Experimental results of strand displacement module

Strand displacement module coupled with T7 transcription

Results

1. Design LBO-Cas and SRO-Cas

1. PAGE electrophoresis verification showed that LBO1-SRO1 and LBO2-SRO2 exhibited high hybridization efficiency, and strand displacement could be initiated upon antibiotic addition.

2. After incorporating the T7 promoter sequence, LBO1-SRO1-T7 and LBO2-SRO2-T7 were hybridized at a 1:1 ratio for quantitative fluorescence analysis. It was found that LBO1-SRO1-T7 demonstrated relatively stable hybridization.

3. To decrease background signal, we performed the ratio hybridization experiment for LBO1-SRO1-T7. The background signal was observed to be lower at an LBO1:SRO1-T7 ratio of 5:1. Although further increasing the ratio could reduce background noise, it would compromise the sensor's sensitivity. Therefore, this ratio was selected for subsequent experiments.

4. At an LBO1:SRO1-T7 ratio of 5:1, quantitative fluorescence analysis was performed after antibiotic addition to investigate the strand displacement effect. It was observed that antibiotic concentrations greater than 10 µM induced concentration-dependent changes in fluorescence intensity, thereby establishing a foundation for coupling with the T7 amplification system.

Strand displacement module coupled with Cas system

Results

1. PAGE electrophoresis analysis confirmed that the LBO1-SRO2-Cas complex exhibits high hybridization efficiency at a 1:1 ratio and undergoes strand displacement upon antibiotic addition. However, the proportion of hybrid complexes participating in strand displacement remains relatively low.

2. When using a 1:1 ratio of LBO1:SRO1-Cas, quantitative fluorescence measurements were performed after antibiotic introduction to evaluate the strand displacement effect. The results demonstrated antibiotic concentration-dependent fluorescence changes at concentrations above 20 µM, establishing the foundation for integration with the Cas amplification system.

3. Comparison of normalized fluorescence intensity results from quantitative fluorescence experiments conducted with and without crRNA confirmed that crRNA enhances the strand displacement reaction.

Experimental results of T7 transcription module

Problem

When the screened LBO1-SRO1-T7 strand displacement system was directly coupled with the transcription system, signal leakage occurred because the T7 promoter sequence in SRO1-T7 remained unblocked. Even when SRO1-T7 was hybridized with LBO1 and not released, the exposed T7 promoter could still initiate transcription. To address this, we explored various methods to block the T7 promoter and reduce signal leakage. The following section documents our some effective attempts and results.

Experimental results of the single-strand system

Overview

We aimed to construct a single-strand aptamer sensor to detect vancomycin through structural switching. Multiple design iterations were carried out, testing variations in sensor length, template concentrations, T7 polymerase levels, and promoter sealing strategies. Although several constructs showed partial improvements, consistent antibiotic-dependent fluorescence responses remain a challenge. Below, we summarize our results and the outcomes.

Experimental Findings

Initial SSS Family Designs

Template and Polymerase Optimization

Results

The SSS family of designs tested whether a single-stranded promoter hairpin could prevent premature transcription while still responding to vancomycin. Among the three variants, SSS-S (short blocking sequence) performed best, showing reduced background fluorescence and a modest but measurable increase (~16.2%) in fluorescence at 100 µM vancomycin compared to the control. This suggests that a shorter blocking sequence provides tighter promoter sealing without completely disrupting the aptamer's ability to switch conformation. In contrast, SSS-M and SSS-L failed to demonstrate any concentration-dependent response, likely due to excessive complementarity that stabilized the hairpin too strongly or interfered with the aptamer binding domain. These results highlighted an important design principle: over-engineering the promoter lock can inadvertently abolish ligand sensitivity, while a carefully balanced short blocker may offer a better trade-off between background control and responsiveness.

Extended Families: 5TS and 3TS Designs

To overcome incomplete promoter sealing and disrupted aptamer binding, we developed two new families:

5TS family: T7 promoter at 5′ end with complementary bulges (3--0 bulges).

3TS family: T7 promoter at 3′ end with mismatch designs (0--2 mismatches).

Key Findings:

5TS variants: Bulge number influenced total fluorescence but not vancomycin response.

3TS variants: More mismatches and mismatches near the inner hairpin increased fluorescence intensity.

Some 3TS constructs showed ~11–13% fluorescence increase in vancomycin-treated samples, but background leakage was high

Summary of Observations:

Incomplete promoter sealing was the main cause of background leakage.

SSS-S and some 3TS constructs showed relatively better vancomycin responsiveness, but the sensitivity was still insufficient.

Template and polymerase concentration adjustments alone did not solve the problem.

NUPACK simulations highlighted that maintaining the aptamer's recognition domain is critical, as design modifications risk disrupting ligand binding.

Analysis & Interpretation:

Our cycles demonstrate that while single-strand promoter-locking is a promising strategy, balancing low background leakage with ligand-dependent switching remains difficult. The trade-off lies between strong promoter sealing (which prevents leakage but can also hinder switching) and weak sealing (which allows switching but suffers from high background).

Lessons Learned

Experimental errors (e.g., in polymerase concentration setup) led to misleading results in early trials, highlighting the importance of replicates and strict operation standards.

Sequence design trade-offs are non-trivial: adding complementary regions risks distorting aptamer binding motifs.

Secondary structure prediction (NUPACK) is essential to pre-validate that recognition domains remain intact before synthesis.

Future Plans

Refined design: Explore hybrid single-/double-strand systems that combine strong promoter sealing with flexible release mechanisms.

Reduce background leakage: Introduce rational bulge/mismatch engineering guided by systematic NUPACK and ViennaRNA simulations.

Application direction: If successful, the platform could be expanded to detect antibiotics beyond vancomycin (e.g., tetracycline, streptomycin), or adapted for food safety and clinical diagnostics.

Transparency: Although results so far are not ideal, we report them in full to remain scientifically honest and to help future teams avoid repeating our pitfalls.

In summary, our single-strand aptamer sensor development showed partial successes (especially in SSS-S and certain 3TS variants), but failed to achieve a stable concentration-dependent response. The lessons from these attempts will guide us toward improved promoter-sealing strategies and more reliable aptamer--antibiotic detection systems.

Experimental results of the extended double-strand system

Overview

To further optimize promoter sealing and antibiotic responsiveness, we explored a double-strand extension strategy. By progressively extending the 5′ or 3′ ends of the LBO strand, we aimed to adjust the degree of promoter--aptamer complementarity and achieve an optimal balance between background suppression and vancomycin-dependent release.

Experimental Findings

1. Initial LBO 5′-Extension

We tested six variants (L1–L6) with 0–19 complementary bases added to the 5′ end of the LBO strand, paired with a constant SRO carrying the T7 promoter (S1).

Among the six designs, S1+L2 struck the best balance: the promoter was partially sealed by four complementary bases, enabling vancomycin to trigger displacement and transcription with ~21% higher fluorescence in the positive group. In contrast, L3–L6 sealed the promoter too tightly, abolishing responsiveness. These results demonstrated that moderate complementarity improves background control while preserving aptamer switching capacity. However, even in the best group, background fluorescence remained high and the overall signal-to-background ratio was unsatisfactory.

2. LBO 3′-Extension

Building on the results above, we modified L4 and L5 by progressively extending their 3′ ends (L4_1–L4_3 and L5_1–L5_4). The aim was to destabilize the LBO–SRO duplex upon vancomycin binding, thereby improving responsiveness.

Key Findings:

All S1+L4_n variants remained insensitive to vancomycin, suggesting that the tested increments overshot the optimal range.

S1+L5_3 (13-base extension) exhibited a clear response, with ~1.35-fold higher fluorescence at 100 µM vs. 0 µM VAN — stronger than S1+L2.

Extensions beyond this point (e.g., L5_4) destabilized the system completely, producing high but non-specific fluorescence.

The L5 series followed a transition from over-sealed → intermediate → unsealed states as more bases were added. At the intermediate state (L5_3), the promoter was sealed enough to suppress background yet still able to open in response to vancomycin. By contrast, L4 variants skipped over this "sweet spot," indicating that our step sizes were too large. These results suggest that fine-tuned base additions (smaller increments) are necessary to locate the optimal trade-off.

3. Template Concentration Effects

To further refine the best-performing construct (S1+L5_3), we varied template concentrations relative to S1 (ratios 0.5–10).

Key Findings:

At a 1:1 template:S1 ratio, vancomycin responsiveness was most pronounced.

At low template concentrations, overall signal was too weak, masking group differences.

At excessively high template concentrations, the template strand displaced S1 directly, overriding vancomycin dependence.

The transcription system displayed classic concentration trade-offs: too little template resulted in insufficient signal, while too much template led to non-specific displacement. A narrow intermediate range allowed antibiotic-dependent differences to be observed. This highlights the importance of carefully balanced stoichiometry in aptamer-based transcription systems.

Summary of Observations

S1+L2 (4-base 5′ extension) and S1+L5_3 (13-base 3′ extension) were the most responsive constructs, though both still suffered from high background.

Too few complementary bases → leakage and weak specificity.

Too many complementary bases → over-sealing and loss of sensitivity.

System performance is strongly influenced by template concentration, which must be optimized within a narrow window.

Analysis & Interpretation

Our double-strand extension strategy confirmed that small, carefully tuned modifications to the aptamer–promoter interface can dramatically alter performance. The results demonstrate a U-shaped trade-off: both insufficient and excessive complementarity abolish responsiveness, while intermediate levels yield the best signal-to-background balance.

Lessons Learned

Even minor base additions (4 vs. 13) can completely shift aptamer performance, underscoring the sensitivity of nucleic acid design.

Using different dyes (DFHBI-1T vs. DFHBSI) improved visualization of subtle changes, suggesting the choice of reporter is critical.

Step increments in base addition were too large in the L4 series, preventing us from capturing the optimal range.

Future Plans

Fine-resolution tuning: Explore smaller step extensions (e.g., +6, +7, +8 bases) to pinpoint the optimal LBO–SRO balance.

Combine strategies: Integrate promoter-sealing principles from the single-strand system with double-strand extension to reduce leakage.

Practical applications: If optimized, this strategy could serve as a modular framework for designing aptamer sensors responsive to a wide range of antibiotics or other small molecules.

Scientific transparency: By reporting both successful and failed constructs, we ensure that future teams can learn from our design space exploration without duplicating ineffective attempts.

In summary, the double-strand extension approach confirmed that promoter sealing can be tuned to improve antibiotic responsiveness. S1+L2 and S1+L5_3 emerged as promising leads, but further refinements are required to reduce background leakage and enhance reliability.

Experimental results of Cas module

Results

1. Design LBO-Cas and SRO-Cas

To implement signal amplification, Cas12a was introduced and used to construct LBO-Cas (BHQ-labeled) and SRO-Cas (6-FAM-labeled). Fluorescence was measured by microplate reader and displacement efficiencies were calculated by normalizing fluorescence against a positive control. Two SRO-Cas / crRNA combinations were designed and tested.

2. Measurement of Fluorescence Intensity After Cas12a Cleavage

In the Cas12a system, once activated, Cas12a non-specifically cleaves all single-stranded DNA in the system. At this point, the pre-incorporated reporter probe (a short ssDNA fragment bearing a fluorescent and quenching moiety) is cleaved. Following cleavage, the separation of the fluorescent group from the quencher group generates a fluorescent signal. The amount of SRO displaced at the front end correlates positively with the rate of fluorescence intensity increase. Therefore, vancomycin concentration can be determined by measuring the rate of fluorescence intensity increase.

1. The figure below shows the fluorescence changes of the system over 30 minutes after the addition of different antibiotic concentrations. It can be observed that antibiotic concentrations greater than 5 µM produce a concentration-dependent increase in fluorescence intensity.

2. To verify whether the introduction of Cas12a can effectively amplify the signal, we designed two experiments. The only difference between the two experiments was the presence or absence of Cas12a protein. As can be seen, the addition of Cas12a protein significantly enhanced the resolution of the sensor.

By comparing two figures, it can be observed that in the uncoupled Cas system, the strand displacement system exhibited concentration-dependent fluorescence changes at antibiotic concentrations greater than 20 µM. In contrast, after coupling with the Cas system, the strand displacement system produced concentration-dependent fluorescence changes at antibiotic concentrations above 5 µM. This demonstrates that the Cas system effectively lowered the minimum detectable concentration of the sensor and served as a signal amplification mechanism.