1.1 Design and Implementation of SRO Non-T7 Sequence Strand Displacement System

Design

The core principle of strand displacement reaction is competitive binding. In this system, vancomycin (Vancomycin, VAN) acts as a small molecule inducer, binding to the specific binding site on the long-strands aptamer LBO, thereby disrupting the pre-formed hybrid double-strand structure between LBO and the short-strands probe SRO. The SRO sequence is designed as short strands without T7 promoter sequences, focusing on the strand displacement function, and avoiding interference from the T7 promoter sequence.

Build

We designed SRO1-8 and their corresponding LBO1-8, whose lengths and sequences were optimized to reduce nonspecific binding. The LBO long strand contains the vancomycin (VAN)-specific aptamer region, with aptamers of varying affinities. The key binding domains within the LBO sequence allow competitive access of VAN. The specific sequences of LBO and SRO, as well as the aptamer affinities [2][3], are shown in the table below. (Complementary base pairs are marked in red)

Complementary base pairs are marked in red in the original NUPACK simulation.

Test

Step 1. Strand hybridization

Our first objective was to obtain a stable LBO–SRO duplex, ensuring that in the absence of antibiotics, the hybridized duplex does not spontaneously dissociate and that free SRO remains minimal in the system.

Procedure

Hybridization mixtures were prepared according to the table below, annealed by cooling from hot water to room temperature, and then analyzed by PAGE for preliminary validation.

Results

According to the electrophoresis results, LBO–SRO1–3 exhibited high hybridization efficiency.

Step 2. Strand displacement upon antibiotic addition

Based on the results from Step 1, LBO-SRO1–3 were selected to perform strand displacement assays in the presence of antibiotics.

Procedure

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

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 5 μM, 10 μM, 20 μM, 50 μM, 100 μM.

2. Place the prepared hybridization strands 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.

Results

LBO1+SRO1 System and LBO2+SRO1 System: As the VAN concentration increased (5→100 μM), the brightness of the hybridization bands significantly decreased, with the bands almost disappearing at concentrations above 50 μM, indicating high disassembly efficiency and potential for sensor construction.

LBO3+SRO3 system: The strip attenuation is relatively small, only slightly weakened at 100 μM, indicating higher hybridization stability.

Step 3. Strand proportional hybridization

In previous experiments, we observed a distinct SRO1 band when the LBO:SRO ratio was 1:1. Therefore, we increased the LBO:SRO ratio in an attempt to achieve higher SRO hybridization efficiency and to reduce the concentration of free SRO.

Procedure

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

2. Place the prepared hybridization strands 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.

Results

The experiment shows that the highest hybridization efficiency (with the best band clarity) is achieved when the molar ratio of LBO:SRO is 2:1 or 2.5:1, and a higher ratio leads to an enhanced background signal.

Learn

The LBO1–SRO1 and LBO2–SRO2 combinations exhibited higher hybridization efficiency and demonstrated superior concentration-dependent strand displacement upon vancomycin stimulation, making them suitable as the foundational framework for subsequent sensor design. Moving forward, the strand displacement system will be integrated with the transcriptional module to achieve signal amplification. Through systematic analysis of these modules, the SRO-based strand displacement system with the incorporated T7 sequence can be further optimized toward higher sensitivity and reduced background leakage, thereby providing a robust foundation for biosensor applications.

1.2 Strand Displacement System with SRO Incorporating a T7 Sequence

Design

LBO1–SRO1 and LBO2–SRO2 exhibited superior stability and stronger concentration–reaction rate correlation in the strand displacement assays, with relatively higher reaction efficiencies. Therefore, LBO1–SRO1 and LBO2–SRO2 are recommended as the core components. Incorporating a T7 promoter sequence further extends their functionality and enables coupling with the transcriptional amplification system.

Build

We added a T7 promoter sequence to the SRO, designated as SRO-T7. The LBO strand was labeled with the quencher group BHQ, while the SRO-T7 strand was labeled with the fluorophore FAM. This design allows fluorescence measurements to quantitatively investigate LBO–SRO hybridization and strand displacement.

NUPACK simulation (Complementary base pairs are marked in red)

Test

Step 1. Strand hybridization

Procedure

1. Preparation of hybrid strands (long strands: short strands = 2:1)

The hybrid strands system was prepared as above, with the concentration diluted from 10μM to 1μM, incubated at 95℃ for 5 min, and then slowly cooled to room temperature in boiling water.

2. RFU measurement of hybrid strands

The reaction system was added to a 384-well plate, with the hybrid strands concentration diluted from 1μM to 100nM. Triplicate measurements were performed for each group, and the microplate reader was set at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

Results

The T7-modified strands showed lower stability, and the binding efficiency between long and short strands was reduced.

As shown in the figure, the fluorescence intensity of the LBO1 and SRO1-T7 system is more stable; therefore, this system was selected for subsequent experiments.

Step 2. Strand proportional hybridization

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.

2. RFU measurement of hybrid strands

The reaction system was added to a 384-well plate, with the hybrid strands concentration diluted from 1μM to 100nM. Each group was diluted according to the table below for fluorescence measurement. Five replicate experiments were performed for each group.

Results

At hybridization ratios of 1:1, 2:1, and 3:1, the hybridization strands were unstable, with fluorescence values increasing over time, indicating strand separation at 37 °C. At ratios of 5:1, 7:1, and 9:1, hybridization was stable, and background fluorescence was low. To ensure sensor sensitivity, a ratio of 5:1 was selected for subsequent experiments.

Step 3. Strand displacement reaction upon antibiotic addition

Procedure

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

2. After preparation, vortex the mixture thoroughly and centrifuge for 10 seconds.

3. Place the prepared hybridization strands 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.

Results

Learn

The LBO1–SRO1-T7 system exhibited relatively stable hybridization and a concentration-dependent response to antibiotics, thereby preparing the system for integration with the transcriptional amplification module.

References

1. Tan, J.H., Fraser, A.G. Quantifying metabolites using structure-switching aptamers coupled to DNA sequencing. Nat Biotechnol (2025).
2. Alexander Shaver, J.D. Mahlum, Karen Scida, Melanie L. Johnston, Miguel Aller Pellitero, Yao Wu, Gregory V. Carr, and Netzahualcóyotl Arroyo-Currás. Optimization of Vancomycin Aptamer Sequence Length Increases the Sensitivity of Electrochemical, Aptamer-Based Sensors In Vivo ACS Sensors 2022 7 (12), 3895-3905
3. Xiaona Fang, Tian Gao , Zhaofeng Luo , Renjun Pei . Efficient selection of vancomycin-specific aptamers via particle display and development of a high-sensitivity fluorescent apta-sensor for vancomycin detection . Sensors and Actuators B: Chemical 2025 8(1),137681

1.3 Cas Strand Displacement

Cycle 1.3.1

Design

In the T7 transcription system of Cycle 2, detection of antibiotics within a certain concentration range was achieved, but it did not demonstrate significant amplification effects. We are still exploring alternative amplification approaches. Through literature review, we learned that in the CRISPR-Cas12a system, activated Cas12a exhibits trans-cleavage activity[1], enabling continuous and non-specific cleavage of single-stranded DNA reporter probes in the system, thereby achieving efficient fluorescence signal amplification.

For this purpose, we designed LBO1-Cas and SRO1-Cas, predicted binding efficiency with NUPACK, and experimentally verified whether vancomycin could displace SRO1-Cas.

Build

NUPACK Simulation

Test

BHQ group was added to LBO1-Cas, and 6-FAM group was added to SRO1-Cas. Fluorescence intensity was measured with a microplate reader to verify whether vancomycin could displace SRO1-Cas. Each group was tested in quintuplicate, with the highest and lowest values discarded before averaging.

The results showed no significant correlation between fluorescence intensity and vancomycin concentration. Even when the final concentration of vancomycin reached 100 μM, there was still no significant difference compared with 0 μM vancomycin.

Learn

Our first sensor design bound too tightly, so even high doses of vancomycin could not displace SRO. Therefore, we shifted the binding site of SRO and LBO so that the hairpin-forming sequence of LBO was positioned in the middle of the LBO–SRO binding region, in an attempt to facilitate SRO release.

Cycle 1.3.2

Design

To address the overly tight binding between LBO and SRO in the previous design, we altered the binding positions of LBO and SRO so that SRO could be more easily displaced.

Build

For this purpose, we designed a new SRO, which we named SRO2-Cas.

NUPACK Simulation

Test

Step 1. PAGE electrophoresis to determine strand displacement upon antibiotic addition

Learning from the previous experiment and to save costs, we first ordered LBO2-Cas and SRO2-Cas without fluorescent or quenching groups. A PAGE gel was used for qualitative analysis of whether vancomycin could displace SRO2-Cas.

Note: Except for the sequences of the strands used, the group settings and operating methods for PAGE electrophoresis are the same as in Cycle 1.1 step 2.

The results showed that vancomycin was able to displace SRO, confirming that our design was effective.

Step 2. Fluorescence quantitative to determine strand displacement upon antibiotic addition

We then ordered LBO1-Cas with a BHQ group and SRO2-Cas with a 6-FAM group. Fluorescence intensity was measured with a microplate reader to quantitatively analyze SRO displacement by vancomycin. Each group was tested in quintuplicate, with the highest and lowest values discarded before averaging.

Procedure

1. Prepare the hybridization chain of LBO1-Cas and SRO2-Cas at a 1:1 ratio, with both LBO1-Cas and SRO2-Cas at a concentration of 1 μM in the system.

2. Construct strand displacement system

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 5 μM, 10 μM, 20 μM, 50 μM, 100 μM.

Learn

By shifting the LBO–SRO binding site into the middle of LBO's self-complementary sequence, we successfully enhanced vancomycin's ability to displace SRO. We ultimately selected LBO1-Cas and SRO2-Cas for subsequent signal amplification experiments.

References

1. Janice S. Chen et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436-439 (2018).

2.1 Transcription system based on the original LBO1 and SRO1-T7

Design

Based on wide research and application, T7 in vitro transcription system is able to product certain RNA molecules with high efficiency, high yield and high fidelity, and exhibits strict specificity for the T7 promoter [1]. Therefore it has potential to be designed as a signal amplification platform.

In the T7 in vitro transcription system, SRO was designed to comprise T7 promoter sequence. When it released and bound with a downstream template, it would activate transcription. The transcription product was an aptamer RNA that enhanced the fluorescence of a specific dye DFBHSI [2], enabling the quantification of antibiotic residues through fluorescence intensity.

Build

We added T7 promoter sequence to SRO1, and named it as SRO1-T7.

Test

Step 1. Attempt to construct a T7 transcription system

First, we used T7 promoter sequences and template to test the T7 in vitro transcription system, investigating the appropriate amounts of substances such as NTPs, T7 enzyme, and dyes for the system [3].

Incubate in hot water bath for 30 minutes (above 95℃)

Results

The kinetics of transcription were measured by monitoring fluorescence intensity within the first two hours of transcription initiation. The results are as follows.

The initial fluorescence intensity of one experimental group reached approximately 1,600 RFU, significantly higher than the previous result (<50 RFU), confirming successful transcription. However, the fluorescence intensity decreased over time. This phenomenon occurred because after T7 RNA polymerase addition, approximately 30 minutes were spent for instrument parameter optimization before measurement began. By the time detection started, transcription had already ceased, and the fluorescence signal had passed its peak. The premature termination of transcription may be attributed to magnesium pyrophosphate precipitation, which could inhibit the reaction.

Step 2. Test on the appropriate enzyme amount for adding pyrophosphatase

Based on previous experimental results, transcription could proceed in the system with a 50 nM T7 promoter, but it stopped within half an hour, with no further increase in fluorescence intensity—suggesting that magnesium pyrophosphate (PPase) deposition may have inhibited transcription [4]. In this experiment, factors such as the T7 promoter and T7 RNA polymerase remained consistent with the previous setup. On this basis, pyrophosphatase was added to investigate whether it affects correct RNA transcription and whether it can improve transcription efficiency.

Results:

Step 3. Test on transcriptional activation at different promoter concentrations

This experiment investigated the transcriptional activation efficiency under gradient promoter strand concentrations (10 nM, 25 nM, 50 nM, 75 nM, and 100 nM).

Results:

The differences in fluorescence ratios among various T7 promoter concentrations were not statistically significant, and the relative ranking of these ratios continually changed over time, indicating unstable results. Further optimization is required.

Step 4. Test on transcriptional activation of SRO1-T7

All operations are the same as in step 3, except replacing the promoter with SRO1-T7.

A concentration-dependent relationship exists for 0, 10 nM, and 20 nM fluorescence intensities. However, a pronounced downward trend appears at 50 nM, 75 nM, and 100 nM, with the value at 100 nM even approaching that of the negative control.

Step 5. Couple strand displacement to T7 in vitro transcription

All operations about strand displacement are the same as in Cycle 1. After strand displacement, the reaction mixture was added to the T7 in vitro system.

We observed signal leakage in the system, where transcription was activated even without antibiotic binding.

Learn

The in vitro transcription system possesses high sensitivity and has the potential for signal amplification. However, due to signal leakage, it cannot yet be utilized. Causes include:

1. The SRO1-T7 sequence formed a hairpin structure, reducing hybridization efficiency and leaving excess free promoter.
2. The promoter sequence was not effectively blocked, allowing activation without release.

In the next cycle, we will work on solutions to decrease signal leakage.

References

1. Diane Imburgio, Minqing Rong, Kaiyu Ma et al. Studies of Promoter Recognition and Start Site Selection by T7 RNA Polymerase Using a Comprehensive Collection of Promoter Variants. Biochemistry 2000, 39, 34, 10419–10430
2. Dao, N.T., Haselsberger, R., Khuc, M.T. et al. Photophysics of DFHBI bound to RNA aptamer Baby Spinach. Sci Rep 11, 7356 (2021).
3. Tianshuo Liu, Shivali Patel, Anna Marie Pyle. Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates. Methods in Enzymology. Volume 691, 2023
4. Milligan, J. F., & Uhlenbeck, O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods in Enzymology, 180(1989), 51-62.

2.2 Three-strand system to decrease the signal leakage

Design

In the previous cycle, we observed significant transcriptional leakage caused by the exposed promoter sequence on sro1-T7. By consulting the literature, we learned that the hybridization and displacement between nucleic acid strands can be regulated by adding an additional regulatory strand to the system [1], or a catalytic strand can be used to open previously blocked domains [2].

This inspired us to consider adding an additional blocker strand to hybridize with the T7 promoter region and prevent premature transcription. Blocker strands with 0, 3, 6 and 9 base truncations at the 5' end were selected for further experiments.

Build

Test

Step 1. Strand displacement upon blocker 4 addition

Each set of experiments was repeated five times, and the fluorescence changes in the system were measured over 30 minutes using a microplate reader at 37°C.

Results

Upon addition of the blocker strand, the system exhibited an antibiotic concentration-dependent strand displacement reaction. However, the presence of the blocker may facilitate hybridization between LBO and SRO, thereby reducing the reaction rate. After antibiotic addition, the strand displacement reaction reached equilibrium at approximately 30 minutes, and the fluorescence intensity of the system stabilized.

Step 2. Test Blocker4 in transcription system

Results

Theoretically, Group 3 should exhibit the lowest fluorescence value, demonstrating that the blocker can effectively reduce signal leakage. However, in practice, the fluorescence intensity of Group 3 is higher than that of Group 4, and the large error bars within the group indicate that the results cannot conclusively prove the blocker's efficacy in reducing signal leakage.

Step 3. Test Blocker2 in transcription system

Possibly due to the excessively short length of Blocker4, it failed to effectively reduce signal leakage. In this step, Blocker2 with only three truncated bases was used for testing.

Results

Theoretically, Group 3 should exhibit the lowest fluorescence value, demonstrating that the blocker can effectively reduce signal leakage. However, in practice, the fluorescence intensity of Group 3 is higher than that of Group 4, and the large error bars within the group indicate that the results cannot conclusively prove the blocker's efficacy in reducing signal leakage.

Learn

The three-strand system did not successfully reduce signal leakage. The blocker strands either failed to bind stably or were displaced too easily. Future designs may require longer blocker sequences or alternative blocking strategies.

References

1. Lee, H., Xie, T., Kang, B. et al. Plug-and-play protein biosensors using aptamer-regulated in vitro transcription. Nat Commun 15, 7973 (2024).
2. Soshu Yasuda, Kunihiko Morihiro, Shuichiro Koga. et al. Amplified Production of a DNA Decoy Catalyzed by Intracellular MicroRNA. Angewandte chemie 2025 4(64), e202424421

2.3 Double-strand system to decrease the signal leakage

2.3.1 Initial Design and Verification

With the core goals of sealing the T7 promoter on the closed SRO1-T7 and suppressing transcriptional leakage in the absence of vancomycin, the design scheme achieves self-sealing by extending the SRO1-T7-2 sequence: a 14-base extension is added to the 3' end of SRO1-T7-2, enabling it to form complementary base pairing with its own T7 promoter region (in strict accordance with the A-T and G-C base pairing rules). This ensures that the T7 promoter is sealed by a double-stranded structure when there is no vancomycin. Meanwhile, the original complementary region between SRO1-T7-2 and LBO1 is retained to ensure that after vancomycin binds to LBO1, SRO1-2 can dissociate normally, unfold the T7 promoter, and initiate transcription. Concurrently, the original unextended SRO1-T7-2 is designed as a negative control to evaluate the sealing effect; the template sequence (temp.1) is modified to include a binding region complementary to SRO1-T7-2, meeting the template requirements for transcription initiation.

Build

Feasible sequences were simulated using Nupack software:

Test

Nupack Simulation Results:

2.3.2 Parameter Optimization and Performance Improvement

The hybridization ratio of SRO1-T7-2 to LBO1 directly affects the assembly efficiency and stability of their complex: a too low ratio may lead to free SRO1-T7-2 (failing to form effective sealing), while a too high ratio may interfere with vancomycin-induced dissociation due to excess LBO1. Both scenarios affect the system's sealing effect and signal response specificity. Therefore, the goal of this stage is to screen the optimal hybridization ratio through gradient ratio experiments, ensuring complete assembly of the complex without interference from free SRO1-T7-2 strands, and clear signal gradients under different vancomycin concentrations. This provides a stable reaction basis for subsequent transcription experiments.

Build

To investigate the hybridization effect of different SRO1-T7-2:LBO1 ratios, the two strands were mixed at molar ratios of 1:1, 1:3, and 1:5, followed by slow annealing at 95℃. Each group was repeated 3 times.

Test

1. SRO1-T7-2:LBO1 = 1:1

2. SRO1-T7-2:LBO1 = 1:3

3. SRO1-T7-2:LBO1 = 1:5

Learn

The optimal hybridization ratio is SRO1:LBO1 = 1:1. At this ratio, the two strands can fully assemble into a stable complex, and regular signal gradients can be observed under induction by different vancomycin concentrations, ensuring the sealing effect.

2.3.3 Transcription Experiment Verification

(Subsequent experiments not conducted due to time limitation)

The concentration of the template strand (temp.1) is a key parameter affecting transcription efficiency: a too low concentration leads to insufficient SRO1-T7-2 binding sites, limiting transcription signal amplification; a too high concentration may cause non-specific binding (e.g., cross-reaction between free template and LBO1), increasing background leakage. The design goal of this stage is to screen the optimal temp.1 concentration. Based on the SRO1:LBO1 hybridization ratio of 1:1, the following must be ensured:

1. No increase in background leakage in the absence of vancomycin;
2. Transcription signals increase clearly with the vancomycin concentration gradient (0 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM) in the presence of vancomycin;
3. The template concentration matches the ratio of SRO1-LBO1 complex formation to avoid resource waste or reaction limitations.

The concentration gradient is set to 0.1 μM, 0.5 μM, 1 μM, and 2 μM (focusing on the low-to-medium concentration range based on the pre-experiment phenomenon that excess template increases background).

The concentration of the T7 promoter and the dosage of pyrophosphatase are also critical parameters for transcription initiation efficiency, signal stability, and persistence, and will be optimized under the same conditions.

2.4 Single-strand system to decrease the signal leakage

2.4.1 Design and Initial Test (SSS-M)

To prevent the T7 promoter from leaking transcription under antibiotic-free conditions, we designed a single-strand aptamer sensor. Based on the conformational change of the LBO strand induced by vancomycin, we added a T7 promoter reverse complementary sequence followed by the forward sequence to the 3′ end of the LBO strand, forming a closed hairpin structure. In the absence of vancomycin, the T7 promoter is sequestered; upon binding, the aptamer undergoes a conformational change that disrupts the hairpin, exposing the T7 promoter and initiating transcription to produce a fluorescence signal.

To reduce nonspecific binding by T7 polymerase, two bulges were introduced in the antisense complementary region to further minimize erroneous recognition.

Build

We designed three variants differing in the length of the 5′ disruptive sequence:

• SSS-S (short)
• SSS-M (medium)
• SSS-L (long)

DNA sequences were ordered from a synthesis company. We first tested SSS-M using an annealing protocol in Grade I water (1 μM), then added directly into the transcription reaction.

Test

Learn

No significant change in fluorescence was observed with increasing vancomycin concentration. Although the hairpin formed, the system failed to show dose dependence. Possible issues include suboptimal template-sensor ratio, annealing method, or polymerase activity.

2.4.2 Template Concentration Optimization

We hypothesized that template concentration affects sensitivity and signal-to-noise ratio.

Test

Learn

A difference between negative and positive groups was only visible at 10 nM template, but with high standard deviation. Higher concentrations showed no distinction. Low template may improve contrast but lacks reliability.

2.4.3 Antibiotic Gradient Test

With template fixed at 10 nM, we tested whether a dose-dependent response could be achieved.

Test

Learn

No clear dose-dependence was observed. Even at low template, the system remained unresponsive, suggesting deeper issues in design or kinetics.

2.4.4 Ratio and Annealing Optimization

The template-to-sensor ratio may deviate from conventional in vitro transcription conditions, so we adjusted the ratio and annealing conditions. Template:SSS-M ratio = 1:1 (0.5 μM : 0.5 μM). DFHBI concentration increased to 10 μM. Annealing was performed with sensor, template, and VAN (500 μM) together.

Test

Learn

Reaction speed significantly increased (peak at ~10 min), but no meaningful difference between +VAN and −VAN groups. Fast kinetics may mask subtle regulatory effects.

2.4.5 Polymerase Concentration Test

To better resolve differences, we reduced T7 polymerase concentration to slow down reactions. Tested: 0.025, 0.25, 1.25, 2, 2.5 μM.

Test

Learn

No consistent vancomycin-dependent response across enzyme levels. At 1.25 μM, −VAN showed higher fluorescence than +VAN—likely due to experimental variability rather than biological effect.

2.4.6 Sub-gradient VAN Test

To verify if the anomaly at 1.25 μM T7 pol was reproducible, we repeated the test with finer VAN gradients.

Test

Learn

The earlier anomaly was not reproducible. The SSS-M system does not generate a genuine antibiotic-dependent response under these conditions.

2.4.7 Evaluation of SSS-S and SSS-L

We tested SSS-S and SSS-L under the same conditions as Cycle 2.4.4 to assess impact of 5′ sequence length.

Test

Learn

SSS-S showed the best performance (~16.2% higher signal at 10 h with VAN), likely due to more effective T7 promoter sealing. However, sensitivity remains low. NUPACK analysis revealed that the vancomycin-binding domain was structurally altered during design, impairing recognition.

2.4.8 Redesign: 5TS and 3TS Families

Upon analysis, poor performance stemmed from two factors: (1) incomplete sealing of the T7 promoter, causing leakage; (2) alteration of the aptamer binding area, preventing vancomycin recognition. To address this, we redesigned two families:

5TS Family (5′-T7 Hairpin)

The principle is similar to the SSS-n design, but in this case, the T7 promoter was linked to the 5′ end of the original LBO sequence, followed by a reverse complementary segment. The overall effect was to form a hairpin structure at the 5′ end of the sequence, such that in the absence of vancomycin, the T7 promoter is locked and transcription cannot be triggered; when vancomycin is present, it induces a conformational change in the aptamer, disrupting the hairpin and releasing the T7 promoter to initiate transcription.

When designing the 5TS family, we drew lessons from the shortcomings of the SSS-n scheme. We used NUPACK to simulate the secondary structures of the aptamer sequences, ensuring that the vancomycin recognition domain maintained its native conformation. In addition, we considered the effect of the number and position of bulges in the T7 complementary strand on promoter sealing. As a result, we designed four variants (5TS-1 to 5TS-4), with their T7 complementary strands containing 3, 2, 1, or 0 bulges, respectively.

3TS Family (3′-T7 Hairpin with Mismatches)

We also designed the 3TS family, whose principle is based on the SSS-n family. At the 3′ end of the sequence, a reverse complementary sequence of the T7 promoter followed by the T7 promoter itself was appended, forming a hairpin structure that blocks the T7 promoter. When vancomycin is present, the aptamer undergoes a conformational change, disrupting the hairpin and releasing the T7 promoter to initiate transcription. We used the NUPACK software to predict the secondary structures of the aptamers, ensuring that the secondary structure of the vancomycin recognition domain remained intact. To explore the effect of T7 promoter closure, we introduced 0–2 groups of base mismatches (3 bp each) at different positions within the complementary strand. In addition, we altered bases at the 5′ end that participate in vancomycin-induced conformational changes, thereby changing the number of base pairs formed in the LBO after the induced hairpin structure is generated, in order to investigate how intrinsic aptamer properties influence performance. These two variables were designed independently, resulting in eight sequences, designated 3TS-1 through 3TS-8. Among them, 3TS-1 to 3TS-4 and 3TS-5 to 3TS-8 form two subgroups, differing in the number of 5′-end bases used to form the antibiotic-induced hairpin structures, while the rest of their sequence designs correspond one-to-one.

Build

Ordered DNA sequences from company.

Test

EWe performed transcription tests on the 3TS-n and 5TS-n sensors in sequence. Each aptamer was first annealed individually in hybridization buffer and then added to the transcription system. The annealing buffer, referred to as the Binding Buffer, was PBS supplemented with 50 mM MgCl₂.ach sensor was tested for vancomycin responsiveness.

The test results for different groups are as follows

Learn

5TS family: Bulge number affected overall fluorescence intensity, but none showed vancomycin responsiveness.
3TS family: Mismatch position and count influenced performance. Sensors with longer 5′ sequences and inner mismatches (e.g., 3TS-6, 3TS-8) showed up to ~13% increase with VAN, indicating partial success. However, background fluorescence was high.
Conclusion: Future designs must balance promoter sealing (low background) with efficient conformational switching (high dynamic range).

References

1. Tan, J.H., Fraser, A.G. Quantifying metabolites using structure-switching aptamers coupled to DNA sequencing. Nat Biotechnol (2025).
2. Xiaona Fang, Tian Gao, Zhaofeng Luo, Renjun Pei. Efficient selection of vancomycin-specific aptamers via particle display and development of a high-sensitivity fluorescent apta-sensor for vancomycin detection. Sensors and Actuators B: Chemical, Volume 436, 2025, 137681.

2.5 LBO modification for reduced signal leakage

2.5.1 Design and Initial Test

To prevent leakage from the T7 promoter, we extended the 5′ end of the LBO strand. The extended segment was designed to be complementary to the T7 promoter on the SRO, so that in the absence of vancomycin, the template strand could not hybridize with the T7 promoter sequence and transcription would not occur. In the presence of vancomycin, however, the LBO undergoes a conformational change to form a hairpin structure, promoting the displacement and release of the SRO strand. The T7 promoter of the freed SRO can then hybridize with the template strand, be recognized by T7 polymerase, and initiate transcription.

Another possible mechanism is that the conformational change of the LBO exposes a T7 sequence toehold originally bound to LBO, which facilitates the displacement of SRO by the template strand and triggers transcription.

Build

In our initial design, the SRO's T7 promoter contained 19 bases with no complementarity to the LBO strand. Here, we extended the 5′ end of the LBO strand by 0, 4, 8, 12, 16, or 19 complementary bases, designated L1, L2, L3, L4, L5, and L6, respectively. The SRO carrying the T7 promoter was designated S1.

Test

Our objective was to identify combinations of L-n and S1 that could respond to the antibiotic. Negative group: 0 μM VAN; Positive group: 100 μM VAN.

Annealing conditions:

(Heated at 95 °C for 5 min, slowly cooled.)

Transcription system:

Learn

The best response was S1+L2 (fluorescence ratio ~1.21:1). S1+L2 validated the strategy, but high background remained. Fluorescence decreased from L1 to L6, indicating stronger sealing, yet responsiveness dropped.

2.5.2 Optimized Design and Performance Evaluation

To improve S1+L4/L5, we extended their 3′ ends to destabilize the duplex and favor hairpin formation upon vancomycin binding.

Build

Test

Used DFHBI-1T dye for higher signal contrast.

Learn

S1+L5_3 showed improved response (1.35:1). Extending the 3′ end transitioned the system from over-sealed to functional. Optimal state lies near 13 added bases.

2.5.3 Template Concentration Optimization

We varied template:S1 ratios (0.5, 1, 5, 10) to optimize signal-to-background.

Test

Learn

Best response at 1:1 ratio. Too low: weak signal; too high: template displaces S1 regardless of vancomycin. Narrow optimal window exists.

References

1. Tan, J.H., Fraser, A.G. Quantifying metabolites using structure-switching aptamers coupled to DNA sequencing. Nat Biotechnol (2025).
2. Xiaona Fang, Tian Gao, Zhaofeng Luo, Renjun Pei. Efficient selection of vancomycin-specific aptamers via particle display and development of a high-sensitivity fluorescent apta-sensor for vancomycin detection. Sensors and Actuators B: Chemical, Volume 436, 2025, 137681.