min → slow cool to RT (e.g., 0.1 °C/s) → hold on ice.
   • This run needs 50 µL of the 1 µM SSS-M for the master mix; preparing 100 µL provides headroom.
2. Transcription Master Mix (Total 750 µL)

   This master mix does not contain VAN, T7 polymerase, or template DNA. Template will be added per well to set the gradient.

   Component	Final conc. (in reaction)	Volume
   5× Transcription buffer	1×	200 µL
   SSS-M (1 µM, annealed)	50 nM	50 µL
   NTP mix (25 mM)	2 mM	80 µL
   DFHBSI (1 mM)	1 µM	1 µL
   DEPC water	---	409 µL
   Pyrophosphatase (0.001 U/µL)	0.00001 U	10 µL
   Total	---	750 µL

   Mix gently, vortex briefly, spin down. This volume supports 20 wells at 37.5 µL/well of master mix.

3. Plate Setup (Per-well totals = 50 µL)

   Distribute 37.5 µL master mix per well, then add VAN, T7 polymerase, and 5 µL template stock as indicated:

   Group	Master Mix (µL)	VAN (µL)	T7 (µL, 50 µM)	Template per well	Notes (Final template in 50 µL)
   1	37.5	5.0 (0 µM, PBS)	2.5	5 µL of 10 µM	~1 µM
   2	37.5	5.0 (1 mM)	2.5	5 µL of 10 µM	~1 µM
   3	37.5	5.0 (0 µM, PBS)	2.5	5 µL of 1 µM	~100 nM
   4	37.5	5.0 (1 mM)	2.5	5 µL of 1 µM	~100 nM
   5	37.5	5.0 (0 µM, PBS)	2.5	5 µL of 100 nM	~10 nM
   6	37.5	5.0 (1 mM)	2.5	5 µL of 100 nM	~10 nM
   Negative	0	0	0	---	50 µL of 1 µM DFHBSI only

   Template working stocks (from 10 µM):

   • 1 µM: 10 µL of 10 µM + 90 µL water.
   • 100 nM: 10 µL of 1 µM + 90 µL water (or 1 µL of 10 µM + 99 µL water).
4. Anneal SSS-M to 1 µM (Step 1).
5. Prepare the 750 µL master mix (Step 2).
6. Dispense 37.5 µL/well master mix to the 384-well plate.
7. Immediately add VAN (5 µL/well) per group (PBS for 0 µM; 1 mM VAN for +VAN groups).
8. Immediately add T7 polymerase (2.5 µL/well, 50 µM).
9. Add template DNA (5 µL/well) using the specified stock for each group (10 µM / 1 µM / 100 nM).
10. Seal the plate. Incubate at 37 °C for 10 h. Record fluorescence every 1 min at Ex/Em =506 / 560nm.

Controls

• 0 µM VAN (PBS) serves as the no-VAN control at each template level.
• Negative wells: 50 µL of 1 µM DFHBSI only for background fluorescence.

Transcription Protocol for SSS-M (Low-template System; T7 in Master)

This experiment followed the same procedure as "Experiments --- SSS-M Aptamer Sensor (Before Annealing-Condition Change)", except that the template strand concentration was diluted to 10 nM to evaluate how lower template input affects transcription efficiency and signal-to-background ratio.

Transcription Protocol for SSS-M (Modified Annealing: Co-anneal with VAN vs PBS; T7 in Master)

Purpose

Assess whether co-annealing SSS-M with the T7 reverse strand in the presence of VAN (vs PBS) changes transcriptional output.

Materials & Reagents

• 10 µM SSS-M DNA
• 10 µM T7 reverse strand DNA
• Vancomycin (VAN) 1 mM (PBS as 0 µM control)
• 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
• Template DNA working solution (2.5 µM) --- added later
• NTP mix 25 mM
• DFHBSI 1 mM
• T7 RNA polymerase 50 µM
• Pyrophosphatase (PPase) 0.001 U/µL
• DEPC-treated water

Procedure

1. DNA Annealing (SSS-M + T7 Reverse, ±VAN)

   Tube A (VAN-annealed "template")

   Component	Volume
   10 µM SSS-M	8 µL
   10 µM T7 reverse strand	8 µL
   1 mM VAN	16 µL
   Total	32 µL

   Tube B (PBS-annealed "template")

   Component	Volume
   10 µM SSS-M	8 µL
   10 µM T7 reverse strand	8 µL
   PBS	16 µL
   Total	32 µL

   Annealing program (both tubes): Hot water bath >95 °C for 5 min, then cool to RT (bench) → keep on ice.

2. In-vitro Transcription System (Master, 300 µL total)

   Do NOT add the 2.5 µM template yet. Prepare the base mix first (300 µL recipe shown below), then split, then add annealed material.

   Base Mix (without template, 240 µL):

   Component	Volume
   5× Transcription buffer	60 µL
   25 mM NTP mix	15 µL
   50 µM T7 polymerase	15 µL
   DEPC water	144 µL
   1 mM DFHBSI	3 µL
   0.001 U/µL PPase	3 µL
   Subtotal	240 µL

   Split the 240 µL base mix into two tubes, 120 µL each.

   Now add template (2.5 µM) + annealed materials:

   • To Tube 1 (VAN condition): add 30 µL of the annealed Tube A material.
   • To Tube 2 (PBS condition): add 30 µL of the annealed Tube B material likewise.

   At this point, each condition tube is ~150 µL (120 µL base + 30 µL template/annealed additions).

3. Plate Setup (per-well totals = 50 µL, 3 replicates/condition)

   For each condition (VAN-annealed vs PBS-annealed), dispense 50 µL/well into 3 wells (total 150 µL per condition). Include a background well if desired.

   Condition	Per-well volume	Replicates
   VAN-annealed (Tube A)	50 µL	3
   PBS-annealed (Tube B)	50 µL	3
   Negative (optional)	50 µL of 1 µM DFHBSI only	≥1

4. Anneal SSS-M + T7 reverse with VAN (Tube A) or PBS (Tube B) as in Step 1.
5. Prepare the 240 µL base mix (Step 2) without template; split 120 µL + 120 µL.
6. Add 30 µL of 2.5 µM template to each tube and incorporate the respective annealed material (VAN or PBS) per your run plan → ~150 µL per condition.
7. Quickly dispense 50 µL/well into a 384-well plate (3 wells per condition).
8. Seal the plate. Incubate at 37 °C for 10 h. Read fluorescence every 1 min (Ex/Em506 /560 nm).

Controls

• PBS-annealed condition serves as the 0-VAN control.
• Optional background wells: 1 µM DFHBSI only (50 µL) for instrument background.

Transcription Protocol for SSS-M (Modified Annealing; T7 Polymerase Concentration Series)

Purpose

Assess how T7 RNA polymerase level affects transcriptional output when SSS-M and the T7 Reverse strand are co-annealed either with 1 mM VAN or with PBS (0 µM).

Materials & Reagents

• 10 µM SSS-M DNA
• 10 µM T7 Reverse strand DNA
• Vancomycin (VAN) 1 mM; PBS (0 µM)
• 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9)
• NTP mix 25 mM
• DFHBSI 100 µM
• Pyrophosphatase (PPase) 0.001 U/µL
• T7 RNA polymerase stocks: 50 µM, 40 µM, 25 µM (and optionally 50 µM, 5 µM, 0.5 µM for the second set)
• DEPC-treated RNase-free water

Procedure

1. DNA Annealing (SSS-M + T7-Reverse, ±VAN)

   Tube A (VAN-annealed duplex)

   Component	Volume
   10 µM SSS-M	24 µL
   10 µM T7 Reverse strand	24 µL
   1 mM VAN	48 µL
   Total	96 µL

   Tube B (PBS-annealed duplex)

   Component	Volume
   10 µM SSS-M	24 µL
   10 µM T7 Reverse strand	24 µL
   PBS	48 µL
   Total	96 µL

   Annealing program (both tubes): Hot water bath >95 °C for 5 min, then allow to cool to room temperature; keep on ice until use.

2. In-vitro Transcription System (Base Master, 900 µL total)

   Prepare in a 1.5 mL tube (no template yet):

   Component	Volume
   5× Transcription buffer	200 µL
   25 mM NTP mix	50 µL
   DEPC water	480 µL
   DFHBSI 100 µM	8 µL
   Pyrophosphatase 0.001 U/µL	10 µL
   Subtotal (no T7, no template)	748 µL

   Now split this 748 µL base into three equal systems (≈ 249--250 µL each).

   Standardize each system to 225 µL (discard/retain excess so each is exactly 225 µL), then add T7 as below:

   System	Base (pre-T7)	T7 polymerase	T7 stock	T7 volume
   Sys-1 (High)	225 µL	+ 15 µL	50 µM	15 µL
   Sys-2 (Mid)	225 µL	+ 15 µL	40 µM	15 µL
   Sys-3 (Low)	225 µL	+ 15 µL	25 µM	15 µL

   Mix each system gently after adding T7.

   Second set: repeat the same workflow with T7 stocks at 50 µM, 5 µM, 0.5 µM (still 15 µL per 225 µL system) to create an additional high/medium/low enzyme series

3. Split per Condition (±VAN duplex "template")

   Do not add "template" earlier. From each T7 system (Sys-1/2/3), split into two tubes of 110 µL (for 0 µM and 1 mM conditions):

   • Sys-X / PBS condition: take 110 µL of the T7-containing system + 27.5 µL of the PBS-annealed duplex (from Step 1, Tube B).
   • Sys-X / VAN condition: take 110 µL of the T7-containing system + 27.5 µL of the VAN-annealed duplex (from Step 1, Tube A).

   This yields six tubes total (High-PBS, High-VAN, Mid-PBS, Mid-VAN, Low-PBS, Low-VAN), each ~137.5 µL.

4. Plate Setup (per-well totals)

   Dispense 50 µL/well to the 384-well plate from each tube.

   At ~137.5 µL/tube, you can fill 2 wells (100 µL) comfortably (with ~37.5 µL dead volume).

   If you need triplicates, scale each split to ≥150 µL (e.g., use 120 µL split + 30 µL annealed duplex) so you can plate 3 × 50 µL per tube.

   Negative control (optional): 50 µL of 1 µM DFHBSI only to measure background fluorescence.

5. Anneal SSS-M + T7-Reverse with 1 mM VAN or PBS (Step 1).
6. Prepare the 900 µL base transcription system (Step 2, no template).
7. Create three T7 systems by adding 15 µL of the specified T7 stock to 225 µL of base (Sys-1/2/3).
8. From each T7 system, split 110 µL + 110 µL; add 27.5 µL of PBS-annealed duplex to one, and 27.5 µL of VAN-annealed duplex to the other (Step 3).
9. Plate 50 µL/well for each tube into the 384-well plate (2 wells per tube as prepared; scale if need 3).
10. Seal the plate; incubate at 37 °C for 10 h. Record fluorescence every 1 min (Ex/Em 506/560 nm).

Transcription Protocol for SSS Sensor: VAN Gradient at 1.25 µM T7

This experiment followed the same conditions as "Experiments --- SSS-M (Modified Annealing; T7 Polymerase Concentration Series)" under the 1.25 µM T7 polymerase setting, but extended the vancomycin gradient to include 0, 5, 20, and 100 µM concentrations to achieve a finer resolution of the dose--response relationship.

Transcription Protocol for SSS-S vs SSS-L

This experiment was conducted under the same conditions as "Experiments --- SSS-M (Modified Annealing: Co-anneal with VAN vs PBS; T7 in Master)", except that the DNA strands were replaced with SSS-S and SSS-L sequences to compare their structural effects on transcription activation and vancomycin responsiveness.

Transcription Protocol for TS Families (5TS-1~4; 3TS-1~8)

Purpose

Evaluate redesigned TS aptamer sensors (bulge/mismatch families) for vancomycin responsiveness under a standardized in-vitro transcription assay. Each batch runs four variants in parallel with VAN = 0 or 1 mM.

Materials & Reagents

• 5× Binding Buffer (PBS containing MgCl₂; your note: 5×, PBS with 10 mM MgCl₂)
• 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9 @ 25 °C)
• T7 Template DNA 10 µM (used in master; target 250 nM in system)
• TS variants: 5TS-1, -2, -3, -4 (and separately 3TS-1 ... -8) --- 10 µM stocks
• NTP mix 25 mM
• DFHBSI 100 µM stock (final 1 µM)
• Pyrophosphatase (PPase) 0.001 U/µL
• T7 RNA polymerase 50 µM
• Vancomycin (VAN): 1 mM stock; PBS for 0 µM
• DEPC-treated RNase-free water

Procedure

1. DNA Annealing (per variant)

   Prepare one annealing tube per variant (works for either 5TS-n or 3TS-n):

   Component	Volume
   10 µM TS variant (5TS-n or 3TS-n)	8.0 µL
   1st-grade water	4.8 µL
   5× Binding Buffer (PBS with Mg²⁺)	3.2 µL
   Total per variant	16.0 µL

   Program: Hot water bath >95 °C for 5 min, then cool to room temperature; keep on ice. (This yields an annealed TS solution you'll add to each system in Step 3.)

2. Transcription Master (Total 1,020 µL)

   (no VAN, no T7 added here; T7 and VAN are added at the plating step)

   Component	Final conc. (system)	Volume
   5× Transcription buffer	---	240 µL
   10 µM T7 Template strand	250 nM	30 µL
   25 mM NTP mix	2 mM	96 µL
   DFHBSI 100 µM	1 µM	12 µL
   DEPC water	---	570 µL
   PPase 0.001 U/µL	0.001 U	12 µL
   Total	---	1,020 µL

3. Split for Four Variants & Spike in Annealed TS

   Split the 1,020 µL master into four tubes: 240 µL × 4 (960 µL used; ~60 µL spare is dead volume)

   To each 240 µL tube, add 15 µL of the corresponding annealed TS from Step 1 → 255 µL per variant system.

   This gives four variant systems (e.g., 5TS-1, -2, -3, -4) each at ~255 µL prior to plating.

   Repeat the same for 3TS in two batches of four variants each (3TS-1~4, then 3TS-5~8).

4. Plate Setup (per-well totals = 40 µL)

   Per your grouping:

   Group	Transcription system (µL)	VAN (µL)	T7 polymerase (µL; 50 µM)	DFHBSI only (µL)
   0 (0 µM VAN)	34	4 (PBS)	2	0
   3 (1 mM VAN)	34	4 (1 mM)	2	0
   Negative	0	0	0	50 (1 µM DFHBSI)

   Quick check: 34 + 4 + 2 = 40 µL / well.

5. Procedure (per batch of four variants)
   • Anneal each TS variant (Step 1).
   • Prepare the 1,020 µL transcription master (Step 2).
   • Split master into 4 × 240 µL and add 15 µL annealed TS to each (Step 3).
   • Plate per well:
     • Add 34 µL of the corresponding variant system to each designated well.
     • Add 4 µL VAN: PBS for Group 0 (0 µM) or 1 mM VAN for Group 3 (100 µM final).
     • Add 2 µL T7 polymerase (50 µM) per well.
   • Seal; incubate at 37 °C for 10 h. Record fluorescence every 1 min at Ex/Em =506 /560 nm.

Controls

• Group 0 (PBS) is the no-VAN control for each variant.
• Negative wells: 50 µL of 1 µM DFHBSI only for instrument/background reference.

Transcription Protocol for Double-Strand Extension (S1:L1--L6)

Purpose

Evaluate the vancomycin responsiveness of the extended hybrid duplex sensors built from S1 + Ln (n = 1...6) under a standardized in-vitro transcription assay. The workflow mirrors the TS runs: prepare a 1,020 µL transcription master, split for variants, spike annealed S1:L duplex, and plate with VAN = 0 or 1 mM.

Materials & Reagents

• S1 strand (10 µM)
• L strands (10 µM): L1, L2, L3, L4, L5, L6
• 5× PBS (so that the annealing mix is 1× PBS in the 32 µL anneal)
• 5× Transcription buffer (200 mM Tris-HCl, 30 mM MgCl₂, 10 mM spermidine, 50 mM DTT, pH 7.9 @ 25 °C)
• T7 Template DNA 10 µM (target 250 nM in the transcription system)
• NTP mix 25 mM
• DFHBSI 100 µM (final 1 µM)
• Pyrophosphatase (PPase) 0.001 U/µL
• T7 RNA polymerase 50 µM
• Vancomycin (VAN) 1 mM stock; PBS for 0 µM
• DEPC-treated RNase-free water

Procedure

1. Annealing (per variant, S1:L with 5× PBS)

   Prepare one annealing tube per L variant (S1:L1, S1:L2, ... S1:L6):

   Component (10 µM stocks)	Volume
   S1	3.2 µL
   L (L1...L6)	6.4 µL
   5× PBS	6.4 µL
   Water (1st-grade)	16.0 µL
   Total	32.0 µL

   Program: Hot water bath >95 °C for 5 min, then cool to room temperature; hold on ice. (6.4 µL of 5× PBS in 32 µL → 1× PBS during annealing.)

2. Transcription Master (Total 1,020 µL; no VAN, no T7)

   Same system as above:

   Component	Final conc. (system)	Volume
   5× Transcription buffer	---	240 µL
   T7 Template (10 µM)	100 nM	12 µL
   NTP mix (25 mM)	2 mM	96 µL
   DFHBSI (100 µM)	1 µM	12 µL
   DEPC water	---	648 µL
   PPase (0.001 U/µL)	0.001 U	12 µL
   Total	---	1020 µL

   Mix gently.

3. Split for Variants & Spike Annealed Duplex

   Split the master into four equal aliquots for a 4-variant batch (repeat the batch for remaining Ls): 225 µL × 4 (uses 900 µL; ~120 µL remains as dead volume).

   To each 225µL tube, add 30µL of the corresponding annealed S1:L duplex (from Step 1) → 255 µL per variant system.

   Batching:

   • Batch A: L1, L2, L3, L4 (4 variants).
   • Batch B: L5, L6 (use the same workflow; you may replicate some variants or allocate more replicates to fill the four-way split if desired).
4. Plate Setup (per-well totals = 40 µL)

   Use the same group volumes as the TS runs:

   Group	Transcription system (µL)	VAN (µL)	T7 polymerase (µL; 50 µM)	DFHBSI only (µL)
   0 (0 µM VAN)	34	4 (PBS)	2	0
   3 (1 mM VAN)	34	4 (1 mM)	2	0
   Negative	0	0	0	50 (1 µM DFHBSI)

   Quick check: 34 + 4 + 2 = 40 µL/well.

5. Procedure (per 4-variant batch)
   • Anneal S1 with each L variant using Step 1 (5× PBS → 1× PBS in 32 µL).
   • Prepare the 1,020 µL transcription master (Step 2).
   • Split into 4 × 225µL; add 30µL annealed S1:L to each to make variant systems (Step 3).
   • Plate per well:
     • Add 34 µL of the variant system to each designated well.
     • Add 4 µL VAN: PBS for Group 0 (0 µM) or 1 mM VAN for Group 3 (100 µM final).
     • Add 2 µL T7 polymerase (50 µM) per well.
   • Seal; incubate at 37 °C for 10 h. Record fluorescence every 1 min (Ex/Em506 /560 nm).

Controls

• Group 0 (PBS) is the no-VAN control for each S1:L variant.
• Negative wells: 50 µL of 1 µM DFHBSI only for background.

Transcription Protocol for S1 : L4, L4-1, L4-2, L4-3, L5, L5-1, L5-2, L5-3, L5-4

This experiment followed the same procedure as "Experiments --- Double-Strand Extension (S1:L1--L6)", except that the L strands were replaced with L4, L4-1, L4-2, L4-3, L5, L5-1, L5-2, L5-3, L5-4, and the fluorescent dye DFHBI-1T was used instead of DFHBSI for improved signal clarity.

Transcription Protocol for S1 + L5_3: Template Concentration Gradient (0.5:1, 1:1, 5:1, 10:1)

This experiment was conducted under the same conditions as "Experiments --- S1 : L4, L4-1, L4-2, L4-3, L5, L5-1, L5-2, L5-3, L5-4", using the S1 + L5_3 duplex, but the template-to-S1 ratio was varied (0.5:1, 1:1, 5:1, 10:1) instead of the original 1:1 ratio to examine the effect of template concentration on transcriptional output.

Cas12a System

The Cas12a system can be used for nucleic acid detection. A typical Cas12a detection system consists of the following components:

• Cas12a protein: Cas12a is a nuclease belonging to the type II CRISPR--Cas system, subtype V.
• crRNA: CRISPR RNA (crRNA) is typically an RNA of 42--44 nucleotides. Unlike Cas9, which requires both crRNA and tracrRNA, the Cas12a system requires only a single crRNA.
• Reporter: A short single-stranded DNA (ssDNA) labeled with a fluorophore at one end and a quencher at the other.

Cas12a recognizes the target DNA through complementary base pairing with the crRNA. Once Cas12a is activated, it non-specifically cleaves all ssDNA in the system. At this point, the pre-introduced reporter probe (short ssDNA labeled with a fluorophore and a quencher) is cleaved. Upon cleavage, the fluorophore separates from the quencher, producing a fluorescence signal.

Procedure

1. Strand hybridization

   Reagents	0	1	2	3	4	5
   10 μM LBO-Cas(μL)	2.6	2.6	2.6	2.6	2.6	2.6
   10 μM SRO-Cas (μL)	2	2	2	2	2	2
   VAN(μL)	0	10	10	10	10	10
   PBS(μL)	15.4	5.4	5.4	5.4	5.4	5.4
   Overall(μL)	20	20	20	20	20	20

   Note: The concentrations of added vancomycin in group 1-5 were 50 μM, 100 μM, 200 μM, 500 μM, and 1 mM respectively, and are diluted ten times in the reaction system, and the final concentrations are respectively 5μM, 10μM, 20μM, 50μM, 100μM.

   6 groups of solution this step contains free SRO-Cas, serving as the target for the Cas system

2. Preparation of master mix: To ensure that each target group reacts in a uniform solution, a master mix is prepared first according to the table below, by pre-mixing all components except the target.

   Component	Volume
   10x Reaction buffer(μL)	72
   1μM reporter(μL)	14.4
   20 x Cas12a(μL)	36
   1μM crRNA(μL)	72
   DEPC water(μL)	453.6
   Overall(μL)	648

3. After preparation, add 18 μL of master mix and 2 μL of target to each tube. Perform the experiment in triplicate for each target concentration. Each group is repeated 5 times. The composition of each 20 μL reaction system is shown in the table below.

   Component	Volume	Final concentrations
   10x Reaction buffer	2 μL
   Target	2 μL
   1μM reporter	2 μL	200 nM
   20 x Cas12a	2 μL	100 nM
   1μM crRNA	2 μL	100 nM
   DEPC water	10 μL

4. Loading sample
   1. Place the centrifuge tubes on an ice tray.
   2. For each sample, add the substituted solution to the pre-mix solution to reach a final volume of 20 μL.
   3. Transfer the mixture into a 384-well plate.
5. Measurement of fluorescence intensity
   • Temperature: 37°C
   • Excitation wavelength: 490 nm
   • Emission wavelength: 520 nm
   • Time: 30 min