Design and optimization of TRHDA for tearing protospacer activated Cas12a

As crRNA might bind with the hairpin DNA from two opposite orientations, forward tearing protospacer (identical to crRNA scaffold, Fig. 1A) and reverse tearing protospacer (opposite to crRNA scaffold, Fig. 2A) are designed to activate Cas12a, respectively. Firstly, the feasibility of the forward tearing protospacer activated Cas12a is verified by fluorescence analysis. As shown in Fig. 1B, in the presence of target miRNA, the fluorescence signal is seven times higher than the blank control (blue curve vs. red curve). This result demonstrates that forward tearing protospacer could enable Cas12a activation. To explore the effect of target-responsive hairpin DNA length on forward tearing protospacer activated Cas12a, a series of hairpin DNAs with different base pairs in the tail region are designed (Fig. 1C). Figure 1D demonstrates that there is a significant decrease in signal-to-background (S/B) ratios with the increasing base pair numbers (0, 3 bp, 6 bp). Consequently, it is not recommended to introduce additional base pairs in the tail region of the hairpin DNA. The region where the hairpin DNA hybridized with the target miRNA encompasses three distinct regions: a toehold region marked as “A”, a transition region marked as “B”, and a seed region marked as “C” (Fig. S1), and the lengths of the above three regions have also been optimized. It is reported that seed region is important for crRNA spacer to bind to protospacer for subsequent Cas12a activation, thus the length of “C” region is firstly investigated [37,38,39]. As shown in Fig. 1E, a series of hairpin DNAs with different base pairs including 4 bp, 6 bp, and 8 bp in the “C” region are conceived. The results suggest that hairpin DNA with 6 bp of “C” region exhibits the highest S/B ratio, thereby establishing the length of the “C” region at 6 bp for subsequent optimization (Fig. 1F). In addition, the 8 bp hairpin possesses a distinct background signal, which is hypothesized to be caused by a suboptimal PAM sequence (TCTG) in the stem segment of this hairpin [40]. Considering the fixed sequence of miRNA binding region, the “A” region and “B” region of hairpin DNA should be parallel optimized. Specifically, nine hairpin DNAs (H1, H2, H3, H4, H5, H6, H7, H8, and H9) with different base pairs in the “A” region and “B” region are synthesized (Fig. 1G). It could be seen that H5 (A12B4C6) with 12 bp of “A” region and 4 bp of “B” region represents the highest S/B ratio (Fig. 1H).

Fig. 1

A Schematic illustration and B feasibility of TRHDA-based forward tearing protospacer activated Cas12a. C Schematic diagram and D optimization of the base pair numbers in the tail region of hairpin DNA. E Schematic diagram and F optimization of the base pair numbers in the “C” region of hairpin DNA. G Schematic diagram and H parallel optimization of the base pair numbers in “A” region and “B” region of hairpin DNAs (H1 → H9). I Molecular dynamics simulations of Cas12a-crRNA-miRNA complex binding to representative five hairpin DNAs (H1, H3, H5, H7, and H9) and the corresponding binding free energies (ΔG, KJ·mol−1). J Plot of the binding free energy changes vs. fluorescence signal changes for five hairpin DNAs (H1, H3, H5, H7, and H9). The data error bars indicate mean ± SD (n = 3)

Fig. 2

A Schematic illustration and B feasibility of TRHDA-based reverse tearing protospacer activated Cas12a. C Schematic diagram and D optimization of the base pair numbers in the “c” region of hairpin DNA. E Schematic diagram and F parallel optimization of the base pair numbers in “a” region and “b” region of hairpin DNAs. The data error bars indicate mean ± SD (n = 3)

Molecular dynamics simulations are implemented to survey the process that Cas12a-crRNA-miRNA complex bond with representative five hairpin DNAs (H1, H3, H5, H7, and H9) and calculate the corresponding binding free energies (Fig. 1I). Remarkably, H5 presents the highest free energy change (− ΔG = 363.72 kJ·mol−1) among the five hairpin DNAs and the change trend of the binding free energies from H1 to H9 is well matched with the change trend of fluorescence signal (Fig. 1J). These results reveal that the process forming the complex of Cas12a-crRNA-miRNA-hairpin DNA may determine the reaction kinetics of the cleavage activities.

Moreover, the reverse tearing protospacer activated Cas12a is also discussed and the schematic illustration of reverse tearing protospacer mode is shown in Fig. 2A. Fluorescence analysis is employed to explore the feasibility of the reverse tearing protospacer activated Cas12a. As presented in Fig. 2B, in the presence of target miRNA, the fluorescence signal is slightly higher than the blank control (blue curve vs. red curve), manifesting that reverse tearing protospacer is not conducive to Cas12a activation. Similarly, the region where the hairpin DNA hybridized with the target miRNA is divided into three distinct regions: a toehold region marked as “a”, a transition region marked as “b”, and a seed region marked as “c” and the lengths of the above three regions have also been optimized. Firstly, three hairpin DNAs are designed, each containing base pairs of 4 bp, 6 bp, and 8 bp in the “c” region (Fig. 2C). Unfortunately, it indicates that all three hairpin DNAs, which has different base pairs in the “c” region, exhibit a low S/B ratio of approximately 1.0 (Fig. 2D). Subsequently, the “a” region and “b” region of hairpin DNA are parallel optimized under “c” region of 6 bp (Fig. 2E). Regretfully, Fig. 2F reveals that all five hairpin DNAs with distinct base pairs in the “a” region and “b” region also exhibit a relatively low S/B ratio of approximately 1.2. These indicate that reverse tearing protospacer activated Cas12a is unavailable.

Based on the above findings, forward tearing protospacer activated Cas12a with A12B4C6 hairpin DNA as an optimal TRHDA is chosen in the subsequent experiments.

Feasibility of TRHDA-mediated Cas12a for miRNA analysis

The upregulation of miR-21 has been observed in various cancer types and is implicated in tumorigenesis, making it a highly promising biomarker for cancer diagnosis [41, 42]. Therefore, miR-21 is selected as target model to evaluate the feasibility of the TRHDA-mediated Cas12a-powered biosensing. As the proof of the principle, miR-21 binds to the optimal hairpin DNA (A12B4C6) facilitating the complementarity between crRNA spacer and protospacer in the stem sequence of A12B4C6, thereby activating the trans-cleavage activity of Cas12a. To validate this design, the PAGE experiment is conducted. As shown in Fig. S2, compared to un-cleaved ssDNA (reporter 2) band in lane 5, the ssDNA (reporter 2) band disappears in lane 6 when miR-21 is added into the mixture of Cas12A-crRNA and hairpin DNA of A12B4C6. This observation confirms that miR-21 binding to A12B4C6 could activate the trans-cleave activity of Cas12a through forward tearing protospacer behavior. Next, the reaction rates of ssDNA activator, dsDNA activator and the TRHDA-mediated Cas12a system were investigated through fluorescence kinetic analysis. By further derivation of the fluorescence kinetic curve to obtain the rate curves, it was determined that ssDNA activator has the highest maximum reaction rate, followed by TRHDA-mediated Cas12a system, with dsDNA activator being the slowest. Of note, the reaction rate of TRHDA-mediated Cas12a system is about twice that of traditional PAM-dependent dsDNA activator (Fig. S3).

Optimization of experimental conditions

To achieve optimal analytical performance for miRNA detection, several experimental conditions such as the reaction temperature of TRHDA-mediated Cas12a system, the concentration of Cas12a-crRNA complex, the type of reaction buffer, and the concentration of A12B4C6 are optimized. Firstly, the TRHDA-mediated Cas12a system is conducted at various reaction temperatures, namely 25 ℃, 33 ℃, 37 ℃, and 41 ℃. As depicted in Fig. S4, with the increase of reaction temperatures, the S/B ratio increases and achieves its peak at 37 ℃, so 37 ℃ is ultimately chose as the optimal reaction temperature for subsequent experiments. Then, a series of concentrations of Cas12a-crRNA complex are explored to obtain the optimum cleavage activity of Cas12a. As shown in Fig. S5, the concentration of Cas12a-crRNA complex is studied from 10 to 100 nM. The S/B ratio undergoes an upward trend from 10 to 50 nM. After that, S/B ratio dramatically decreases. Therefore, 50 nM is selected as the optimal concentration of Cas12a-crRNA complex in the subsequent research. It is well-known that enzymatic reactions may be significantly affected by reaction buffers. Therefore, the trans-cleavage activity of TRHDA-mediated Cas12a system reacted in NEBuffer r2.1, NEBuffer 3, NEBuffer 4, and rCutsmart buffer is investigated, respectively. As illustrated in Fig. S6, although the highest fluorescence signal is observed in NEBuffer 4, it has a relatively high background signal. Obviously, the highest S/B ratio is received in NEBuffer 3, which is selected to utilize in follow-up experiments. As everyone knows, the activated Cas12a could indiscriminately trans-cleavage ssDNA, which causes that the loop of hairpin A12B4C6 and the F-Q reporter could be trans-cleaved in this system. If the concentration of hairpin A12B4C6 is excess, the excessive hairpin A12B4C6 could compete with F-Q reporter to be trans-cleaved by activated Cas12a, resulting in signal inhibition. Thus, we optimize the concentration of hairpin A12B4C6 to improve the detection performance and results are depicted in Fig. S7. The hairpin A12B4C6 concentration-dependent S/B ratio increases till it reaches highest peak at 25 nM, indicating that 25 nM of hairpin A12B4C6 is enough to guarantee the target miRNA binding for subsequent reaction. After that, the S/B ratio gradually decreases, which is attributed to the hairpin A12B4C6 is competitive with F-Q reporter to be non-specifical trans-cleavage by Cas12a. Therefore, the favorable concentration of the hairpin A12B4C6 at 25 nM is adopted in the following experiments.

Analytical performance of TRHDA-mediated Cas12a system for universal miRNA assay

As a proof-of-concept TRHDA-mediated Cas12a system, the designed input-responsive hairpin DNA contains the specific target miRNA binding site, which further engineers different hairpin DNAs for universal miRNA detection (Fig. 3A). Here, three hairpin DNAs (A12B4C6) with distinct sequences are constructed to individually detect miR-21, let-7a, and miR-30a under the optimized experimental conditions. Figure 3B shows that the fluorescence intensities are increased with the increasing target miR-21 concentrations in the range of 50 pM-15 nM. In addition, the ΔFluorescence intensity (F-F0), which is the difference in fluorescence intensity between the target miRNA and blank, represents a good linear relationship over the dynamic range of 50 pM to 15 nM. The correction equation is ΔF = 640.60C + 207.95 with the correlation coefficient (R2) of 0.9766 (Fig. 3C). The limit of detection (LOD) is calculated to be 13.9 pM according to the rule of 3 times standard deviation over the blank response. Furthermore, the developed TRHDA mediated Cas12a-powered method for miR-21 detection has been compared to other reported sensing assays in terms of linearity and LOD. As shown in Table S2, the designed miR-21 biosensing method has a satisfactory analytical performance, which is attributed to exceptional trans-cleavage activity of TRHDA-mediated Cas12a as a signal amplifier.

Fig. 3

Analytical performance of TRHDA-mediated Cas12a-powered universal assays for three different miRNAs (miR-21, let-7a, and miR-30a) detection. A Principle diagrams of universal assays for miR-21, let-7a, and miR-30a detection with TRHDA-mediated Cas12a system. Fluorescence spectrum showing different concentrations of B miR-21 (0.05–15 nM), E let-7a (0.1–15 nM), and H miR-30a (0.1–15 nM). Linear relationship between the fluorescence intensity and C miR-21 concentration in the range of 0.05–15 nM, F let-7a concentration in the range of 0.1–15 nM, I miR-30a concentration in the range of 0.1–15 nM. Selectivity of the TRHDA-mediated Cas12a based assays for D miR-21 detection, G let-7a detection, and J miR-30a detection against the interference miRNAs and a blank control. The data error bars indicate mean ± SD (n = 3)

Selectivity is equally important to the proposed fluorescent assay for miRNA detection. To explore the selectivity of the approach for miR-21 analysis, a variety of cancer-associated miRNAs, including miR-122, miR-141, miR-155, miR-378, and let-7a, are added to the detection system, each at the concentration of 10 nM. Weak fluorescence signals are observed in the presence of the interfering miRNAs, which is approximate to the signal of blank control. However, when miR-21 is present alone or together with the other five interfering miRNAs, a strong fluorescence signal is obtained (Fig. 3D). These results validate that target-binding driven forward tearing protospacer behavior to activate cleavage activities of TRHDA-mediated Cas12a, thereby ensuring the high selectivity of the proposed method for miR-21 detection.

Meanwhile, the TRHDA-mediated Cas12a-powered methods for sensitivity and selectivity of general detection of let-7a and miR-30a are also investigated. For both target let-7a and miR-30a, the fluorescence intensities are in line with the law that miRNA concentration is proportional from 100 pM to 15 nM (Fig. 3E and H). As displayed in Fig. 3F and I, their correction equations for let-7a and miR-30a detection are ΔF = 551.63 C + 351.42 with the correlation coefficient (R2) of 0.9683 and ΔF = 500.52 C + 234.83 with the correlation coefficient (R2) of 0.9855, respectively. According to the 3-times standard deviation of the blank response, the LODs of let-7a and miR-30a detection are calculated to be 26.6 pM and 32.4 pM, respectively. Similarly, several irrelevant interfering miRNAs are used to estimate the selectivity of the proposed methods for let-7a and miR-30a detection. In Fig. 3G and J, the fluorescence signals of nonspecific groups closely resemble the blank control, whereas the addition of target let-7a or miR-30a only induces a strong fluorescence signal. These findings demonstrate that the proposed methods effectively discriminate miRNAs based on the well-design of target-responsive hairpins in the TRHDA.

Identification of single-base mismatches and homologous miRNA family in TRHDA-mediated Cas12a activation mode

The ability to distinguish single-base mismatches and highly homologous miRNA families poses a significant challenge in clinical molecular diagnostics. To investigate the discrimination of single-base mutations by the designed TRHDA-mediated Cas12a activation mode in targets compared to ssDNA activation mode, we introduce single-point mutations into miR-21 and ssDNA activator to activate Cas12a for comparison (Fig. 4A). The specificity of two activators-mediated Cas12a activation modes is evaluated by the discrimination factor (DF) which is calculated using formula (1).

$$\text=\frac}_]}}}_]}}$$

(1)

where RFU[Target] means the fluorescence intensity for ssDNA target or miR-21 target and RFU[Target Mut] means the fluorescence intensity for ssDNA target mutant or miR-21 target mutant. As depicted in Fig. 4B and C, it is observed that all ssDNA activators in positions Mut1-Mut19 are able to activate Cas12a, indicating that the ability of ssDNA activation mode to distinguish single-base mutations is relatively poor, which is consistent with the previous works [23]. However, miR-21 mutations in positions Mut4, Mut10, Mut13, and Mut19 significantly reduce the trans-cleavage activity of TRHDA-mediated Cas12a, suggesting the differentiation of single-base mutations in TRHDA is location-dependent. Clearly, compared to traditional ssDNA activators, TRHDA-mediated Cas12a activation mode exhibits improved discriminatory capacity for target mutations, as expected.

Fig. 4

A Schematic of ssDNA activator vs. TRHDA for the detection of a target nucleic acid. SsDNA activator and miR-21 are designed with single-base mutations across the length of the activator. The mutation location is identified by ‘Mut’ following the nucleotide number where the base has been changed. B The heat map represents the fluorescence intensity of different mutation activators. C Comparison of DF of ssDNA activators and TRHDA containing different single-base mutations in different positions. D Fluorescence signals of different members of the let-7 family. E Fluorescence signals of different members of the miR-30 family. The data error bars indicate mean ± SD (n = 3)

To further assess the specificity of the designed TRHDA-mediated Cas12a activation mode in miRNA assays, we utilize let-7 family and miR-30 family as model targets, which exhibit high sequence homology and some of them even differing by only one base. In Fig. 4D, it is evident that the fluorescence signal of let-7a surpass that of other let-7 members significantly. Notably, even the most indistinguishable let-7c, let-7e, and let-7f could be distinguished from let-7a. Similarly, in Fig. 4E, the fluorescence signal of miR-30a exhibits superiority over other miR-30 members. These findings elucidate that the designed TRHDA demonstrates exceptional specificity when dealing with highly homologous miRNA families.

Intracellular imaging of miR-21 via TRHDA-mediated Cas12a system

Benefiting from the excellent in vitro analytical performance of the designed TRHDA-mediated Cas12a sensing for miRNA detection, the applicability of TRHDA-mediated Cas12a system for miR-21 intracellular imaging is further explored (Fig. 5A). Here, MCF-7 cell line with high expression levels of miR-21 and MCF-10A cell line with low expression levels of miR-21 are used for intracellular imaging. To demonstrate the capability of TRHDA-mediated Cas12a system for miR-21 sensing in cells, the two cell lines (MCF-7 and MCF-10A) are treated with three experimental conditions (group I: only Cas12a-crRNA, group II: Cas12a-crRNA and F-Q reporter, group III: all of Cas12a-crRNA, F-Q reporter and hairpin A12B4C6) in parallel, respectively. As shown in Fig. 5B, no significant green fluorescence in the MCF-10A cells is observed in the three groups. Besides, no green fluorescence in the MCF-7 cells is observed in the two control groups (group I and II), whereas bright green fluorescence in the MCF-7 cells is observed in experimental group (group III), as expected. The corresponding fluorescence intensity quantified by Image J software is evaluated in Fig. 5C. These results suggest that the strong green fluorescence is caused by target miR-21 binding to TRHDA to activate the Cas12a for cleaving F-Q reporter, rather than by non-specific degradation of the F-Q reporter.

Fig. 5

The TRHDA-mediated Cas12a sensing of miRNA in cells. A Schematic illustration of TRHDA-mediated Cas12a system for miR-21 imaging in cell. B Confocal microscopic images and C the corresponding fluorescence intensities of each cell with different treatments. The scale bar is 25 μm. The data error bars indicate mean ± SD (n = 3)

Considering the differential expression levels of miRNA in various cellular contexts, it is imperative to assess the applicability of TRHDA-mediated Cas12a sensing for visualizing miR-21 in alternative cells, including MCF-7 cells, HepG2 cells, HeLa cells, and MCF-10A. The fluorescence images in Fig. 6A clearly demonstrate distinct green fluorescence observed in MCF-7, HepG2, and HeLa cells. Conversely, MCF-10A cells exhibit minimal green fluorescence. The quantification of mean fluorescence intensities for four distinct cell types in Fig. 6B demonstrates a sequential increase in fluorescence intensity, highlighting the robust capability of the TRHDA-mediated Cas12a system to detect miR-21 across various tumor cell lines. To further illustrate the strong correlation between FAM fluorescence intensity and miR-21 expression, we conduct qRT-PCR analysis to examine the expression levels of miR-21 in above-mentioned cell lines. Remarkably, the results are in complete concordance with those yielded by the designed TRHDA-mediated Cas12a sensing assay (Fig. 6B vs. C). Collectively, these findings unequivocally demonstrate that the proposed TRHDA-mediated Cas12a sensing holds immense potential for detecting endogenous miRNAs within cells.

Fig. 6

A Confocal microscopic images and B the corresponding fluorescence intensities of TRHDA-mediated Cas12a system for miR-21 imaging in MCF-10A, HeLa, HepG2, and MCF-7 cells. The mean fluorescence intensities of the corresponding groups (n = 3) are displayed in histograms. C The qRT-PCR results of the relative miR-21 expression levels in MCF-10A, HeLa, HepG2, and MCF-7 cells. The scale bar is 25 μm. The data error bars indicate mean ± SD (n = 3)