A programmable ribozyme for RNA signal transduction

Domain: chemistry
Published: 2026-01-10
Editorial outcome: accept

Abstract
RNA detection applications can be augmented if a sensed RNA can be directly functionally transduced. However, there is no generalisable approach that allows an RNA trigger itself to directly activate diverse non-coding RNA effectors. Here, we report engineering of a programmable, RNA trigger-activated, dual-site self-cleaving ribozyme with modular sensing domain and cleavage product. This platform, UNlocked by Activating RNA (UNBAR), is entirely encoded within one RNA strand. The ribozyme can be designed to be almost completely inactive in absence of trigger, and to exhibit single-nucleotide trigger specificity. UNBAR ribozymes carry out cell-free sensing and protein-free amplification of microRNA and viral RNA sequences, and trigger-dependent release of ncRNA effectors sgRNA, shRNA and aptamer. We demonstrate RNA detection and functional transduction by a cleaved aptamer, whose fluorescence can be directly readout as a function of trigger RNA. We further engineer the ribozyme for function in cells, and demonstrate trigger-dependent regulation of CRISPR-Cas9 editing by sgRNA-embedded ribozymes in zebrafish embryos and human cells. UNBAR is a first-in-class modality with potential to be developed into a versatile platform for synthetic biology, diagnostics and gene regulation.

Introduction
The versatility of RNA has inspired engineering of RNA-sensing technologies based on various RNA modalities, including fluorescent aptamers that light up in response to RNA triggers, guide RNAs for CRISPR-Cas9-mediated gene editing modified to respond to RNA triggers or ligands via antisense blocking sequences and toehold-mediated strand displacement, gene regulatory machinery that can be targeted by microRNA (miRNA) (e.g. miRNA-directed Ago2 cleavage and release of sgRNA) and miRNA-regulated Cas9 or other mRNA. Switch-based approaches that utilise antisense blocking sequences on the functional RNA are usually restricted in sequence due to their design, while methods based on sensing miRNA and/or recruitment of RNAi machinery are miRNA- and RNAi-specific. Methods that use RNA sensing to control designer mRNAs in cells have also been developed (e.g. and reviewed in ref. 25). However, to our knowledge, no general approach currently exists that can convert a sensed RNA into a second, sequence-independent RNA signal, which can take the form of diverse non-coding RNA (ncRNA) effectors with specific functional outputs.

Ribozymes are RNA molecules with catalytic activity. Auto-catalytic ribozymes have been used to precisely trim guide RNAs for CRISPR-Cas9 editing, and have been adapted for various cellular applications aimed at regulating mRNA function, including to enable stabilising RNA circularisation, to temporally control expression of AAV-delivered mRNAs, and to join up gene therapy payloads.

Here, we describe the development of an innovative modular RNA signal transduction platform based on a trigger-activated, conditional self-cleaving ribozyme. We iteratively engineer this ribozyme to comprise an RNA-sensing domain and two cleavage sites, between which can be embedded a releasable RNA cleavage product (e.g. a ncRNA effector). This is, to our knowledge, the first example of a single ribozyme that can cleave at two distinct sites to release almost any short RNA product of choice. The sequences of both the sensing domain and the cleavage product can be independently and modularly varied. Upon complementary binding of an RNA trigger, ribozyme cleavage is activated to release the embedded RNA cleavage product from ribozymes in cis and in trans. Hence, the ribozyme alone acts simultaneously as an RNA signal detector, transducer and amplifier. We employ 2D and 3D computational modelling techniques to understand how trigger binding activates ribozyme catalysis and permits the novel second-site cleavage we observed.

We develop ribozymes programmed to release short RNA, sgRNA, hairpin RNA and a fluorescent aptamer in an RNA sequence-specific, trigger-dependent manner in cell-free assays. These ribozymes can carry out protein-free RNA signal detection and functional transduction, as demonstrated by direct fluorescent readout of cleaved aptamer. We further engineer the ribozyme for function in cells by identifying variants that can cleave in cellular ionic conditions. We provide evidence that an sgRNA-embedded ribozyme can cleave to release the sgRNA in a trigger-dependent manner in cells to activate CRISPR-Cas9 editing. This versatile platform can potentially be explored for a range of synthetic biology and diagnostic applications.

Results
Engineering trigger-activated tandem ribozymes
We previously developed a miRNA sensor whose fluorescence is activated upon binding of a specific target miRNA, by altering the structure of the fluorescent RNA Spinach2, so that hybridisation of a target miRNA to its cognate sensor backbone is required for fluorescent complex stabilisation. We hypothesised that an analogous approach could be used to modify a catalytic RNA, reasoning that a ribozyme with sensing functions could be engineered from two or more RNA molecules if its stable catalytic conformation required reassembly of these RNAs. Specifically, one of these RNAs should comprise sequences complementary to a trigger RNA so that base-pairing between the ribozyme sensor and its trigger RNA would reconstitute an active ribozyme. We further hypothesised that tandem-linkage of two such ribozymes could result in trigger-activated self-cleavage at two sites and release of an embedded RNA molecule from the tandem ribozymes, allowing for signal amplification if one trigger could activate self-cleavage of multiple ribozymes.

After considering the structural characteristics of various ribozymes, we selected the hairpin ribozyme (HpRz) as a proof-of-concept based on two observations. First, previous studies have shown that two HpRzs linked in tandem exhibit dual cleavage, releasing a cleavage product. Second, mutagenesis studies of the well-studied HpRz structure revealed the presence of a stem structure, helix 4 (H4), which is structurally required for ribozyme catalysis but whose sequence could be varied. We hypothesised that self-cleaving activity could be assembled from several RNA molecules coming together at H4 to reconstitute HpRz structures. The HpRz comprises two independently folding stems A and B, each consisting of a loop containing key catalytic nucleotides, flanked by two helices (H1–H4). The two loops dock prior to cleavage, and A38 and G8 are critical to formation of the transition state. The phosphodiester cleavage reaction then occurs between the N−1 and guanine (G+1) nucleotides in loop A. The sequence requirement at the cleavage site has been systematically studied; sequences with activity >20% of wild-type are shown in Fig. 1A.

Fig. 1B shows a schematic of our original design approach. Two HpRzs, both modified to contain sequences complementary to a trigger miRNA, are arranged in tandem, one in a wild-type configuration (‘right’ side) and the other in a reverse-joined configuration (‘left’ side). In the absence of an intact H4 and without the trigger RNA, loop B was predicted to be conformationally unstable and hence unable to fold into a catalytically active conformation. The binding of the RNA trigger is predicted to stabilise loop B, activating cleavage and product release. This strategy required the original tandem-linked ribozymes to undergo three modifications. First, we would circularly permute the structure so that H4 would no longer be a closed stem loop; instead, the 5′ and 3′ ends would reside within both H4s (Fig. 1Biii; different junction configurations are possible). The tandem-linked ribozymes would consist of two strands, one containing the cleavage sites and cleavage product (strand 1; S1) and the second comprising a non-cleaved complementary strand (strand 2; S2). Next, we would introduce two sequence-variable stem loops that branch off from both H4s (IN-form; Fig. 1Biv), mimicking the structure formed by the binding of a trigger. Finally, we would assemble self-cleaving tandem ribozymes from three RNAs (S1, S2 and trigger RNA) that are sequence-complementary at the new branched H4s (OUT-form; Fig. 1Bv). Such a ribozyme is predicted to produce several different fragments when cleaved: the S2 strand (S2), the full-length S1 strand (FL), a ‘left’-cleaved fragment that includes the cleavage product (R+C), a ‘right’-cleaved fragment that includes the cleavage product (C+L), a ‘left’-cleaved fragment without the cleavage product (L), a ‘right’-cleaved fragment without the cleavage product (R), and finally, the dual-cleaved product (C). We systematically tested the feasibility of this strategy.

We selected as a trigger the Drosophila microRNA bantam-5p (ban-5p), which could be effectively sensed by an analogous sensing domain. As an example cleavage product, we chose a 29-nucleotide (nt) RNA (29nt-clvRNA). The first step was to circularly permute the tandem structure; this retained self-cleavage activity to release the embedded 29-nt cleavage product. For the trigger-activated cleavage strategy to work, it is important that cleavage is inhibited by destabilising H4; indeed, while ribozymes with an 8 bp H4 released the cleavage product, shortening this helix (and thereby reducing its stability) reduced product release.

The initial designs incorporated a 3-way junction (3WJ) in an HHH configuration, and dual-cleavage activity was low. We took two approaches to boost cleavage. First, we tested alternative junctional configurations—2WJ, 3WJ (HHS H) or 4WJ—that were previously shown to cleave better than the 3WJ (HHH). In addition, since re-ligation rates increase when the cleaved product is highly complementary within H1, we varied the degree of base-pairing between the cleavage product and S2. 3WJ (HHS H) and 4WJ unpaired at H1 showed the highest amounts of dual-cleaved cleavage product. However, fully unpaired H1 resulted in unexpected bands, perhaps due to alternative ribozyme folding. Therefore, we partially restored cleavage product complementarity to the 4WJ (HHHS H). A configuration of two paired nucleotides alternating with three or four unpaired nucleotides was effective at releasing the cleavage product.

Next, we introduced branched stems into H4 to mimic an RNA-binding trigger region (Trigger-IN form, where the trigger RNA forms part of the ribozyme; Fig. 1Biv). We used the optimised 4WJ tandem ribozymes with alternating base-pair configuration. As H4 longer than 4 bp released dual-cleaved product in the absence of trigger, we tested H4 of 1 to 4 bp. IN-form ribozymes could self-cleave S1 to release substantial 29-nt cleavage product when H4 was at least 3 bp long, with increased H4 length associated with greater product release. This was corroborated using a probe for 29nt-clvRNA to label S1 and cleavage products comprising the 29-nt. Therefore, our combined modifications—circularly permuting the tandem ribozymes, reducing complementarity of the cleavage product, introducing a 4-way junction, and shortening helix 4 and altering it to be complementary to a trigger RNA—allowed the possibility of trigger-activated tandem ribozymes.

The sequence of the trigger region replacing H4 could be varied, allowing us to encode sequences complementary to trigger RNAs into the ribozymes. We therefore hypothesised that we could reconstitute ribozyme activity from three RNAs: a two-strand tandem-ribozyme with ban-5p-binding domain (OUT-form, Fig. 1Bv) and a short RNA (ban-5p). In the OUT-form, when H4 was 1–2 bp in length, the ribozyme released little dual-cleaved product even in the presence of trigger, and substantial S1 remained uncleaved. A ribozyme with H4 of 3 bp released more cleavage product with the cognate trigger vs. a non-specific control trigger, and its corresponding A38mut catalytic mutant ribozyme did not cleave. Increasing H4 to 8 bp led to high background cleavage in the absence of trigger, and a probe for the 29nt-clvRNA showed that cleavage activity correlated with H4 length. A second ribozyme, triggered by miRNA hsa-let-7f, also released a cleavage product in a trigger-dependent and cleavage-dependent manner. These data show that we have developed a tandem ribozyme structure with two cleavage sites, two loops B and two trigger-binding domains, which is activated by trigger RNA to release an embedded RNA cleavage product.

Development of a programmable dual-site single-stranded ribozyme
To understand the cleavage mechanism of tandem ribozymes, we mutated catalytic nucleotides in each loop B. A38 is a critical nucleotide that stabilises the transition state during catalysis, and its mutation abolishes cleavage. To our surprise, A38U mutation in either the ‘right’ wild-type or the ‘left’ reverse-joined loop B did not substantially reduce release of the cleavage product, and mutation of both was required to abolish cleavage. This suggested that either single loop B was sufficient for dual cleavage.

To investigate this further, we designed ribozymes with a single wild-type loop B, as we reasoned that this conformation could be rationally optimised based on the abundant functional studies that have been carried out on this canonical structure. This ribozyme could now be encoded by a single strand of RNA, which, in a cleavage assay, would be predicted to produce: the full-length ribozyme (FL), a proximal-cleaved fragment that includes the cleavage product and distal fragment (C+D) a distal-cleaved fragment that includes the proximal fragment and cleavage product (P+C), a distal-cleaved fragment without the cleavage product (D), a proximal-cleaved fragment without the cleavage product (P), and finally, the dual-cleaved product (C). Single-loop B ribozymes triggered by miRNAs (hsa-mir-451a, dme-mir-184, dme-mir-252, dme-mir-263a or hsa-let-7f) or by fragments of the E-gene and Orf1ab transcripts of the SARS-CoV-2 genome could all self-cleave at both cleavage sites to release the cleavage product. This confirms that one loop B is sufficient for dual cleavage. A U mutation in the single loop B abolished cleavage. This is, to our knowledge, the first example of a single HpRz capable of cleaving at two different cleavage loops, a phenomenon not previously described. Henceforth, ribozymes are named based on their trigger (T) RNA and cleavage (Cl) product; e.g., a ribozyme triggered by RNA ban-5p to cleave out a 29-nt cleavage product is named T-ban-5p_Cl-29nt-clvRNA.

Even though we observed dual cleavage of the single-loop B ribozymes, cleavage appeared to be trigger-independent. To improve trigger-activated cleavage, we aimed to identify the optimal length and sequence for the H4 stem, which we predicted would function as a trigger-responsive communication module (CM). Ribozymes with H4 > 5 bp cleaved in a trigger-independent manner, while one with H4 = 2 bp did not release dual-cleaved product even in the presence of trigger. We systematically tested various CMs of 2 and 3 bp and identified two 3 bp motifs, 5’-ACG/CGU-3’ (3bp-v1) and 5’-ACG/CGA-3’ (3bp-v2), which greatly improved the signal-to-noise ratio of E-gene-triggered ribozymes. We tested the same panel of CMs for two other different trigger RNAs, mir-451a and SARS-CoV-2 S-gene, and found that 3bp-v1 and 3bp-v2 CMs also improve ribozymes triggered by these different RNAs. We named this trigger-activated, single-stranded dual-site self-cleaving ribozyme platform: UNlocked By Activating RNA (UNBAR).

Our data show that cleavage can occur at a second site distal to the canonical (proximal) loop A. Such dual-cleaving activity at separate cleavage loops has not, to our knowledge, been previously observed in any natural or engineered ribozyme and was unexpected here, as ribozymes fold into specific conformations for catalysis. Since loop A docks with loop B prior to cleavage, and G8 in loop A is critical for stabilising the transition state, distal cleavage could occur via: (1) conformational rearrangement and ‘sliding’ of the distal G+1 cleavage site onto the proximal G8, followed by canonical HpRz docking between loop B and proximal loop A; or (2) a novel docking event between loop B and the distal loop A. Mutation of G8 at the distal loop A would affect cleavage at the distal G+1 in the second, but not the first, scenario. When we mutated G8 in either or both loops A, we observed that the distal G8 was essential for cleavage at the distal G+1 but not for the proximal site, and vice versa. Therefore, distal cleavage occurs via a novel docking interaction between loop B and distal loop A.

Protein-free RNA signal transduction and amplification
Many methods for RNA detection require signal amplification to enhance the turn-on signal, or the fold-increase in detected signal of a target RNA compared to background. For example, in RT-qPCR, RNA is first copied into complementary DNA (cDNA) by enzyme-mediated reverse transcription, and the cDNA is subsequently amplified by gene-specific primers. Of note, this copied cDNA sequence is invariable as it depends on the RNA template. Therefore, we next asked whether UNBAR ribozymes alone could directly transduce one sensed trigger into amplified release of multiple copies of a sequence-independent RNA cleavage product. Keeping T-E-gene_Cl-29nt-clvRNA ribozyme concentration constant while increasing trigger RNA concentration, we observed that the cleavage product had a maximum turn-on signal of at least ~55-fold in presence vs. absence of trigger. To determine whether one trigger could cause amplified release of multiple cleavage products, we kept trigger concentration constant while increasing ribozyme concentration. We observed that when trigger amount is kept constant (50 nM), the amount of cleaved 29-nt cleavage product increases as more ribozyme is added. At the lowest concentration (200 nM) of ribozyme, the amount of cleavage product released relative to the fixed amount of trigger was about 1:1. As more ribozyme is added, the amount of cleavage product released from the self-cleaving ribozyme gradually exceeds the fixed amount of trigger, demonstrating that the original trigger signal has been amplified into released cleavage product. Quantification of the amount of cleavage product vs. trigger showed that the ribozyme exhibits up to ~7.4-fold amplification at 16-fold more ribozyme than trigger. Hence, a trigger RNA can cause the release of multiple cleavage products with programmable sequence, allowing the ribozyme to act as a protein-free signal detector, transducer and sequence-independent amplifier.

We next investigated the specificity of UNBAR ribozymes by introducing sequence changes into the test/trigger RNA. An unrelated RNA (SARS-CoV-2 S-gene fragment) was unable to trigger cleavage product release by T-E-gene_Cl-29nt-clvRNA. The effects of single-nucleotide mismatches depended on their locations, with position 4 (from the 5′ end) reducing cleavage product release by ~90%. These results suggest that UNBAR ribozymes can be optimised to distinguish between similar trigger sequences. For UNBAR ribozymes to be useful in mixed RNA samples, they should also identify their triggers within complex mixtures. We found that T-E-gene_Cl-29nt-clvRNA ribozyme could detect trigger RNA spiked into a mixture comprising 100-fold excess of competing mammalian cell RNA.

Finally, we asked whether an UNBAR ribozyme can dock with and cleave a cleavage site in a separate UNBAR ribozyme molecule (trans-cleavage). A wild-type T-E-gene_Cl-29nt-clvRNA ribozyme was incubated in a cleavage reaction with a second, catalytically inactive (A38 mutant) ribozyme triggered by a different RNA trigger (let-7f) to release a 113-nt cleavage product (T-let-7f_Cl-113nt-clvRNA_A38mut). When only E-gene fragment was provided in the cleavage reaction, we observed cleavage and release of both the short and long cleavage products, providing evidence that the E-gene-bound and activated ribozyme can dock upon and trans-cleave the catalytically inactive second ribozyme to release its long cleavage product. Mutations of the cleavage site G+1C, G8C in loop A and C25G in loop B of the E-gene-triggered ribozyme confirmed that a trigger-bound ribozyme can trans-cleave another UNBAR ribozyme as long as its loop B is catalytically active. Therefore, a trigger-bound UNBAR ribozyme can trans-cleave other, trigger-free ribozymes, potentially allowing signal amplification.

Computational modelling of ribozyme structural properties
An E-gene-triggered ribozyme with a 3 bp-v1 H4 exhibited substantially increased cleavage activity in the trigger-bound (+trigger) vs. trigger-free (-trigger) state. We used 2D and 3D computational modelling techniques to understand the effect of trigger binding on this ribozyme compared to other E-gene-triggered ribozymes with 2, 5 and 8 bp H4s. Combined k-means clustering of predicted ±trigger thermodynamic secondary structure ensembles of the 3 bp-v1 ribozyme showed marked reduction in conformational variability (fewer, more compact clusters) in +trigger vs. -trigger structures. Examination of predicted structures showed high probability base-pairing between trigger-binding arms and catalytically critical 33, 38 and highly conserved 41 nucleotides A38 and C25 in the absence of trigger. This stable interaction likely creates an auto-inhibitory state with a significant energetic barrier to catalytic loop formation, offering a structural rationale for the limited background cleavage observed in the absence of trigger for the 3 bp-v1 ribozyme. High-affinity binding of trigger RNA releases the catalytic core residues. Although -trigger structures with +trigger homology could be found in predominantly +trigger clusters, these structures are outliers (±trigger ensembles are statistically different [P≈0.05], with low probability of overlap in Cartesian space). Trigger binding, therefore, shifts ensembles away from a diverse set of structures where A38 and C25 residues are “trapped” through association with the trigger arms and towards a defined set of conformations with greater catalytic potential. These conformational properties may underlie the low leakiness and high signal of the 3 bp-v1 ribozyme.

Lengthening H4 to 5 bp or 8 bp resulted in greater homology between -trigger and +trigger structures. For the 8 bp H4 construct, there was substantial overlap (p≈0.61) of pairwise distances between ensembles of ±trigger states; this was exemplified by their highly similar centroid structures. Hence, H4 formation appears important for stabilisation of the active catalytic loop. Without trigger, increased H4 length can shift ensembles towards an active conformation: 8 bp H4 -trigger structures exhibited intact H4 and ipsilateral A38 and C25 residues within a catalytic loop-like structure, similar to those observed in crystallography experiments. Therefore, we next asked whether the probability of H4 formation correlated with experimental cleavage efficacy.

Ribozyme sequences with varying H4 lengths and in both ±trigger states were used to sample 100,000 secondary structures from the Boltzmann ensemble. Resulting statistical ensembles were searched for fully paired H4. Unsurprisingly, increasing H4 length corresponded with increasing probability of reconstituted H4: in the absence of trigger, ~79% of 8 bp H4 structures contained reconstituted H4 (similar to +trigger percentages), while trigger-bound 2 bp H4 exhibited only ~0.31% H4 formation. Furthermore, ±trigger H4 reconstitution rates were predictive of the respective cleavage efficacies in cell-free assays, suggesting that H4 stability is integral to dual-site ribozyme activation. Therefore, an ideal H4 should not base-pair in the absence of a trigger but should stably form when a trigger is present.

To study the cleavage reaction at the distal site required analysis of UNBAR ribozymes in multiple cleaved states. We used 3D modelling and molecular dynamic simulation techniques to deduce the conformational preconditions that prompted the novel observation of distal site cleavage. A tertiary structure model derived from minimum free energy (MFE) secondary structure constraints was used to generate uncleaved and proximal site-cleaved (proximal G+1 phosphate removed) models. For each state, average A38-G+1 distances to both sites were comparable and in a narrow range (~4.0-5.0 nm). This suggests that both sites are similarly accessible to the catalytic loop, supporting dual-site cleavage.

Understanding the degree to which single-site cleavage affects ribozyme dynamics may help predict the structural context for subsequent cleavage. We measured pairwise distances of stem B residues over 1 μs. The distance between H4 and catalytic loop residues in the uncleaved simulation reached a minimum at around 368 ns with limited fluctuation thereafter, suggesting that the catalytic loop adopts a stable conformation before initial cleavage. After proximal-site cleavage, the H4-catalytic loop region appears less stable, as indicated by larger distance fluctuations between H4-catalytic loop residues throughout the entire simulation trajectory. Throughout the cleaved simulation, binding of trigger RNA helped to maintain catalytic loop integrity even after proximal-site cleavage, likely allowing exploration of conformations susceptible to distal site cleavage.

Functional RNA as cleavage products: single guide RNA, hairpin RNA and an aptamer
To explore whether UNBAR ribozymes can transduce sensed RNA inputs into functional RNA outputs, we first asked whether RNA effectors with more complex secondary structures could be embedded as cleavage products. We engineered ribozymes that comprise a CRISPR single guide RNA (sgRNA), a short hairpin RNA (shRNA) or an aptamer (Red Broccoli). To allow for cleavage, three and two nucleotides were left in the cleavage ‘scars’ at the 5′ and 3′ ends of the cleavage products, respectively. The functional relevance of the cleavage scars will depend on the nature of the cleavage product and require functional testing, which we carry out in the next section for a sgRNA-releasing ribozyme. A previous study found that a non-canonical N3 in the catalytic loop can be compensated by mutation of A7 in the wild-type cleavage loop to base-pair with N3, a finding that we replicated. This reduces cleavage scar limitations.

For these ribozymes, we chose let-7f as the trigger RNA. The let-7 family is highly conserved, with let-7f one of its most abundantly expressed members. The concentration of let-7 in mouse mesenchymal stem cells was previously estimated at ~42,000 copies per cell or ~46 nM, which is within the range detectable by UNBAR ribozymes in our cell-free assays (>10 nM). In addition, there is data to suggest that at least 50% of let-7a is stably expressed but not bound to Ago. We assayed T-let-7f_Cl-sgRNA, T-let-7f_Cl-shRNA and T-let-7f_Cl-Red-Broccoli for trigger-activated cleavage in cell-free assays, and observed substantially more cleavage product released in the presence of trigger than without. Quantitative analyses showed that no cleavage was observed with non-specific triggers for the sgRNA and shRNA ribozymes or with their respective non-cleaving catalytic mutants. The Red-Broccoli-releasing ribozyme also tended to show increased cleavage with specific trigger compared to non-specific trigger. Therefore, UNBAR ribozymes can be triggered to release RNA with more complex structures, including sgRNA, shRNA and aptamers.

For a majority of these ribozymes, we observed background, trigger-independent cleavage at the site proximal to loop B. However, the distal site was not cleaved without trigger. We hypothesised that this was due to its increased distance from loop B. To test this, we lengthened H2 of T-let-7f_Cl-sgRNA from 4 bp to 8 and 12 bp. This decreased trigger-independent cleavage at the proximal site. We similarly altered the shRNA-embedded ribozyme and observed a comparable effect. Since loops A and B dock prior to catalysis, we hypothesised that H3 length can also influence cleavage efficacy. Lengthening of H3 also decreased trigger-independent background cleavage at the proximal site; we note that the larger shRNA ribozyme fragments occasionally migrate at larger sizes, likely due to dsRNA formation of the highly self-complementary fragments even in a denaturing gel. Therefore, helices 2 and 3 can be individually altered to refine cleavage regulation by the trigger RNA. This led us to ask whether we could identify an optimal combination of altered H2 and H3 that applies across different types of RNA cleavage products. We tested 30 combinations of helices 3 and 2, where H3 was varied from 2 to 7 bp and H2 was varied from 1 to 6 bp. We found that a ribozyme with a 7 bp H3 paired with a 5 bp H2 (7H3, 5H2), as well as (6H3, 3H2), (5H3, 3H2), (4H3, 3H2), (3H3, 5H2) and (3H3, 3H2), showed strong trigger-dependent cleavage with minimal trigger-independent background cleavage. We then tested ribozymes embedded with other cleavage products with the best-performing helix combinations and found that (7H3, 5H2) and (5H3, 3H2) ribozymes cleaved with a high signal-to-noise ratio across modalities.

Since UNBAR ribozymes can directly transduce detection of a trigger RNA into amplified release of a second RNA by self-cleavage, they have the potential to form a novel class of diagnostics. We hypothesised that RNA trigger-activated cleavage and signal amplification can be functionally read out if the cleavage product is a fluorescent aptamer, e.g. Broccoli. To test this, we designed ribozymes triggered by a fragment of the SARS-CoV-2 E-gene, to self-cleave and release Broccoli. In-gel staining of cleavage assay products showed strong fluorescence only of the dual-cleaved Broccoli cleavage product, with increased dual cleavage and release of Broccoli as trigger concentration was increased. Increasing fluorescence with increasing trigger concentration could be detected on a plate reader, with turn-on signals of up to ~10-fold (up to 20-fold could potentially be reached with a higher concentration of ribozyme) and sensitivity of at least 25 nM. Therefore, UNBAR ribozymes can be used to directly detect trigger RNAs, and transduce and report their concentration via fluorescence of the released aptamer cleavage product.

UNBAR function in cells
We next asked if UNBAR could function in cellular contexts by investigating whether the let-7f-triggered ribozyme embedded with an sgRNA against GFP could regulate CRISPR-Cas9 editing. We tested this ribozyme in the zebrafish model, as let-7 is present in the early zebrafish embryo, and ribozyme-Cas9 ribonucleoprotein complexes can be directly microinjected into embryos. In fish, let-7a, -c and -f account for ~72% of the let-7 miRNA family. A let-7f-triggered ribozyme could distinguish let-7b, let-7i and mir-98 from the rest of the let-7f family, demonstrating that UNBAR ribozymes can be designed to distinguish between some members of related miRNA sequences. Injection of WT or catalytic mutant T-let-7f-Cl-sgRNA-GFP ribozymes with Cas9 protein into transgenic GFP embryos led to strong GFP down-regulation in embryos injected with the WT ribozyme but not the mutant ribozyme, demonstrating that ribozyme cleavage was required for editing.

We next used DNA fragment analysis to obtain a more quantitative measurement of editing levels. Injection of positive control sgRNAGFP with Cas9 protein led to high rates of editing at the target locus (~92%). Injection of equimolar amounts of ribozyme T-let-7f_Cl-sgRNAGFP with Cas9 also led to robust editing, although ~50% lower than the sgRNA alone, which could be due to incomplete dual-cleavage of the ribozyme within the embryo. To determine whether editing was trigger-dependent, we introduced a morpholino antisense inhibitor against let-7f. Compared to co-injection with a control inhibitor, co-injection of the let-7f inhibitor significantly reduced ribozyme-induced editing in the fish, although the presence of other members of the let-7 family may result in incomplete inhibition. There was no difference in the survival rates of the let-7f vs. control inhibitor samples (80% vs. 75%, respectively). In cell-free assays, the inhibitor disrupted trigger-induced cleavage by a trigger-dependent ribozyme, but did not disrupt cleavage of a non-trigger-dependent (auto-cleaving) ribozyme with an 8 bp H4, demonstrating that the inhibitor can compete with the ribozyme for the trigger, and that it inhibits ribozyme cleavage by trigger-binding rather than by disrupting overall ribozyme structure.

These experiments provided evidence that UNBAR ribozymes can potentially regulate sgRNA function in a trigger-dependent manner in cellular contexts.

We next investigated sgRNA-embedded ribozymes in human cells. To allow for functional normalised assessment of sgRNA editing levels, we made use of HEK293T-Stoplight+ spCas9+ reporter cells (Stoplight cells), which initially express only mCherry but switch to expressing both mCherry and eGFP post-editing with sgRNASTOPLIGHT but not a non-targeting sgRNACTL. We embedded sgRNASTOPLIGHT within a ribozyme triggered by a fragment of the SARS-CoV-2 E-gene (T-E-gene_Cl-sgRNASTOPLIGHT). This viral sequence trigger was chosen as it exhibits high specificity and signal-to-noise ratio. Since the concentrations of Mg2+ and polyamines are lower in cells (~1 mM Mg2+ and <0.01–0.05 mM free polyamine) than in the buffer we used for cell-free cleavage assays in this study (7 mM Mg2+ and 5 mM spermine), we first assayed UNBAR cleavage in conditions of lowered Mg2+ and spermine. We found that cleavage of the wild-type ribozyme was substantially lower in buffer conditions that mimic cellular ionic conditions. It was previously reported that mutations A7G, A10G and U39C can improve HpRz cleavage. Hence, we tested these variants singly or in combination. We found that UNBAR ribozymes with either U39C alone or all three mutations combined showed trigger-dependent cleavage rates in cellular ionic conditions that were comparable to that in the original cell-free cleavage buffer, whilst keeping trigger-independent background cleavage low.

We incorporated a U39C mutation into a T-E-gene_Cl-sgRNASTOPLIGHT ribozyme to study its cleavage and function in human cells. This ribozyme exhibited robust trigger-dependent cleavage in cell-free assays only with cognate and not control trigger, and its corresponding A38C catalytic mutant did not cleave. Using this ribozyme, we systematically investigated: (1) Effect of ribozyme cleavage scars on sgRNA efficacy; (2) Whether embedding the sgRNA within a ribozyme scaffold was sufficient to block sgRNA function; (3) The maximal functional turn-on signal in human cells if cleavage occurs at rates similar to cell-free assays, (4) Efficacy of partially cleaved UNBAR fragments on gene editing; (5) Whether we can visualise trigger-dependent cleavage product release in cells; and (6) Whether trigger-induced cleavage of ribozymes in cells can produce a switch in functional CRISPR-Cas9 editing output.

Ribozyme cleavage occurs at specific sequences. In the case of UNBAR, the cleavage product retains 5′-GUC and 3′-CA scars. While guide RNAs beginning with a GUC would be able to cleave within UNBAR without additional sequences, in the case of sgRNASTOPLIGHT, a 5′ GUC was appended to enable cleavage. To investigate the effect of cleavage scars on the functional activity of sgRNASTOPLIGHT released from UNBAR, we measured editing by sgRNASTOPLIGHT with no cleavage scars, compared to the same sgRNA with 5′, 3′ or both 5′ and 3′ cleavage scars, as well as a non-targeting control sgRNA (sgRNACTL) with cleavage scars. An sgRNA with a 3′ cleavage scar edited as efficiently as the sgRNA with no scar (>70% double-positive cells; p>0.05), while sgRNAs with a 5′ or both 5′ and 3′ cleavage scars exhibited reduced editing (~40% double-positive cells; p~0.05 or p<0.01, respectively). The non-targeting sgRNACTL did not edit. Nonetheless, a ~40% editing activity of sgRNA with both a 5′ and 3′ cleavage scar, still provided a sufficient dynamic range to study regulation of gene editing by UNBAR ribozymes.

Next, we asked whether the ribozyme was able to sequester embedded sgRNA within its scaffold to prevent its editing activity. Embedding sgRNASTOPLIGHT within a mutant ribozyme reduced editing in mammalian cells (~2.8% double-positive cells) compared to the sgRNA alone (~43.0% double-positive cells; p<0.0001), which is a ~15-fold repression of eGFP turn-on by the sgRNA when it was embedded within the ribozyme compared to free sgRNA. Therefore, embedding the sgRNA within the ribozyme efficiently blocks its editing efficacy in mammalian cells. We then investigated the maximal eGFP turn-on that could be achieved by trigger-activated sgRNA released from UNBAR ribozyme, if ribozyme cleavage occurred to the same extent in the cell as was observed in cell-free cleavage assays. To test this, we purified and transfected RNA of mutant and wild-type UNBAR ribozymes pre-cleaved in low Mg2+/spermine buffer into Stoplight cells. Flow cytometry analyses showed a significant ~4-fold increase in eGFP levels induced by wild-type cleaved UNBAR ribozyme (~10.9% double-positive cells) compared to its catalytic mutant (~2.8%; p=0.044).

To investigate the effects of partially cleaved T-E-gene_Cl-sgRNASTOPLIGHT on gene editing, we transcribed RNA fragments that mimic partial cleavage at either the distal or proximal cleavage site. SgRNA corresponding to dual-cleaved UNBAR (with cleavage scars) exhibited the highest editing efficiency (~40%), non-significantly higher than partially distal-cleaved fragment (P+C; with a 5′ overhang), which also robustly activated gene editing (~25%), while partially proximal-cleaved fragment (C+D; with a 3’ overhang) had a much lower editing efficacy (~6%; p<0.01). Therefore, partially cleaved T-E-gene_Cl-sgRNASTOPLIGHT ribozymes can also contribute to gene editing signal.

Next, to visualise trigger-dependent cleavage within cells, we co-transfected T-E-gene_Cl-sgRNASTOPLIGHT ribozyme with control or cognate E-gene trigger. We observed only a small amount of dual-cleaved product released in HEK293T cells. Northern blotting of the transfection mix sample at 24 h showed no ribozyme cleavage. Hypothesising that spermine, a naturally occurring polyamine, may aid cleavage, we asked whether supplying spermine to cells would improve ribozyme cleavage within cells. This approach also allowed us to definitively test whether cleavage could occur within cells.

HEK293T cells were transfected with T-E-gene_Cl-sgRNASTOPLIGHT ribozyme and either control or cognate trigger. After 24 h, all media and transfection mix were removed from the culture wells, so that any ribozyme remaining must have entered cells to be protected from the media removal. At this point, we incubated the cells with either normal or spermine-supplemented replacement media. Ribozymes from cells replaced with normal media exhibited limited cleavage by 72 h. In contrast, ribozymes from cells replaced with spermine-supplemented media exhibited robust cleavage, with a marked increase in cleavage product released just 2 h after spermine addition, at 26 h, with a further increase at the 48 h mark. Notably, RNA in the transfection mix alone at 24 h did not show detectable cleavage product, in contrast to RNA from spermine-treated cells tested just 2 h later. These results demonstrate that substantial ribozyme cleavage occurred in cells after media with transfection mix was removed at 24 h, only with cognate trigger and not control trigger.

Since cleavage can be observed in HEK293T supplemented with spermine, this allowed us to test ribozyme cleavage and function in HEK293T-Stoplight+ spCas9+ reporter cells with certainty that cleavage could only have occurred post-spermine incubation, within cells. We examined editing by sgRNASTOPLIGHT, wild-type T-E-gene_Cl-sgRNASTOPLIGHT and its corresponding catalytic mutant ribozyme co-transfected with control or cognate E-gene trigger into Stoplight cells using flow cytometry. While E-gene co-transfection had no effect on editing by sgRNASTOPLIGHT alone or mutant ribozyme (p>0.05), its co-transfection with wild type T-E-gene_Cl-sgRNASTOPLIGHT ribozyme caused ~24% increase in editing rates compared to those incubated with control trigger (p<0.01). As an additional control, we examined the effect of co-incubation of control vs. cognate E-gene trigger on wild-type and mutant ribozymes that comprise a non-targeting sgRNACTL, and observed no editing. Therefore, ribozymes can cleave in a trigger-dependent manner in human cells to regulate gene editing.

In summary, northern blotting and functional data provide evidence for trigger-dependent cleavage and CRISPR-Cas9 gene editing by UNBAR ribozymes in cellular contexts. With further engineering, UNBAR ribozymes have the potential to be developed for RNA sensing-dependent functions of embedded ncRNA effectors for regulation of gene expression.

Methods
Ethical statement
Our research complies with all relevant ethical regulations. Our study protocols were approved by the Institutional Biosafety Committee at Institute of Molecular and Cell Biology, A*STAR, Singapore. All experiments with zebrafish embryos were approved by the Singapore National Advisory Committee on Laboratory Animal Research (Protocol number: 221702).

PCR amplification of the template and in vitro transcription of RNA
DNA templates and primers were ordered from Integrated DNA Technologies, USA, and PCR amplification was performed using Phusion High-Fidelity PCR Master mix (#F531L, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. PCR products were purified using QIAquick PCR Purification kit (#28106, Qiagen, Germany), or gel-extracted using Zymoclean Gel DNA Recovery Kit (ZYR.D4008, Zymo Research, USA), and used as templates for in vitro transcription using AmpliScribe T7-Flash transcription kit (#LGLC-ASF3507, Lucigen, USA) according to the manufacturer’s instructions. For some templates, DNA gene fragments, oligopools or clonal genes (Twist Bioscience, USA) were used for in vitro transcription. GGGA would be added to the 5’ end of every transcript during T7 transcription. After the in vitro transcription reaction and DNase I treatment as per manufacturer's instructions, 280 μL of RNase-free water (#SH30538.02, HyClone, USA) was added to 20 μL of the in vitro transcription reaction. The RNA was purified by adding 300 μL of acid-phenol:chloroform, pH 4.5 (with IAA, 125:24:1) (#AM9722, Invitrogen, USA), mixed, and centrifuged at 18,000×g for 3 min at room temperature. The aqueous phase was transferred to a new tube, and 300 μL of chloroform (#07278-00, Kanto Chemical, Japan) was added. The mixture was centrifuged at 18,000×g for 3 min at room temperature. The aqueous phase was transferred to a new tube and the RNA was precipitated with 1/10 volume (25 μL) of 3 M sodium acetate (pH 5.2) and 2.5× volume (625 μL) of absolute ethanol. After overnight precipitation at −20 °C, the samples were centrifuged at 16,000×g for 20 min at 4 °C. The supernatant was discarded and the RNA pellet was washed with cold 75% ethanol and centrifuged at 16,000×g for 20 min at 4 °C. After centrifugation, the supernatant was removed completely, and the pellet was allowed to air-dry. The RNA was then resuspended in 50 μL of RNase-free water, and its concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific).

Cell-free cleavage assays
Assays were performed with in vitro transcribed ribozyme RNA (200 nM unless otherwise described) and trigger RNA (50 nM unless otherwise described) (Integrated DNA Technologies, USA) in 1× cleavage buffer. For tandem ribozyme cleavage assays: composition of the cleavage buffer used is 50 mM Tris, 12 mM MgCl2, 5 mM Spermine, pH 7.4. Reaction conditions were as follows: 60 °C for 1 min, 56.5 °C for 1 min, 53 °C for 1 min, 49.5 °C for 1 min, 46 °C for 1 min, 42.5 °C for 1 min, 39 °C for 1 min, 37 °C, 15 mins; Steps 1 to 8 were repeated for 9 cycles.

For single ribozyme cleavage assays: cleavage buffer contained (10 mM Tris, 7 mM magnesium chloride, 5 mM spermine, 2 mM sodium chloride, pH 6.4). For conditions with lowered Mg2+ and spermine, 1× cleavage buffer comprised pH 6.4 with 10 mM Tris, 1 mM magnesium chloride, 0.05 mM spermine, and 2 mM sodium chloride. 200 nM of an inert 40-nt RNA sequence (5′-GGGACAUGGAAGUCACACCUUCGGGAACGUGGUUGACCUA-3′) was spiked into the reaction as a loading control in the cleavage assay master mix. Reactions were incubated at 37 °C for 4 h.

Detection and visualisation of cleavage product
Cleavage reaction product was directly mixed with 2× RNA Loading Dye (#B0363S, New England Biolabs, USA), then heat-denatured at 70 °C for 10 min and separated on a 10% denaturing polyacrylamide gel (#EC-833, National Diagnostics, USA) in 1× TBE (Tris/Boric Acid/EDTA) buffer (#1610770, Bio-Rad, USA) at 200 V for 45 min to 1 h, or until the dye front migrated to the bottom of the gel. Low range ssRNA ladder (#N0364S, New England Biolabs) was loaded as a size marker, and 25 ng of an RNA oligonucleotide, e.g. 29-nt oligo (5′-GUCCUUAGUCGAAAGUUUUACUAGAGUCA-3′) (Integrated DNA Technologies), or an in vitro transcribed RNA sequence corresponding to the size of the expected cleavage product, was spiked into the ladder as an additional size marker. Where appropriate, 25 ng each of the in vitro transcribed ribozyme, trigger and/or spike-in sequence were also added into the ladder as size markers. Gels were stained with SYBR Gold at 1:10,000 dilution (#S11494, Invitrogen), and were visualised using a ChemiDoc MP Imaging System and analysed with Image Lab software (Bio-Rad), or using an iBright FL1500 and analysed with iBright Analysis software (Thermo Fisher Scientific).

Densitometry analyses and calculation of fraction dual-cleaved
Gel images stained with SYBR gold were analysed using the Image Lab software. The adjusted volume intensity was measured for the full-length band, the partially cleaved fragments (C+D, P+C), and the dual-cleaved product (C). To account for fragment size, each measured intensity was normalised to the corresponding fragment length. The fraction of ribozyme dual-cleaved was then calculated as the ratio of the normalised intensity of the dual-cleaved fragment to the total normalised intensity of all fragments (full length (FL), the partially cleaved fragments (C+D, P+C), and the dual-cleaved fragment (C)).

Northern blotting
RNA was transferred onto a Hybond-N+ membrane (#RPN303B, GE Healthcare, USA) using the Transblot SD semi-dry transfer cell (Bio-Rad), in 1× TBE at 10 V and 300 mA for 1 h at 4 °C. The membrane was cross-linked using a UV crosslinker (Analytik Jena, USA), and pre-hybridised in PerfectHyb Plus Hybridisation Buffer (#H7033-1L, Merck, USA) at 45 °C for 5 min with rotation in a hybridisation oven. 5′ Alexa Fluor 647- or Cy5-labelled DNA probe (Integrated DNA Technologies, see Sequence Listing in Supplementary Data 1) against the cleavage product was added to the pre-hybridisation solution and further incubated at 45 °C for 3 h with rotation. Following hybridisation, membranes were washed progressively with increasing stringency of wash buffer (low stringency: 2X SSC, 0.1% SDS; high stringency: 0.5X SSC, 0.1% SDS; ultra-high stringency: 0.1X SSC, 0.1% SDS). All washing steps were performed at 45 °C in a hybridisation oven with 20 min rotation (except for low stringency wash, which was performed at room temperature with 5 min rotation). Membranes were visualised using a ChemiDoc MP Imaging System and analysed with Image Lab software (Bio-Rad), or using an iBright FL1500 and analysed with iBright Analysis software (Thermo Fisher Scientific).

Broccoli sensor experiments
Cloning and digest of template plasmid for IVT. The DNA gene fragment (Twist Bioscience) containing the T7 promoter sequence followed by the ribozyme sequence for T-E-gene_Cl-Broccoli was amplified with the Phusion High Fidelity PCR Master Mix with HF Buffer (#F531L, Thermo Fisher Scientific) using primers SK63 (5′-TAAGCATCTAGACACCTGCCGGATGAAGTATAATACGACTCACTATAGGGA-3′) and SK64 (5′-TGCTTAACCGGTCACCTGCTCCGTTCGGAAGAGTCGTGTTT-3′) to include XbaI_PaqCI and PaqCI_BshTI sites on each DNA end, respectively (Integrated DNA Technologies). The PCR product was purified with the FavorPrep™ GEL/PCR Purification Kit (#FAGCK 001-1, Favorgen, Taiwan) according to the manufacturer’s protocol. The amplicon and the plasmid backbone, pcDNA3.3_eGFP (Addgene plasmid #26822; http://n2t.net/addgene:26822; RRID:Addgene_26822), were digested with FD XbaI and FD BshTI (#FD0684 and #FD1464, Thermo Fisher Scientific) before being gel-isolated with the FavorPrep™ GEL/PCR Purification Kit. Digested backbone and insert were ligated with T4 DNA Ligase (#M0202L, New England Biolabs) and transformed to One Shot TOP10 Chemically Competent cells (#C404006, Invitrogen). Plasmids from positive transformants were prepared with a QIAprep Spin Miniprep Kit (#27106, Qiagen) and verified with Sanger sequencing (Bio Basic Asia Pacific, Singapore). To obtain the linearised DNA template for IVT, the sequence-verified plasmid was digested with PaqCI (#R0745L, New England Biolabs, USA) and gel-isolated with the FavorPrep™ GEL/PCR Purification Kit.

Ribozyme cleavage reaction and DFHBI-1T-based fluorescence reading
T-E-gene_Cl-Broccoli ribozyme was synthesised through in vitro transcription using AmpliScribe T7-Flash transcription kit with either plasmid-derived linearised DNA template or DNA gene fragment (Twist Bioscience) before purification with a Monarch® Spin RNA Cleanup Kit (#T2040L, New England Biolabs). Cell-free cleavage assays were performed with 1 or 2.5 μM of ribozyme RNA and varying amount of trigger RNA (5′-UUCGGAAGAGACAGGUACGU-3′; Integrated DNA Technologies) in 1× cleavage buffer (10 mM Tris, 7 mM MgCl2, 5 mM spermine, 2 mM sodium chloride, pH 7.0). To this end, IVT RNA prepared using DNA gene fragment was used for this experiment with 2.5 μM ribozyme, and IVT RNA prepared using template from digested plasmid was used for experiments with 1 μM ribozyme. The reaction was incubated at 37 °C for 4 h, and the cleavage product was then purified with the Monarch® Spin RNA Cleanup Kit and eluted in 20 μL of RNase-free water.

For DFHBI-1T-based fluorescence reading, 16.25 μL of either RNase-free water or purified cleavage product was mixed to obtain a final reading solution with 10 μM DFHBI-1T (unless otherwise stated; Lucerna Technologies, USA), 40 mM HEPES pH 7.4, 125 mM KCl and 1 mM MgCl2. Reactions were loaded into a 384-well polypropylene F-bottom plate (#781209, Greiner Bio-One, Germany) and incubated at 37 °C for 1 h. Fluorescence signal was recorded using a BioTek Synergy Neo2 fluorescence microplate reader (Agilent, USA) with excitation wavelength 460/10 nm, emission wavelength 501/10 nm, and extended dynamic range. Experiments were carried out in duplicate for each data point, and fluorescence fold change was reported as the ratio between absolute fluorescence signal from the sample over the mean of absolute fluorescence signal from the no-trigger cleavage product.

In-gel DFHBI-1T staining
After RNA samples were separated on a 10% denaturing polyacrylamide gel, the gels were washed in RNase-free water thrice for 5 min each. Gels were subsequently soaked with a staining buffer containing 10 μM DFHBI-1T, 40 mM HEPES pH 7.4, 100 mM KCl and 1 mM MgCl2 in the dark for 30 min. Gels were then visualised using a ChemiDoc Imaging System under the AF488 channel. Before a subsequent SYBR Gold staining, gels were washed in RNase-free water thrice for 5 min each.

Zebrafish micro-injection and genomic extraction
Wild-type, heterozygous and homozygous foxj1b:GFP (T2BGSZ10 Gt(foxj1b:GFP)) transgenic zebrafish were maintained under standard conditions of fish husbandry. All experiments with zebrafish embryos were approved by the Singapore National Advisory Committee on Laboratory Animal Research (Protocol number: 221702).

Ribozyme RNA for injection into fish was in vitro transcribed from T7 promoter-driven DNA templates using MEGAscript T7 Transciption Kit (#AM1334, Thermo Fisher Scientific, USA). Ribonucleoprotein mixture (RNP) containing 300 ng/μL of in vitro transcribed ribozyme RNA and 0.5 μg/μL of Cas9 protein (#TGEN_CP2, ToolGen/nSAGE Inc., South Korea) was incubated at 37 °C for 10 min and allowed to cool to room temperature. Each embryo was injected with 0.75 nL of RNP mixture (~0.22 ng or 2.5 fmol of ribozyme per embryo). hsa-let-7f (mA/ZEN/mA mCmUmA mUmAmC mAmAmU mCmUmA mCmUmA mCmCmU mC/3ZEN/) or negative control (NC1) morpholino (Integrated DNA Technologies) were introduced into the RNP mix at a final concentration of 8 μM.

Genomic DNA was extracted from healthy single embryos 24 h after micro-injection. Each embryo was first rinsed with 1× PBS (#10010023, Gibco, USA), followed by addition of 20 μL alkaline lysis solution (25 mM sodium hydroxide, 0.2 mM EDTA) and incubation at 95 °C for 30 min. The tubes were vortexed to check for complete lysis of embryos and then incubated at 95 °C for another 10 min. Twenty microliters of neutralisation buffer (40 mM Tris-HCl, pH 8.0) was added to each tube.

miRNA inhibitor
hsa-let-7f inhibitor (mA/ZEN/mA mCmUmA mUmAmC mAmAmU mCmUmAmCmUmAmCmCmUmC/3ZEN/) and control inhibitor NC1 (mG/ZEN/mCmGmUmAmUmUmAmUmAmGmCmCmGmAmUmUmAmAmCmG/3ZEN/) were purchased from Integrated DNA Technologies.

DNA fragment analysis of genome editing
Two microliters of extracted genomic DNA from fish was used as the template for PCR amplification of GFP using primers SAW907_GFPF2 (5′-GTGGTGCCCATCCTGGTC-3′) and SAW908_GFPR2 (5′-CTTGTAGCAGCTCGTCCATGC-3′). The GFP band was gel-isolated (Zymoclean Gel DNA Recovery kit #D4002, Zymo Research, USA) and used as the template for a second PCR using fluorophore-labelled primers (FW+ M13 adaptor (5′-TGTAAAACGACGGCCAGTACGTAAACGGCCACAAGT-3′) and RV (5′-TGAAGAAGATGGTGCGCTC-3′)). Capillary electrophoresis was run on an ABI 3730xl DNA Analyzer (Applied Biosystems, USA). The fluorescently labelled DNA fragments were sized by comparison to a size standard (#4322682, Applied Biosystems). Data analysis was performed using GeneMapper (Applied Biosystems).

Maintenance of cell cultures
HEK293T (cell line received from Yan Chuan’s lab, IMCB) and HEK293 Stoplight+ SpCas9+ (cell line received from Pieter Vader's lab, University Medical Center Utrecht) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM/High Glucose) with L-glutamine (#SH30022.01, HyClone) supplemented with 10% heat-inactivated foetal bovine serum (#10270106, Gibco) and 1% penicillin/streptomycin (#15140122, Gibco). For the HEK293 Stoplight+ SpCas9+ cells, the medium was further supplemented with 1000 μg/mL G418 (#10131027, Thermo Fisher Scientific) and 5 μg/mL Blasticidin S HCl (#A1113903, Thermo Fisher Scientific).

Transfection of ribozymes
For Fig. 5C, RNAs were first synthesized through in vitro transcription using AmpliScribe T7-Flash transcription kit with DNA gene fragment (Twist Bioscience) before purification with Monarch® Spin RNA Cleanup Kit (#T2040L, New England Biolabs). Cell-free cleavage assays were performed in 1x cleavage buffer (10 mM Tris, 7 mM MgCl2, 5 mM spermine, 2 mM sodium chloride, pH 7.0). The reaction was incubated at 37 °C for 4 h, and the cleavage product was then purified with the Monarch® Spin RNA Cleanup Kit and eluted in 30 μL of RNase-free water. HEK293T Stoplight+ SpCas9+ cells were seeded at a density of 2×104 cells per well in a 96-well culture plate. After 24 h post-seeding, 3 pmol equivalent amount of ribozyme RNA was transfected using 3 μL of LipofectamineTM RNAiMAX Transfection Reagent (#13778150, Invitrogen) in Opti-MEMTM I Reduced Serum Medium (#31985070, Gibco) following the manufacturer’s protocol. After 48 h post-transfection, cells were collected and prepared for flow analysis.

For Fig. 5D, HEK293T Stoplight+ SpCas9+ cells were similarly seeded and after 24 h post-seeding, 3 pmol of ribozyme RNA and 3 pmol of trigger RNA were transfected using 3 μL of LipofectamineTM RNAiMAX Transfection Reagent in Opti-MEMTM I Reduced Serum Medium following the manufacturer’s protocol. After 24 h post-transfection, the medium was removed, and replaced with HEK293 Stoplight+ SpCas9+ maintenance culture media without foetal bovine serum (#10270106, Gibco), but with spermine supplementation at 800 uM. After 48 h of spermine treatment, cells were collected and prepared for flow analysis.

For Supplementary Fig. 5J, HEK293T cells were seeded at a density of 1×105 cells per well in 12-well culture plate. After 24 h, 30 pmol of ribozyme RNA and 30 pmol of the trigger RNA were transfected using 3 μL of LipofectamineTM RNAiMAX Transfection Reagent (#13778150, Invitrogen) in Opti-MEMTM I Reduced Serum Medium (#31985070, Gibco) following the manufacturer’s protocol. At 24 h post-transfection, the medium was replaced with fresh medium either without or with 800 uM spermine supplementation. Cells without spermine were harvested at 24 h, 48 h and 72 h post-transfection, whereas spermine-treated cells were collected at 2 h, 24 h and 48 h after spermine addition. Total RNA was extracted using Trizol (#15596018, Invitrogen) according to the manufacturer’s protocol. To detect ribozyme cleavage in the cells, 10 μg of the extracted total RNA was loaded onto a 10% denaturing gel, and as a control, 8 μl of the 24-h transfection mix only sample was also loaded and northern blotting was performed using a sequence-specific fluorescent probe.

For Supplementary Fig. 5L, the sgRNA-embedded catalytic mutant UNBAR and the sgRNA cleavage product (CsgRNA) were cloned under the pLKO.1 puro construct driven by U6 promoter (Addgene plasmid # 8453; http://n2t.net/addgene:8453; RRID:Addgene_8453) and transfected into HEK293T cells using Lipofectamine 3000 (#L3000015, Invitrogen). 1.5×105 cells per well were seeded into a 12-well culture plate. After 24 h, 1 μg of plasmid expressing UNBAR was transfected along with 30 pmol of specific trigger RNA /52MOErT/i2MOErG/i2MOErA/rGrGrUrArGrUrArGrArUrUrGrUrArUrA/i2MOErG/i2MOErT/32MOErT/ (Integrated DNA Technologies, USA). 1 μg of U6-driven CsgRNA was also transfected.. Forty-eight hours post-transfection, total RNA was isolated using Trizol (#15596018, Invitrogen) according to the manufacturer’s protocol. Twelve micrograms of the extracted total RNA was loaded onto each lane in the 10% denaturing gel and northern blotting was done using specific fluorescent probe.

Flow cytometry
Cells in 96-well plates were dislodged from the well with 0.05% Trypsin-EDTA (1X), phenol red (#25300054, Gibco) and neutralised with complete media. The entire volume from each well was transferred to a U-bottom 96-well plate. The plate was spun down at 300×g for 4 min at 4 °C, and the supernatant was discarded. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain (Blue, Invitrogen, L34961 was used for HEK293T Stoplight+ spCas9+ cells) diluted at 1:1000 in 1× PBS (#10010023, Gibco, USA) for 15 min at room temperature. After staining, the plate was spun down at 300×g for 4 min at 4 °C, and the supernatant was discarded. Cells were re-suspended in 100 μL 1× PBS (#10010023, Gibco) per well and analysed on a BD FACSymphony™ A5 flow cytometer. A figure exemplifying the gating strategy is provided in Supplementary Fig. 6. Briefly, cell debris was first gated out, and cells were selected using SSC and FSC. Then, single cells were selected by plotting FSC-A vs. FSC-H. Cells were stained with LIVE/DEAD Fixable Dead Cell Stain (Blue, Invitrogen, L34961) for HEK293T Stoplight+ spCas9+ cells. Therefore, viable cells were selected by plotting FSC-A vs. UV9-A plot for (high signal excluded). For HEK293T Stoplight+ spCas9+ cells, mCherry signal was selected using the yellow-green laser (561 nm) and eGFP signal was similarly assessed using blue laser (488 nm). The same gates were used for all samples and conditions within the same experiment. Data analysis was carried out using FlowJo (BD Biosciences, USA).

Confocal imaging
Transfected Stoplight cells were fixed with 4% paraformaldehyde (#15710, Electron Microscopy Sciences, USA) in 1× PBS (#20012-027, Gibco, USA) for 10 min at room temperature and rinsed twice with 1× PBS (#20012-027, Gibco, USA). Following fixation, cells were stained with DAPI (#62248, Thermo Scientific, USA) 1:1000 in 1× PBS (#20012-027, Gibco, USA) for 10 min at room temperature, rinsed with 1× PBS and then mounted in VECTASHIELD (H-1000, Vector Laboratories, USA). Confocal images were acquired using Olympus FV3000RS Upright confocal microscope with a 20× objective. Image annotations were performed using ImageJ.

Data, statistics and reproducibility
All experiments in the main figures were carried out at least twice, with consistent results. Statistical analyses were carried out using Graphpad Prism (GraphPad Software, Inc., CA, US).

For experiments where data from multiple experiments were pooled, the rate of editing was normalised to the control. For Fig. 5E, the editing rate for the cognate trigger + sgRNA or ribozyme sample was normalised to that for the respective control trigger. The experiment was carried out twice and the normalised data from the two experiments were pooled. For the fish experiment (Fig. 5A), the editing rate for each sample was normalised to the median of the ribozyme-only-injected sample for each experiment. The experiment was carried out three times, and the normalised data from the three experiments were pooled.

For statistical analyses with two samples, a non-parametric Mann–Whitney test was used. For experiments with 3 or more samples, non-parametric one-way ANOVA with Dunn’s multiple comparisons test post hoc test was used to determine which were statistically significant outcomes. For the determination of the fraction dual-cleaved, a Friedman test was used to match bands from the same gel. For all others, a Kruskal–Wallis test was used.

Uncropped and unprocessed scans are compiled in the Source data file.

Molecular modelling
Secondary structure prediction and structure ensemble generation. RNA folding experiments were conducted with RNAstructure (version 6.4). Pairing probabilities were calculated from a given UNBAR ribozyme sequence during the initial analysis step. This was achieved by running partition-smp on T-SARS-CoV-2E-gene_Cl-29nt-clvRNA UNBAR ribozyme constructs with varying H2 or H4 sequences in both + and -trigger-bound states. Calculated pairing probabilities were then used to sample 1000 structures from the Boltzmann ensemble with stochastic-smp. Folding free energy changes for each structure within the representative ensemble were calculated with efn2-smp. MFE structures for a given UNBAR construct were calculated using Fold-smp with the -mfe flag on.

Scaling, secondary structure clustering and centroid structure determination. Scaling and clustering procedures used in this study were adapted from Ding et al. Prior to distance calculations and dimensionality reduction, duplicate structures were removed from the ensemble such that N unique structures comprise a separate ‘filtered’ ensemble. Any unique secondary structure I of a given UNBAR ribozyme of length n can be expressed as an n×n matrix of binary base-pairing indicators Iij where Iij = 1 if the ith and jth base are paired and Iij = 0 if the ith location is single stranded. Differences between all secondary structure pairs were used to construct an N×N pairwise distance matrix. The distance between any two pairs of structures can be defined by a distance metric D(I1, I2) = Σ1≤i<j≤n |I1ij - I2ij| where I1 = I1 and I2 = I2. Multidimensional scaling was then performed on the resulting distance matrix using the scikit-learn package (version 1.2.0) to yield the coordinates of two-dimensional embeddings required for clustering analysis and plotting.

K-Means was selected for clustering analyses after comparing the performance of all available algorithms in the scikit-learn.cluster module. To determine the optimal number of clusters for a given set of embedded coordinates, K-Means was run iteratively over a range of 2–20 such that for each iteration, k number of clusters was a value in this range. Final distortion values for each run were calculated with scipy.cdist (version 1.10.0) and used to construct an elbow plot from which the optimal cluster number was determined via the kneedle algorithm (kneed package version 0.8.1). Each centroid structure was selected by calculating the point nearest the cluster centre with the pairwise_distances_argmin_min method from scikit-learn.

Embedding coordinates for each construct were used to calculate bivariate kernel density estimations (KDEs) for both + and -trigger states and plotted as contours. The degree of overlap between + and -trigger KDEs can be used to estimate the probability of finding both states within a given cluster. Volumes for + and -trigger kernels within each cluster, and within the entire structure ensemble, were acquired by calculating the volume of an irregular solid V = 1/2 s2h. Overlapping volume is determined as the sum of minimum volumes between either kernel at each position and reported as probability (P) between 0.0–1.0. Clusters that are a composite of both + and -trigger structures show high values for P while clusters that are predominantly + or -trigger have lower values.

3D modelling of UNBAR ribozymes. The FARFAR2 algorithm was utilised for de novo prediction of a given ribozyme’s global fold. MFE base-pair identities were supplied as secondary structure constraints to guide fragment assembly, and -minimize_rna was passed as an option to ensure high-accuracy modelling solutions.

Molecular dynamic preparation. FARFAR2 output was used to prepare MD simulations of trigger-bound UNBAR ribozymes. The 3D model of T-SARS-CoV-2E-gene_Cl-29nt-clvRNA + trigger is designated as ‘Native’. To produce a ribozyme model that mimics a proximal site-only cleaved state, the scissile phosphate at residue position 70 (equivalent to the canonical G+1 site) was manually removed from the ‘Native’ model and prepared separately as a ‘Proximal Site Cleaved’ model. RNA interactions were modelled using the CHARMM36 force field. All systems were solvated in a 0.1 M sodium chloride and 0.05 M magnesium chloride solution containing approximately 104,000 TIP3 water molecules. Solvation resulted in rectangular box sizes of approximately 14.5×14.5×14.5 nm.

Simulation setup. All systems were equilibrated by performing 10,000 steps of steepest descent minimisation followed by 100 ps NpT ensemble simulations with gradually decreasing position restraints on the RNA heavy atoms. All simulations were performed using GROMACS 2019.3 from Bekker et al. Electrostatic interactions were described using particle mesh Ewald. Van-der-Waals and Ewald cut-offs were set to 1.2 nm. Bonds to hydrogen atoms were constrained with the LINCS algorithm, allowing an integration time step of 2 fs. Temperature was controlled for distinct coupling groups of solvent and solute using separate v-rescale thermostats at 303.15 K, using a coupling constant τ of 1 ps. Anisotropic Berendsen barostat maintained a pressure of 1 atm, using a coupling constant τ of 12 ps. Following equilibration, all systems were simulated for 1,000 ns in the NpT ensemble. Frames were saved every 50 ps, yielding a single, continuous trajectory of 20,000 frames for each system.

Simulation analysis. All trajectories were corrected for periodicity and aligned to the reference structure using trjconv from the GROMACS 2019.3 package with the -fit and -rot+trans flags on. Phosphate backbone root-mean-square deviation was then calculated with rms and used to determine the time point at which each system reached equilibrium. Applying this time as a starting point, the conformational change of Stem B residues was evaluated with mdmat, and the resulting pair-wise distance matrix indicated which residue pairs undergo the largest change in distance over the course of the simulation. The pair of residues identified (positions 15 and 164) was further evaluated with dist to observe Stem B stability as a function of ribozyme cleavage. Three-dimensional representations were prepared with the PyMOL molecular visualisation program.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript of Lim, Tan, and Subhramanyam describes a novel ribozyme structure adapted from the hairpin ribozyme that can mediate a double cleavage upon binding of a trigger RNA. They use this property to release non-coding RNA effectors such as sgRNAs and aptamers from a single ribozyme structure.\nThe manuscript is very detailed and provides compelling evidence for their claims. I acknowledge that this new molecular tool could benefit many fields of research and improve diagnosis. However, I believe that it could be further improved by addressing the following:\n- The authors appear to have missed some works that detail the release of sgRNAs upon ribozyme cleavage, such as PMID 24373158 or PMID 27832146 (which also features zebrafish work). I acknowledge that their method is more straightforward, as they use a single conditional ribozyme. However, it is not clear if their ribozyme can also be genetically encoded in a transgene. This would greatly improve the range of applications for their new ribozyme in vivo.\n-Beyond the release of the cleavage product, discussion about differences in partial cleavage is often omitted and could impact the action of the tool (extension of the sgRNA spacer for example, or application in prime editing, where the template is on the tail of the sgRNA).\n-Beyond the understanding of the ribozyme action, it is not clear if the modeling (Fig. 3) also provides clues to further improve the ribozyme sequence.\n-When engineering the ribozyme to include sgRNA, shRNA, and aptamers, it is not clear what are the impacts of the 5’ and 3’ ends of the cleavage products. Additional explanations would be required.\n-Representative images of the eGFP knock-out in zebrafish and the Stoplight conversion in cells should be provided.\n-In Fig 5B, it is not clear what the mutations have changed for future experiments in the text.\n-In fig 5C, the use of the mutant ribozyme makes a good case about the release-dependent action of the sgRNA. It would be necessary to use the same control in the zebrafish experiments.\n-It would be useful to assess whether the engineered ribozyme can be placed in arrays. Particularly for the multiplexing of sgRNA or shRNA targeting.\n-In the zebrafish experiments, it would be useful to detail which kind of template was used for the in vitro synthesis. Moreover, details about the efficiency of let-7f morpholino could be added (if splice morpholino, a RT-PCR can be performed).\n-Last, the figures are quite crowded and it is sometimes difficult to understand when figure items start/end. Adding some spacing or moving some items to the supplementary would help.", "reviewer_2": "In this study, Lim et al. developed a ribozyme, termed UNBAR, exhibiting self-cleaving activity that depends upon binding to a single-stranded RNA (ssRNA) trigger. This cleavage results in the release of an embedded short RNA molecule. To engineer an RNA device with dual cleavage sites, the authors tandemly assembled two hairpin ribozymes (HpRzs). Unexpectedly, removal of one ribozyme still yielded a construct capable of dual cleavage activity. The inducibility of the ribozyme’s activity was conferred by conditional formation of a stable stem structure (H4), which was designed to be dependent on the interaction with the trigger RNA.\nThe RNA device was thoroughly optimized to maximize signal-to-noise ratio, resulting in a ribozyme construct with strong trigger-dependent activity and minimal background (trigger-independent) cleavage. UNBAR ribozymes are modular: both the sensor domain (responsible for binding the trigger RNA) and the embedded RNA payload can be replaced, allowing the system to respond to a range of RNA triggers and to release functional RNA molecules with different biological roles, such as sgRNAs or fluorescent aptamers (e.g., red Broccoli).\nThis type of modular, inducible ribozyme that conditionally releases a functional non-coding RNA molecule represents a compelling synthetic biology tool with promising applications in microbial and biomedical biotechnology. The extensive in vitro optimization yielded ribozymes that qualitatively perform in cleavage reactions under cell-free conditions. However, the evidence supporting the functionality of these ribozymes in cellular environments remains limited and requires further development to be fully convincing.\nDespite the commendable and substantial effort put into the in vitro optimization, the analysis is somewhat limited in terms of quantitative rigor. Most of the evidence is presented in the form of gel images or northern blots, with little to no quantification, making it difficult to assess reproducibility and statistical significance. Additionally, the in vitro part of the ms is quite stuffed and the storyline is at times zigzagging, which hampers the extraction of key conclusions. For example, essential controls are sometimes missing in initial experiments and only introduced later (e.g., non-cleaving HpRz mutants are not included in Figures S1 and 1 but appear later as of Fig.2; the same applies for specificity controls using an unrelated trigger); certain figure panels are not referenced in the main text (e.g., Figures S4 Aii and Aiii), and others are cited out of sequence (e.g., lines 416–417). On the other hand, the experiments in cultured mammalian cells are not clarifying: the evidence is limited and the experimental design complicate the interpretation of the results (see below in specific comments).\nThus, although the results are promising, I do not think the manuscript is acceptable for publication in Nature Communications. This assessment could be reconsidered if an extensive revision of the manuscript were carried out in accordance with the recommendations outlined below. I acknowledge that such an profound revision would effectively result in a substantially different manuscript.\nRecommendations:\n1. Streamline the in vitro results by focusing on the most critical experiments, moving the rest to supplementary materials, and incorporating quantitative analyses with appropriate statistical validation.\n2. Strengthen the section of the manuscript concerning experiments in cultured cells. To unambiguously demonstrate the functionality of UNBAR in a cellular context, it is essential to first show that changes in the functional output can be unequivocally attributed to alterations in ribozyme activity induced by the trigger RNA. To this end, appropriate negative controls should be included for each component of the RNA device: an unrelated trigger RNA, a catalytically inactive ribozyme, and a non-functional RNA output. Furthermore, to establish the relevance, modularity, and transferability of the system, it would be desirable to present these results using at least two different ribozymes and in two distinct model systems with biotechnological relevance—namely, bacteria and mammalian cells.\n________________________________________\nSpecific Comments:\nFigures:\nIntroduce a consistent nomenclature for cleavage products across all ribozymes. Given that product lengths vary between constructs, a name is more intuitive and informative than a number.\nSection: RNA-triggered cleavage in cell-free assays (Lines 189–203):\nA specificity control is missing. Does the ribozyme self-cleave in the presence of an unrelated or non-binding RNA under your assay conditions? Quantification is also needed for Figures 1D, 2A, 2C, and S2A.\nStatistical analysis is missing for all in vitro cleavage results shown throughout the ms, and only for a few of them data quantification was shown (Fig. S1A and D, Fig. 2F-G, Fig. S2C and Fig. 4J). Conclusions should be based on quantitative and reproducible results.\nSignal-to-noise ratio optimization (Lines 231–239; Figure 2D):\nOptimization was carried out using the T-E56gene_Cl-29nt-clvRNA construct. Is this enhancement generalizable to other UNBAR variants with different trigger RNAs? Demonstrating improved signal-to-noise ratios across various triggers would better support claims of modularity.\nSignal amplification (Lines 255–272; Figures 2F–G):\nThe observed linear relationship between trigger RNA concentration and ribozyme activity does not demonstrate true signal amplification (i.e., one trigger molecule catalyzing the cleavage of multiple ribozymes). To support this claim, quantify both trigger RNA and cleaved products and show that product amounts exceed the trigger RNA concentration.\nLines 297, 412–423, 533, 538:\nGiven the lack of evidence for signal amplification, this claim should be removed or rephrased to reflect current data.\nModeling of ribozymes:\nWhile structurally interesting, the modeling did not contribute meaningfully to the subsequent experimental design or analysis. The in silico predictions would have been more valuable if they had informed the rational design that guided in vitro optimization. Moreover, the modeling results are not referenced in the discussion.\nLines 437–438:\nThe statement \"embedding a sgRNA within the ribozyme can reduce its editing functionality\" should also consider that the amount of sgRNA released may be insufficient to achieve an extent of editing comparable to that obtained through direct sgRNA delivery. An appropriate positive control for this experiment would be to inject exactly the same RNA molecule that is expected to be released from the ribozyme.\nLines 426–445; Figure 5A:\nThe results show a trend without an statistical analysis that endorses the conclusion. The dispersion of the data and the unclear differences might likely be due to the complexity of the experimental system: triggering cleaving activity in vivo with a highly-expressed miRNA. A clearer, although less physiological, approach might involve using a let-7 knockout background and testing cleavage upon introduction of let-7f versus an unrelated miRNA. Alternatively, ectopic expression of let-7f under an inducible promoter in mammalian cells would provide a more controlled setting. Logically, cellular experiments should precede more complex systems in the manuscript.\nFigure 5C–D:\nThe co-transfection setup raises concerns about extracellular ribozyme activation. Cellular experiments should ensure cleavage occurs strictly intracellularly. Consider using stable cell lines expressing both T-E-gene and a catalytically inactive mutant (e.g., A38) and transfecting them either with plasmids expressing the E-gene or the let-7f (as control) triggers under an inducible promoter. This allows to assess the sgRNA-mediated gene editing before and after induction. Include additional control ribozymes, with embedded mutant sgRNAs incapable of guiding editing. Moreover, to demonstrate modularity, use a different UNABAR, with distinct sensing capability and functional output.\nIntroduction, Line 60:\nThe list of processes—circular RNA, riboswitches, suppressor tRNAs—might be all ascribed to post-transcriptional regulation and culd be presented under this unified category for clarity.\nSection Title: Trigger-activated self-cleaving tandem ribozymes (Line 107):\nThis section focuses on construct optimization rather than inducible activity via trigger RNA binding. The title should reflect that.\nLine 258:\n\"In RT-qPCR, RNA is first amplified and copied into complementary DNA\" is incorrect. RNA is reverse transcribed to cDNA without amplification. A single RNA molecule cannot participate in multiple cDNA synthesis rounds as the RNA and cDNA remain hybridized."}, "author_response": "Comment 1:\nThe manuscript of Lim, Tan, and Subhramanyam describes a novel ribozyme structure adapted\nfrom the hairpin ribozyme that can mediate a double cleavage upon binding of a trigger RNA.\nThey use this property to release non-coding RNA effectors such as sgRNAs and aptamers\nfrom a single ribozyme structure.\nThe manuscript is very detailed and provides compelling evidence for their claims. I\nacknowledge that this new molecular tool could benefit many fields of research and improve\ndiagnosis. However, I believe that it could be further improved by addressing the following:\nResponse 1:\nWe sincerely thank the reviewer for their kind feedback and constructive suggestions to improve\nour manuscript. We agree with the important points that they have highlighted, and have revised\nour manuscript with suggested additional experiments and explanations to improve its quality.\nComment 2:\n- The authors appear to have missed some works that detail the release of sgRNAs upon\nribozyme cleavage, such as PMID 24373158 or PMID 27832146 (which also features zebrafish\nwork). I acknowledge that their method is more straightforward, as they use a single conditional\nribozyme.\nResponse 2:\nThank you for bringing these studies to our attention. PMID 24373158 and PMID 27832146\ndescribe the use of autocatalytic ribozymes to precisely trim guide RNAs when expressed from\nDNA constructs. This is an important use of ribozymes, and we now describe and cite these\nstudies in Lines 80-81,as follows:\n“Auto-catalytic ribozymes have been used to precisely trim guide RNAs for CRISPR-Cas9\nediting26, 27”\nComment 3:\nHowever, it is not clear if their ribozyme can also be genetically encoded in a transgene. This\nwould greatly improve the range of applications for their new ribozyme in vivo.\nResponse 3:\nThank you for this important question. In response, we tried to express the sgRNA-embedded\nUNBAR ribozymes under a U6 promoter, but observed that the ribozymes exhibited ~5x lower\nexpression compared to sgRNA expressed from the same construct (Response Figure 1),\nsuggesting that it was unstable or being degraded. This would be interesting to follow up upon\nin a future study; however, because of this large difference in expression between the sgRNA\nand sgRNA-embedded Rz, it would be very difficult to interpret functional flow results.\nResponse Figure 1: Expression of ribozyme is ~5x lower under U6 promoter compared to\nsgRNA alone.\nWe have now added the following sentence in Discussion in Lines 675-678:\n“UNBAR ribozyme could also potentially be genetically encoded in a transgene to enable a\nfurther range of applications; this would require study of ribozyme integrity, trigger-dependent\ncleavage and cleavage-dependent function when expressed in this manner.”\nComment 4:\n-Beyond the release of the cleavage product, discussion about differences in partial cleavage is\noften omitted and could impact the action of the tool (extension of the sgRNA spacer for\nexample, or application in prime editing, where the template is on the tail of the sgRNA).\nResponse 4:\nThank you for bringing attention to this important point. Indeed, since UNBAR has two cleavage\nsites, the impact of partial cleavage products should be directly investigated and discussed. In\nresponse, we investigated the effects of partially cleaved UNBAR on gene editing (see\nResponse Figure 2 below, which is now included in Figure 5D and described in Lines 552-559).\nWe also discuss the impact of these data and in Lines 653-663 (see end of this response).\nLines 552-559:\n“To investigate the effects of partially cleaved T-E-gene_Cl-sgRNASTOPLIGHT on gene editing, we\ntranscribed RNA fragments that mimic partial cleavage at either the distal or proximal cleavage\nsite. SgRNA corresponding to dual-cleaved UNBAR (with cleavage scars) exhibited the highest\nediting efficiency (~40%), non-significantly higher than partially distal-cleaved fragment (P+C;\nwith a 5’ overhang), which also robustly activated gene editing (~25%), while partially proximal-\ncleaved fragment (C+D; with a 3’ overhang) had a much lower editing efficacy (~6%; ; p <\n0.01)(Figure 5D).Therefore, partially cleaved T-E-gene_Cl-sgRNASTOPLIGHT ribozymes can also\ncontribute to gene editing signal.”\nResponse Figure 2 and New Figure 5D: Flow cytometry analysis of eGFP-reporter turn-on in\nHEK293T Stoplight+ spCas9+ reporter cells transfected with sgRNASTOPLIGHT dual-cleaved,\npartially distal cleaved or proximally cleaved out of T-Egene_Cl-sgRNASTOPLIGHT. (n = 8 for all\nsamples). Data represents eight biological replicates with error bars showing mean and SEM;\nstatistical significance was assessed by one-way ANOVA (Kruskal Wallis) with Dunn’s multiple\ncomparisons test (** = p < 0.01).\nWe also discuss these data and include their impact in Lines 653-663 as follows:\n“However, the impact of cleavage scars and partially cleaved fragments will impact efficacy and\nselection of UNBAR cleavage product design and applications. In the case of cleavage scars, it\nwould be preferred to screen for cleavage products that function efficiently with 5’GUC and 3’-\nCA cleavage scar overhangs. For partial cleavage, while in the case of the Broccoli-releasing\nribozyme, only the dual-cleaved product exhibited fluorescence (Figure 4I, J), however, for\nsgRNAs, partial cleavage products were able to activate gene editing (Figure 5D). Nonetheless,\nby pairing the flow cytometry results with northern blot analysis, we can confidently attribute\ngene-editing activity to the specific combination of cleaved UNBAR fragments present.\nTherefore, partial cleavage must be considered and tested for each modality. It would be\npreferred to improve engineering of UNBAR to optimize candidates for robust trigger-dependent\ndual-site cleavage.”\nComment 5:\n-Beyond the understanding of the ribozyme action, it is not clear if the modeling (Fig. 3) also\nprovides clues to further improve the ribozyme sequence.\nResponse 5:\nThank you for asking this question and giving us the opportunity to clarify this in the manuscript.\nThe computational modeling presented in our work provides mechanistic insight into the\nconformational dynamics that govern the system’s performance. In particular, the models\nhighlight the finely balanced energetic stability of the H4 helix, which plays a key role in\nachieving the observed signal-to-noise characteristics.\n[Redacted]\nBuilding upon the current study, we are actively using these models to guide the refinement of\nsystem variants and to further explore the interplay between the trigger-binding site and H4\nstructural features. The present study allowed us to design and validate the presented functional\nmodel which will inform next-generation designs with improved performance.\nTo clarify this in the manuscript, we now discuss the following in Lines 633-641:\n“Computational modelling identified principles that underlie optimal ribozyme activation by\ntrigger-binding, including an auto-inhibitory effect by unbound trigger arms that results in low\nleakiness and a large turn-on signal for the best-performing ribozymes. The models we created\naccurately reproduce the behaviour observed experimentally, particularly the sensitivity of\nsignal-to-noise ratio on H4 stability.\n[Redacted]\nComment 6:\n-When engineering the ribozyme to include sgRNA, shRNA, and aptamers, it is not clear what\nare the impacts of the 5’ and 3’ ends of the cleavage products. Additional explanations would be\nrequired.\nResponse 6:\nWe believe that the reviewer is referring to the cleavage scar left behind in the cleavage product\nafter the ribozyme cleaves. Thank you for asking this important question. In response, we\ncarried out experiments to investigate the functional effect of cleavage scars on sgRNA\ncleavage products.\n(See Response Figure 3 below, which is now included in Figure S5H and described in Lines\n525-535). We also discuss the impact of these data in Lines 651-655 (see end of this response).\nLines 525-535:\n“To investigate the effect of cleavage scars on the functional activity of sgRNASTOPLIGHT released\nfrom UNBAR, we measured editing by sgRNASTOPLIGHT with no cleavage scars, compared to the\nsame sgRNA with 5’, 3’ or both 5’ and 3’ cleavage scars, as well as a non-targeting control\nsgRNA (sgRNACTL; Figure S5H)60 with cleavage scars. An sgRNA with a 3′ cleavage scar edited\nas efficiently as the sgRNA with no scar (>70% double-positive cells; p > 0.05), while sgRNAs\nwith a 5′ or both 5’ and 3’ cleavage scars exhibited reduced editing (~40% double-positive cells;\np ~0.05 or p < 0.01 respectively). The non-targeting sgRNACTL did not edit (Figure S5H).\nNonetheless, a ~40% editing activity of sgRNA with both a 5′ and 3’ cleavage scar, still provided\na sufficient dynamic range to study regulation of gene editing by UNBAR ribozymes.”\nResponse Figure 3 and New Figure S5H: Impact of ribozyme cleavage scars on sgRNA activity.\nA 5′ cleavage scar on sgRNASTOPLIGHT reduces gene-editing efficiency, while a 3′ cleavage scar\ndoes not. No editing is observed with non-targeting sgRNA sgRNACTL. Data represent eight\nbiological replicates with error bars showing mean and SEM. Statistical significance was\nassessed by one-way ANOVA (Kruskal Wallis) with Dunn's multiple comparisons test (** = p <\n0.01, ns = p > 0.05).\nWe also discuss the impact of these findings in Lines 651-655:\n“However, the impact of cleavage scars and partially cleaved fragments will impact efficacy and\nselection of UNBAR cleavage product design and applications. In the case of cleavage scars, it\nwould be preferred to screen for cleavage products that function efficiently with 5’GUC and 3’-\nCA cleavage scar overhangs.”\nComment 7:\n-Representative images of the eGFP knock-out in zebrafish and the Stoplight conversion in cells\nshould be provided.\nResponse 7:\nThank you for this helpful suggestion to improve the clarity of our data. We now include these\nimages in Response Figure 4 below, and Supplementary Figures S5B and S5F. We refer to\nthese figures in Lines 490-493, and Lines 578-580 as follows:\nLines 490-493:\n“To allow for functional normalised assessment of sgRNA editing levels, we made use of\nHEK293T-Stoplight+ spCas9+reporter cells (Stoplight cells), which initially express only mCherry\nbut switch to expressing both mCherry and eGFP post-editing with sgRNASTOPLIGHT but not a\nnon-targeting sgRNACTL 60 (Figure S5F).”\nResponse Figure 4 and New Figure S5F. Confocal images of HEK293T Stoplight⁺ SpCas9⁺\ncells transfected with sgRNASTOPLIGHT or a non-targeting sgRNACONTROL, demonstrates CRISPR-\nCas9–mediated editing of the Stoplight reporter locus and permanent eGFP expression\nalongside constitutive mCherry, only by sgRNASTOPLIGHT not the control sgRNA. Scale bar\nrepresents 50 µm.\nLines 461-464:\n“Injection of WT or catalytic mutant T-let-7f-Cl-sgRNA-GFP ribozymes with Cas9 protein into\ntransgenic GFP59 embryos led to strong GFP down-regulation in embryos injected with the WT\nribozyme but not the mutant ribozyme (p < 0.0001; Figures S5B, C), demonstrating that\nribozyme cleavage was required for editing.”\nResponse Figure 5 and New Figure S5H: Representative confocal images showing GFP\nexpression in the kidney tubules of Gt(foxj1b::GFP) zebrafish embryos at 25hpf. The GFP-\npositive cell population is unaffected in uninjected and A38C mutant T-let-7f-Cl-sgRNA-GFP +\nCas9-injected embryos, whereas wild-type T-let-7f-Cl-sgRNA-GFP + Cas9-injected embryos\nshowed strong GFP down-regulation. Scale bar represents 30 µm.\nComment 8:\n-In Fig 5B, it is not clear what the mutations have changed for future experiments in the text.\nResponse 8:\nThank you for highlighting this and we apologise for this oversight. The U39C mutation has\nbeen incorporated into the ribozyme that we study in Figures 5C-E.\nWe now include data that compares the U39C and triple mutants for this ribozyme to explain\nthis clearly, and describe this in Lines 510-514:\n“We incorporated a U39C mutation into a T-E-gene_Cl-sgRNASTOPLIGHT ribozyme to study its\ncleavage and function in human cells. This ribozyme exhibited robust trigger-dependent\ncleavage in cell-free assays (Figure S5Gi) only with cognate and not control trigger (Figure\nS5Gii, iii; p < 0.05), and its corresponding A38C catalytic mutant did not cleave (Figure S5Gii).”\nResponse Figure 6 and new Fig S5Gi: Cleavage assays of T-E-gene_Cl-sgRNASTOPLIGHT\nribozymes with U39C mutation alone or triple mutations (U39C + Dual-A10G + Distal A7G) in\nlow Mg2+/spermine buffer.\nComment 9:\n-In fig 5C, the use of the mutant ribozyme makes a good case about the release-dependent\naction of the sgRNA. It would be necessary to use the same control in the zebrafish\nexperiments.\nResponse 9:\nThank you for this suggestion, as this is indeed an important control. Due to these being very\nlaborious experiments (hundreds of embryos for each construct had to be injected, then\nindividually extracted for DNA, PCR’d for the GFP locus, then sequenced by fragment analysis),\nwe had focused on replicating the let-7 vs control inhibitor experiments, which showed trigger-\ndependent gene regulation, and which we thought was a critical feature of the ribozyme (Fig.\n5A).\nWe seek the reviewer’s understanding that we are unable repeat such extensive fish fragment\nanalysis experiments due to a lack of funding and personnel, as these experiments were done\nin a different lab by a student/co-author who has since graduated. However, what we were able\nto do in response to this suggestion was to inject WT and mutant T-let-7f-Cl-sgRNA-GFP\nribozyme, and score them for GFP fluorescence in the embryo. We found that mutant ribozyme\nwas unable to knockdown GFP when injected in complex with Cas9, while WT ribozyme\nshowed strong GFP down-regulation (Response Figure 7). These data are described in Lines\n461-464 as follows:\n“Injection of WT or catalytic mutant T-let-7f-Cl-sgRNA-GFP ribozymes with Cas9 protein into\ntransgenic GFP59 embryos led to strong GFP down-regulation in embryos injected with the WT\nribozyme but not the mutant ribozyme (p < 0.0001; Figures S5B, C), demonstrating that\nribozyme cleavage was required for editing.”\nResponse Figure 7 and new Figure S5C - Quantification of GFP fluorescence in uninjected (n =\n28), T-let-7f_Cl-sgRNAGFP-WT-injected (n = 31), or T-let-7f_Cl-sgRNAGFP-mutant-injected (n =\n34) Gt(foxj1b::GFP) zebrafish embryos. Statistical significance was assessed using a two-tailed\nbinomial test (P < 0.0001). Wilson/Brown confidence intervals were calculated to compare the\nobserved distribution with the expected distribution, assuming the expected distribution\nresembles that of uninjected and mutant ribozymes.\nComment 10:\n-It would be useful to assess whether the engineered ribozyme can be placed in arrays.\nParticularly for the multiplexing of sgRNA or shRNA targeting.\nResponse 10:\nTo our understanding, this refers to placing multiple different ribozymes consecutively, in a\nhead-to-tail configuration, perhaps separated by RNA processing elements that can separate\nthe ribozymes post-transcription. For sgRNAs, this often involves express multiple sgRNAs from\na single construct, so that Cas9 (or another CRISPR nuclease) can target several sites at once.\nThank you for this thought-provoking suggestion. We did not assess this, but due to the ability of\ntrigger-bound ribozymes to trans-cleave (Fig. 2J), the ribozyme in its current iteration cannot be\nmultiplexed to differentiate between different triggers to release different cleavage products.\nWe now discuss multiplexing of different ribozymes in Lines 618-620 as follows:\n“Amplification occurs at least partly through trans-cleavage, a unique property of ribozymes.\n[Redacted] cannot be multiplexed to differentiate between different triggers to release\ndifferent cleavage products. ”\nComment 11:\n-In the zebrafish experiments, it would be useful to detail which kind of template was used for\nthe in vitro synthesis.\nResponse 11:\nThank you for pointing out this oversight. We now describe the template and in vitro\ntranscription kit used in Lines 885-887, as follows:\n“Ribozyme RNA for injection into fish was in vitro transcribed from T7 promoter-driven DNA\ntemplates using MEGAscript T7 Transciption Kit (#AM1334, Thermo Fisher Scientific, USA). ”\nComment 12:\nMoreover, details about the efficiency of let-7f morpholino could be added (if splice morpholino,\na RT-PCR can be performed).\nResponse 12:\nThank you for suggesting this important control. Morpholinos used in zebrafish injections were\ncommercially designed and synthesized by Integrated DNA Technologies (IDT, USA). In\nresponse, we first carried out Taqman microRNA RT-qPCR to to quantify let-7f levels in\nembryos injected with let-7f inhibitor (dre-let-7f shares the same sequence as hsa-let-7f).\nHowever, let-7f morpholino-treated samples did not show a reduction in let-7f expression\ncompared to controls. We realised that inhibitors do not reduce the mature miRNA’s abundance,\nbut rather block their function. Hence, TaqMan miRNA assays may show little/no change in\nmiRNA Ct values even when inhibition worked.\nWe then carried out RT-qPCR for a reported target of let-7f in zebrafish, lin-41 (Lin et al.,\nhttps://doi.org/10.1093/molbev/msm195). However, we did not see an increase in lin-41 levels,\nbut rather a very slight decrease. Since some miRNAs block translation without mRNA decay,\nwe were unable to fully conclude through this second experiment whether or not the inhibitor\nworked.\nWe agree that it is important to establish that the inhibitor is working within cells, but in light that\nboth of our approaches did not show inhibition, yet did not fully rule it out either, we then asked\nwhether we could establish the function of the inhibitor in cell-free cleavage assays. This\nexperiment is described in Lines 476-481 and Response Figure 8 as follows, and we hope that\nthis demonstrates that the inhibitor is able to strongly inhibit ribozyme cleavage by disrupting let-\n7f trigger binding.\n“In cell-free assays, the inhibitor disrupted trigger-induced cleavage by a trigger-dependent\nribozyme (Figure S5Ei), but did not disrupt cleavage of a non-trigger-dependent (auto-cleaving)\nribozyme with an 8 bp H4 (Figure S5Eii), demonstrating that the inhibitor can compete with the\nribozyme for the trigger, and that it inhibits ribozyme cleavage by trigger-binding rather than by\ndisrupting overall ribozyme structure.”\nResponse Figure 8 and new Figure S5E. Let-7f morpholino inhibitor blocks cleavage by a let-7\ntrigger-dependent T-let-7f_Cl-sgRNAGFP ribozyme with 3bp H4 (i), but not a trigger-\nindependent ribozyme (with 8bp H4)(ii).\nComment 13:\n- Last, the figures are quite crowded and it is sometimes difficult to understand when figure\nitems start/end. Adding some spacing or moving some items to the supplementary would help.\nResponse 13:\nThank you for this suggestion to improve the clarity of the figures. We have now increased the\nspacing around figure panels in all the figures, and also removed parts of Figure 1B and C that\nare not critical nor further referenced or discussed, as well as moved to supplementary\nmaterials:\nPart of Figure 1C to S1D\nPart of FIgure 2H to S2D\nWe hope that this has improved the clarity of the figures.\nReviewer #2 (Remarks to the Author):\nComment 1:\nIn this study, Lim et al. developed a ribozyme, termed UNBAR, exhibiting self-cleaving activity\nthat depends upon binding to a single-stranded RNA (ssRNA) trigger. This cleavage results in\nthe release of an embedded short RNA molecule. To engineer an RNA device with dual\ncleavage sites, the authors tandemly assembled two hairpin ribozymes (HpRzs). Unexpectedly,\nremoval of one ribozyme still yielded a construct capable of dual cleavage activity. The\ninducibility of the ribozyme’s activity was conferred by conditional formation of a stable stem\nstructure (H4), which was designed to be dependent on the interaction with the trigger RNA.\nThe RNA device was thoroughly optimized to maximize signal-to-noise ratio, resulting in a\nribozyme construct with strong trigger-dependent activity and minimal background (trigger-\nindependent) cleavage. UNBAR ribozymes are modular: both the sensor domain (responsible\nfor binding the trigger RNA) and the embedded RNA payload can be replaced, allowing the\nsystem to respond to a range of RNA triggers and to release functional RNA molecules with\ndifferent biological roles, such as sgRNAs or fluorescent aptamers (e.g., red Broccoli).\nThis type of modular, inducible ribozyme that conditionally releases a functional non-coding\nRNA molecule represents a compelling synthetic biology tool with promising applications in\nmicrobial and biomedical biotechnology. The extensive in vitro optimization yielded ribozymes\nthat qualitatively perform in cleavage reactions under cell-free conditions. However, the\nevidence supporting the functionality of these ribozymes in cellular environments remains\nlimited and requires further development to be fully convincing.\nResponse 1:\nWe would like to sincerely thank the reviewer for their detailed feedback and helpful\nsuggestions. We agree with the important points that they have highlighted, and, in response,\nhave carried out additional experiments, which have now been incorporated into the manuscript\nto improve its quality.\nComment 2:\nDespite the commendable and substantial effort put into the in vitro optimization, the analysis is\nsomewhat limited in terms of quantitative rigor. Most of the evidence is presented in the form of\ngel images or northern blots, with little to no quantification, making it difficult to assess\nreproducibility and statistical significance.\nResponse 2:\nWe thank the reviewer for their emphasis on reproducibility. We agree that experimental\nreplicability is important, and the data shown are from experiments that have been carried out at\nleast twice and successfully replicated. We now include the following in Lines 921-922:\n“All experiments were carried out at least twice, with consistent results. Statistical analyses were\ncarried out using Graphpad Prism (GraphPad Software, Inc., Ca, US).”\nWe also repeated the experiments shown in\nFigures 1D, 1D - 3 bp, S1F, 2Cii, 2G, 2H, 4D, 4E, 4F, 4I, 4J, (old figure panels)\nand quantified them. These new data include representative gels and quantification of\nreplicates. We describe and detail all these new experiments and quantitation in Response 17.\nWe selected the experiments most critical for our final interpretations for replication as to repeat\nevery experiment for quantification would significantly extend the revision time while not\nsubstantially improving or altering the conclusions of the study. Hence, we would like to seek\nthe reviewer’s understanding that it was not practicable to repeat every cleavage experiment to\nobtain enough replicates for quantification. We would like to emphasize that the cleavage\nassays are highly replicable as they are simple systems consisting of in vitro transcribed RNA in\nbuffers.\nComment 3:\nAdditionally, the in vitro part of the ms is quite stuffed and the storyline is at times zigzagging,\nwhich hampers the extraction of key conclusions.\nResponse 3:\nThank you for this suggestion to improve the clarity of our manuscript. In response, we have\nstreamlined the data, as detailed in Response 10.\nComment 4:\nFor example, essential controls are sometimes missing in initial experiments and only\nintroduced later (e.g., non-cleaving HpRz mutants are not included in Figures S1 and 1 but\nappear later as of Fig.2;\nResponse 4:\nThank you for pointing out that the initial experiments did not include non-cleaving HpRz\nmutants. We agree that it would be useful to include these controls. In response, we have\nrepeated key initial experiments (Previous Figures S1F and Figure 1D 3bp) as well as other key\nribozymes shown in (Figures 4D, 4E and 5D) with non-cleaving HpRz mutants (A38m mutants),\nand these new data are shown below in Response Figures 1-5 and included in the manuscript.\nOverall, all of the A38 mutants tested exhibited little to no cleavage, and hence we seek the\nreviewer’s understanding that we did not repeat all experiments to include a catalytic mutant.\n4.1. New experiment with non-cleaving A38m mutants for 3bp ribozyme shown in old Figure 1D:\nLines 201-204:\n“A ribozyme with H4 of 3 bp released more cleavage product with the cognate trigger vs. a non-\nspecific control trigger, and its corresponding A38mut catalytic mutant ribozyme did not cleave\n(Figure S1Gi; p < 0.05).”\nResponse Figure 1 and new Figure S1Gi:\nCleavage assay for wild-type and A38U mutant ban-5p-triggered OUT-form tandem ribozymes\nwith 3 bp H4, with specific and non-specific triggers. Sp = Specific trigger; NS = Non specific\ntrigger\n4.2. New experiment with non-cleaving A38m mutant for ribozyme shown in Old Figure S1F\n(new Figure S1H), of dual-cleaving, let-7f-triggered_Cl-29nt ribozyme:\nLines 206-208:\n“A second ribozyme, triggered by miRNA hsa-let-7f, also released cleavage product in a trigger-\ndependent and cleavage-dependent manner (Figure S1H; p < 0.05).”\nResponse Figure 2 and new Figure S1Hi:\nCleavage assay for wild-type and A38U mutant let-7f-triggered OUT-form tandem ribozymes\nwith 3 bp H4, with specific and non-specific triggers. Sp = Specific trigger; NS = Non specific\ntrigger\n4.3. New experiment with non-cleaving A38m mutant for ribozyme shown in Figure 4D (New\nFigure S4Bi)\nLines 406-409:\n“Quantitative analyses showed that no cleavage was observed with non-specific triggers for the\nsgRNA and shRNA ribozymes (Figures S4Bi-ii; p < 0.05) or with their respective non-cleaving\ncatalytic mutants (Figures S4Bi-ii).”\nResponse Figure 3 and new Figure S4Bi:\nCleavage assays for wild-type and A38C mutant T-let-7f_Cl-sgRNAGFP ribozymes with specific\ncognate Let7f or non-specific trigger. Sp = Specific trigger; NS = Non specific trigger.\n4.4. New experiment with non-cleaving A38m mutant for ribozyme shown in Figure 4E (New\nFigure S4Bii)\nLines 406-409:\n“Quantitative analyses showed that no cleavage was observed with non-specific triggers for the\nsgRNA and shRNA ribozymes (Figures S4Bi-ii; p < 0.05) or with their respective non-cleaving\ncatalytic mutants (Figures S4Bi-ii).”\nResponse Figure 4 and new Figure S4Bii:\nCleavage assays for wild-type and A38C mutant T-let7f_Cl-shRNAGFP ribozymes with specific\ncognate Let7f or non-specific triggers. Sp = Specific trigger; NS = Non specific trigger.\n4.5. New experiment with non-cleaving A38m mutant for the T-E-gene_Cl-sgRNASTOPLIGHT\nribozyme (not in previous manuscript - now shown in new supplemental Figure S5Gii):\nLines 510-514:\n“We incorporated a U39C mutation into a T-E-gene_Cl-sgRNASTOPLIGHT ribozyme to study its\ncleavage and function in human cells. This ribozyme exhibited robust trigger-dependent\ncleavage in cell-free assays (Figure S5Gi) only with cognate and not control trigger (Figure\nS5Gii, iii; p < 0.05), and its corresponding A38C catalytic mutant did not cleave (Figure S5Gii).”\nResponse Figure 5 and new New Fig. S5Gii.\nCleavage assays of wild-type and A38C mutant T-E-gene_Cl-sgRNASTOPLIGHT-U39C, with\nspecific cognate E-gene (Sp) or non-specific triggers (NS), in low Mg2+/spermine buffer. Sp =\nSpecific trigger; NS = Non specific trigger.\nComment 5:\nthe same applies for specificity controls using an unrelated trigger;\nResponse 5:\nThank you for highlighting that trigger specificity is an important feature of the ribozyme. While\nwe have demonstrated that the ribozyme is indeed very specific, as it can distinguish between\ndifferent triggers at up to single nucleotide specificity (Fig. 2H), non-specific trigger controls\nwere usually not included (partly due to the high cost of purchasing RNA triggers). However, we\nagree that these are important controls, and hence, in response, we have now repeated\nribozymes in key figures (previous figure labelling) Figures S1G, Figure 1D 3bp, 4D, 4E, new T-\nE-gene_Cl-sgRNASTOPLIGHT-U39C, with a non-specific trigger control.\nThese are described and detailed in Response 15.\nComment 6:\ncertain figure panels are not referenced in the main text (e.g., Figures S4 Aii and Aiii),\nResponse 6:\nThank you for pointing out this omission and we apologise for the oversight. We now reference\nthese figures as follows in Lines 393-395:\n“A previous study found that a non-canonical N in the catalytic loop can be compensated by\n3\nmutation of A in the wild-type cleavage loop to base-pair with N 53 (Figure S4A-i), a finding that\n7 3\nwe replicated (Figure S4A-ii)-.”\nComment 7:\nand others are cited out of sequence (e.g., lines 416–417).\nResponse 7:\nThank you for pointing out this discrepancy. This was due to an error citing Figure 4Eii instead\nof Figure 4Hii. We have now fixed this error as follows in Lines 436-437:\n“To test this, we designed ribozymes triggered by a fragment of the SARS-CoV-2 E-gene, to\nself-cleave and release Broccoli (Figure 4Hii).”\nComment 8:\nOn the other hand, the experiments in cultured mammalian cells are not clarifying: the evidence\nis limited and the experimental design complicate the interpretation of the results (see below in\nspecific comments).\nResponse 8:\nThank you for your specific comments and suggestions to improve the clarity of the experiments\nin cultured mammalian cells. In response, we have carried out further experiments, as detailed\nin Responses 11, 12, 14, 24, 25, 26, 27, 28 and 29.\nComment 9:\nThus, although the results are promising, I do not think the manuscript is acceptable for\npublication in Nature Communications. This assessment could be reconsidered if an extensive\nrevision of the manuscript were carried out in accordance with the recommendations outlined\nbelow. I acknowledge that such an profound revision would effectively result in a substantially\ndifferent manuscript.\nResponse 9:\nThank you again for your detailed comments and suggestions to improve the quality and clarity\nof our manuscript. In response, we have carried out a large number of new experiments and\nanalyses, and extensively revised the manuscript, and we believe that this has vastly improved\nthe quality of the study. We hope that it is now acceptable for publication.\nRecommendations:\nComment 10:\n1. Streamline the in vitro results by focusing on the most critical experiments, moving the rest to\nsupplementary materials, and incorporating quantitative analyses with appropriate statistical\nvalidation.\nResponse 10:\nThank you for this suggestion to improve the experimental flow and clarity. In response, we\nhave:\nRemoved parts of Figure 1B and C referring to one orientation of the ribozyme that is not further\nreferenced or discussed\nMoved to supplementary materials:\nPart of Figure 1C to S1D\nPart of FIgure 2H to S2D\nWe have also incorporated quantitative analyses with statistical validation for the critical\nexperiments shown in 1D, S1F, 2G, 2H, 4D, 4E, 4F, 4I, 4J, (old figure panels),\nquantified them and carried out statistical analyses. These new data include representative gels and\nquantification of replicates, and are now shown below in Response Figures 21 - 31, and\nincluded in the manuscript.\nComment 11:\n2. Strengthen the section of the manuscript concerning experiments in cultured cells. To\nunambiguously demonstrate the functionality of UNBAR in a cellular context, it is essential to\nfirst show that changes in the functional output can be unequivocally attributed to alterations in\nribozyme activity induced by the trigger RNA.\nResponse 11:\nWe agree with the reviewer that the key novel feature of the UNBAR ribozyme is its trigger-\ndependent self-cleaving ribozyme activity, and hence it is essential to show that changes in the\nfunctional output of the cleavage product can be directly attributed to trigger-induced ribozyme\ncleavage in cells. Hence, we have carried out additional experimentation as suggested below in\nResponse 12, to test this in a systematic manner in order to rule out other interpretations.\nComment 12:\nTo this end, appropriate negative controls should be included for each component of the RNA\ndevice: an unrelated trigger RNA, a catalytically inactive ribozyme, and a non-functional RNA\noutput.\nResponse 12 (also in response to Comment 29):\nThank you for this suggestion to improve the robustness of our experiments. While we\npreviously included an unrelated trigger RNA (NC1 control trigger), we now carried out further\nexperiments to include a catalytically inactive ribozyme and a non-functional guide RNA (a non-\ntargeting sgRNA from the original Stoplight reporter paper). These data are shown below in\nResponse Figures 6-8 and are New Figures 5E, S5H and S5K in the manuscript, described in\nthe text as follows:\n12. 1. Inclusion of a catalytically inactive ribozyme:\nLines 592-597:\n“We examined editing by sgRNASTOPLIGHT, wild-type T-E-gene_Cl-sgRNASTOPLIGHT and its\ncorresponding catalytic mutant ribozyme co-transfected with control or cognate E-gene trigger\ninto Stoplight cells using flow cytometry. While E-gene co-transfection had no effect on editing\nby sgRNASTOPLIGHT alone or mutant ribozyme (p > 0.05), its co-transfection with wildtype T-E-\ngene_Cl-sgRNASTOPLIGHT ribozyme caused ~23% increase in editing rates compared to those\nincubated with control trigger (p < 0.01) (Figure 5E).”\nResponse Figure 6 and updated Figure 5E.\nFlow cytometry analysis of sgRNASTOPLIGHT, wildtype T-E-gene_Cl-sgRNASTOPLIGHT and its A38C\ncatalytic mutant, co-delivered in the same transfection mix with control or cognate trigger RNA\n(E-gene). Data were pooled from two experiments, editing rate was normalized to the editing\nrate of each RNA with control trigger in the respective experiment. (n = 13-14 for all samples)\n. Error bars showing mean and SEM. Statistical significance was assessed by non-parametric\none-way ANOVA (Kruskal Wallis) with Dunn’s multiple comparisons test (** = p < 0.01, ns = p >\n0.05).\n12.2. Inclusion of a non-functional guide RNA and its embedding within wildtype and mutant\nribozymes:\nLine 532:\n“The non-targeting sgRNACTL did not edit (Figure S5H).”\nResponse Figure 7 and New Figure S5H.\nImpact of ribozyme cleavage scars on sgRNA activity. A 5′ cleavage scar on sgRNASTOPLIGHT\nreduces gene-editing efficiency, while a 3′ cleavage scar does not. No editing is observed with\nnon-targeting sgRNA sgRNACTL. Data represent eight biological replicates with error bars\nshowing mean and SEM. Statistical significance was assessed by one-way ANOVA (Kruskal\nWallis) with Dunn's multiple comparisons test (** = p < 0.01, ns = p > 0.05).\nLines 597-600:\n“As an additional control, we examined effect of co-incubation of control vs cognate E-gene\ntrigger on wild-type and mutant ribozymes that comprise a non-targeting sgRNACTL 58, and\nobserved no editing (Figure S5K).”\nResponse Figure 8 and New Figure S5K.\nFlow cytometry analysis of HEK293T Stoplight+ spCas9+reporter cells transfected with 5’scar-\nsgRNASTOPLIGHT- 3’scar, 5’scar- sgRNACTL- 3’scar, or wildtype or catalytic A38C mutant T-E-\ngene_Cl-sgRNACTL that releases non-functional sgRNA, co-delivered in the same transfection\nmix with control or cognate trigger RNA (E-gene). Data represent eight biological replicates with\nerror bars showing mean and SEM. The non-targeting guides do not show eGFP-reporter turn\non (no double-positive cells).\nComment 13:\nFurthermore, to establish the relevance, modularity, and transferability of the system, it would\nbe desirable to present these results using at least two different ribozymes and in two distinct\nmodel systems with biotechnological relevance—namely, bacteria and mammalian cells.\nResponse 13:\nThank you for recognising that the UNBAR platform can have potential applications in different\nmodel systems. In our manuscript, we have already shown potential applications in three\ndifferent systems: a cell-free system, an in vivo zebrafish system and in vitro cell culture system:\n1) Cell-free diagnostic sensor with a direct fluorescent readout (Fig. 4H-J);\n2) Trigger-activated sgRNA editing in zebrafish embryos (Fig. 5A)\n3) Trigger-activated sgRNA editing in human cultured cells (Fig. 5C-E).\nWe believe that these strongly demonstrate its modularity and broad applicability:\nWhile it would indeed be very interesting to explore further use of UNBAR in additional,\nbiotechnologically relevant model systems like bacteria, this would require expertise that is not\ncurrently present in our labs and would significantly extend the timeline of this revision. We hope\nthat getting this publication out in front of other researchers with expertise in these different\nmodels would allow for a much more rapid investigation of compatibility in these systems.\n________________________________________\nSpecific Comments:\nFigures:\nComment 14:\nIntroduce a consistent nomenclature for cleavage products across all ribozymes. Given that\nproduct lengths vary between constructs, a name is more intuitive and informative than a\nnumber.\nResponse 14:\nThank you for this astute suggestion to improve the clarity of our ribozyme nomenclature in\norder to simplify interpretation of our cleavage assays. In response, we now introduced a\nconsistent nomenclature for cleavage products across ribozymes, as follows:\n14.1. Consistent nomenclature for tandem ribozymes\nLines 154-159:\n“Such a ribozyme is predicted to produce several different fragments when cleaved (Figure 1C):\nThe S2 strand (S2), the full-length S1 strand (FL), a “left”-cleaved fragment that includes the\ncleavage product (R+C), a “right”-cleaved fragment that includes the cleavage product (C+L), a\n“left”-cleaved fragment without the cleavage product (L), a “right”-cleaved fragment without the\ncleavage product (R), and finally, the dual-cleaved product (C)(Figure 1C). ”\nResponse Figure 9 and New Figure 1C: Nomenclature for each cleaved fragment that can result\nfrom cleavage of tandem ribozymes. Colours are consistent with the example shown in Figure\n1Bv.\n14.2. Consistent nomenclature for the single ribozyme\nLines 223-229:\n“This ribozyme could now be encoded by a single strand of RNA, which, in a cleavage assay,\nwould be predicted to produce: the full-length ribozyme (FL), a proximal-cleaved fragment that\nincludes the cleavage product and distal fragment (C+D) a distal-cleaved fragment that includes\nthe proximal fragment and cleavage product (P+C), a distal-cleaved fragment without the\ncleavage product (D), a proximal-cleaved fragment without the cleavage product (P), and finally,\nthe dual-cleaved product (C)(Figure 2Bii).”\nResponse Figure 10 and New Figure 2Bii: Nomenclature for each cleaved fragment that can\nresult from cleavage of tandem ribozymes. Colours are consistent with the example shown in\nFigure 2Bi.\nWe now include these nomenclature in all the cleavage assay figures, while retaining a cartoon\nof an uncleaved ribozyme annotated with the lengths of the cleavage fragments for reference.\nComment 15:\nSection: RNA-triggered cleavage in cell-free assays (Lines 189–203):\nA specificity control is missing. Does the ribozyme self-cleave in the presence of an unrelated or\nnon-binding RNA under your assay conditions?\nResponse 15 (Also in response to Comment 5):\nThank you for highlighting that trigger specificity is an important feature of the ribozyme. While\nwe have demonstrated that the ribozyme is indeed very specific, as it can distinguish between\ndifferent triggers at up to single nucleotide specificity (Fig. 2H), we did not use a non-specific\ntrigger control for all the experiments due to the cost of these short RNA triggers. However, we\nagree that a non-specific control is more robust than a water-only control, and hence in\nresponse, we have now repeated key figures (Old Figure panels S1F, 1D 3bp, 4D, 4E, 4F, and\nthe ribozyme used in 5E) with a non-specific trigger control, which are shown in Response\nFigures 11-18 below, and have been added into the manuscript. Overall, you can see that the\nnon-specific trigger behaves very similarly to the water-only control, and hence we seek the\nreviewer’s understanding that we did not repeat all experiments to a non-specific trigger.\n15.1. New experiment with a non-specific trigger control for ribozyme shown in previous Figure\nS1F (new Figure S1H):\nLines 206-208:\n“A second ribozyme, triggered by miRNA hsa-let-7f, also released cleavage product in a trigger-\ndependent and cleavage-dependent manner (Figure S1H; p < 0.05).”\nResponse Figure 11 and new Figure S1Hi:\nCleavage assay for wild-type and A38U mutant let-7f-triggered OUT-form tandem ribozymes\nwith 3 bp H4, with specific and non-specific triggers. Sp = Specific trigger; NS = Non specific\ntrigger\n15.2. New experiment with a non-specific trigger control for ribozyme shown in previous Figure\n1D (3bp)(new Figure S1Gi):\nLines 202-204:\n“A ribozyme with H4 of 3 bp released more cleavage product with the cognate trigger vs. a non-\nspecific control trigger, and its corresponding A38mut catalytic mutant ribozyme did not cleave\n(Figure S1Gi; p < 0.05).”\nResponse Figure 12 and new Figure S1Gi.\nCleavage assay for wild-type and A38U mutant ban-5p-triggered OUT-form tandem ribozymes\nwith 3 bp H4, with specific and non-specific triggers. Sp = Specific trigger; NS = Non specific\ntrigger\n15.3. New experiment with a non-specific trigger control for ribozyme shown in previous Figure\n4D (new Figure S4Bi):\nLines 406-409:\n“Quantitative analyses showed that no cleavage was observed with non-specific triggers for the\nsgRNA and shRNA ribozymes (Figures S4Bi-ii; p < 0.05) or with their respective non-cleaving\ncatalytic mutants (Figures S4Bi-ii).”\nResponse Figure 13 and new Figure S4Bi.\nCleavage assays for wild-type and A38C mutant of T-let7f_Cl-sgRNAGFP ribozymes with specific\ncognate let-7f or non-specific triggers. Sp = Specific trigger; NS = Non specific trigger.\n15.4. New experiment with a non-specific trigger control for ribozyme shown in previous Figure\n4E (new Figure S4Bii):\nLines 406-409:\n“Quantitative analyses showed that no cleavage was observed with non-specific triggers for the\nsgRNA and shRNA ribozymes (Figures S4Bi-ii; p < 0.05) or with their respective non-cleaving\ncatalytic mutants (Figures S4Bi-ii).”\nResponse Figure 14 and new Figure S4Bii.\nCleavage assays for wild-type and A38C mutant of T-let7f_Cl-shRNAGFP ribozymes with specific\ncognate let-7f or non-specific triggers. Sp = Specific trigger; NS = Non specific trigger\n15.5. New experiment with a non-specific trigger control for ribozyme shown in previous Figure\n4F (new Figure S4Biii):\nLines 410-411\n“The Red-Broccoli-releasing ribozyme also tended to show increased cleavage with specific\ntrigger compared to non-specific trigger (Figure S4Biii).”\nResponse Figure 15 and new Figure S4Biii.\nCell-free cleavage of T-let7f_Cl-Red Broccoli wild-type ribozyme tested with both specific and\nnon-specific trigger RNAs. Sp = Specific trigger; NS = Non specific trigger\n15.6. New experiment with a non-specific trigger control for ribozyme used in previous Figure\n5D (new Figure S5Gii):\nLines 511-514:\n“This ribozyme exhibited robust trigger-dependent cleavage in cell-free assays (Figure S5Gi)\nonly with cognate and not control trigger (Figure S5Gii, iii; p < 0.05), and its corresponding\nA38C catalytic mutant did not cleave (Figure S5Gii).”\nResponse Figure 15 and new Figure S5Gii.\nCleavage assays of wild-type and A38C mutant T-E-gene_Cl-sgRNASTOPLIGHT-U39C, with\nspecific cognate E-gene (Sp) or non-specific triggers (NS), in low Mg2+/spermine buffer. Sp =\nSpecific trigger; NS = Non specific trigger.\nComment 16:\nQuantification is also needed for Figures 1D, 2A, 2C, and S2A.\nResponse 16:\nWe agree that it is important to quantify these data. We have now added quantification data for\nfigures 1D, 2A and 2Cii (previous panel labels) and these are detailed below.\nWe seek the reviewer’s understanding that we were unable to locate the RNA triggers for Figure\nS2A and they would be too expensive to re-order, and hence were not able to replicate these for\nstatistical analyses. However, these tandem ribozymes mostly dual-cleaved and we were able\nto replicate two of these ribozymes in 2Cii (See Response Figure 18 below)\n16.1. Quantification of Old panel 1D - New Panel 1E\nLines 199-201:\n“In the OUT-form, when H4 was 1-2 bp in length, the ribozyme released little dual-cleaved\nproduct even in the presence of trigger, and substantial S1 remained uncleaved (Figures 1E\nand S1F).”\nResponse Figure 16, and new panel S1F.\nGraph showing the fraction dual-cleaved by the OUT-form of tandem ribozymes when the length\nof H4 was varied from 1 bp to 8 bp. Data represent three biological replicates with error bars\nshowing mean and SEM; No statistical significance was observed among any -/+ Trigger pairs\nwhen analysed using non-parametric one-way ANOVA (Friedman test) with Dunn's multiple\ncomparisons test (p > 0.05). Refers to Figure 1Ei.\n16.2. Quantification of previous panel 2A\nLines 215-218:\n“To our surprise, A U mutation in either the “right” wild-type or the “left” reverse-joined loop B\n38\ndid not substantially reduce release of the cleavage product, and mutation of both was required\nto abolish cleavage (Figures 2A, S2A).”\nResponse Figure 17 and new Figure S2A.\nDensitometry analysis of the fraction dual-cleaved by wildtype and A38U mutant tandem\nribozymes (Fig. 2A) provides evidence that mutation of both Loops B is required to abolish dual-\ncleavage.\n16.3. Quantification of Old panel 2Cii:\nLines 229-234:\n“Single-loop B ribozymes triggered by miRNAs (hsa-mir-451a, dme-mir-184, dme-mir-252, dme-\nmir-263a or hsa-let-7f) or by fragments of the E-gene and Orf1ab transcripts of the SARS-CoV-2\ngenome could all self-cleave at both cleavage sites to release the cleavage product (Figures 2Ci\nand S2Bi); however, dual cleavage appeared to be trigger-independent (Figures 2Cii, p > 0.05;\nand S2Bii).”\nResponse Figure 18 and new panel 2Cii.\nDensitometry analysis of the fraction dual-cleaved by the respective ribozymes in (i) shows no\nsignificant release of cleavage product with trigger. Gel analysis data represents three biological\nreplicates with error bars showing mean and SEM. Statistical significance was assessed by\nnon-parametric one-way ANOVA (Friedman test) with Dunn’s multiple comparisons test (ns =\np > 0.05).\nComment 17:\nStatistical analysis is missing for all in vitro cleavage results shown throughout the ms, and only\nfor a few of them data quantification was shown (Fig. S1A and D, Fig. 2F-G, Fig. S2C and Fig.\n4J). Conclusions should be based on quantitative and reproducible results.\nResponse 17: (also in reply to Comments 2 and 16)\nWe thank the reviewer for their emphasis on reproducibility. We agree that experimental\nreplicability is important, and all the data shown in this study are from experiments that have\nbeen carried out at least twice and successfully replicated.\nWe now include the following in Line 926:\n“All experiments were carried out at least twice, with consistent results.”\nWe also have now repeated key experiments from Figures 1D, S1F, 2G, 2H, 4D, 4E, 4F, 4I, 4J,\n(old figure panels), quantified them and carried out statistical analyses. These new data include\nrepresentative gels and quantification of replicates, and are now shown below in Response\nFigures 19 - 29, and included in the manuscript.\n17.1. Replication and statistical analyses for Old Figure 1D-3bp (updated as Figures 1E and\nS1Gi).\nQuantitative statistics are now described in Lines 202-204 as follows:\n“A ribozyme with H4 of 3 bp released more cleavage product with the cognate trigger vs. a non-\nspecific control trigger, and its corresponding A38mut catalytic mutant ribozyme did not cleave\n(Figure S1Gi; p < 0.05).”\nResponse Figure 19 and new Figure S1Gii:\nDensitometry analysis of the fraction dual-cleaved by wildtype tandem ribozymes in (i) shows\nsignificant release of cleavage product with specific but not non-specific trigger. Data represent\nthree biological replicates with error bars showing mean and SEM. Statistical significance was\nassessed using non-parametric one-way ANOVA (Friedman test) with Dunn's multiple\ncomparisons test (* = p < 0.05).\n17.2. Replication and statistical analyses for Old Figure S1F (updated as Figure S1H).\nQuantitative statistics are now described in Lines 206-208 as follows:\n“A second ribozyme, triggered by miRNA hsa-let-7f, also released cleavage product in a trigger-\ndependent and cleavage-dependent manner (Figure S1H; p < 0.05).”\nResponse Figure 20 and new Figure S1Hii:\nDensitometry analysis of the fraction dual-cleaved by wildtype tandem ribozymes in (i) shows\nsignificant release of cleavage product with specific but not non-specific trigger. Data represent\nthree biological replicates with error bars showing mean and SEM. Statistical significance was\nassessed using non-parametric one-way ANOVA (Friedman test) with Dunn's multiple\ncomparisons test (* = p < 0.05).\n17.3. Replication and statistical analyses for Old Figure 2Cii (Dual cleavage by a single-\nstranded, single-sided ribozyme), is new Figure S2Bii:\nLines 229-234:\n“Single-loop B ribozymes triggered by miRNAs (hsa-mir-451a, dme-mir-184, dme-mir-252, dme-\nmir-263a or hsa-let-7f) or by fragments of the E-gene and Orf1ab transcripts of the SARS-CoV-2\ngenome could all self-cleave at both cleavage sites to release the cleavage product (Figures 2Ci\nand S2Bi); however, dual cleavage appeared to be trigger-independent (Figures 2Cii, p > 0.05;\nand S2Bii).”\nResponse Figure 21 and new Figure S2Bii:\nDensitometry analysis of the fraction dual-cleaved in (i) provides evidence that all ribozymes\nanalysed can self-cleave at dual sites.\n17.4. Replication and statistical analyses for Old Figure 2G (UNBAR amplification)\nLine 287-289:\n“Quantification of the amount of cleavage product vs trigger showed that the ribozyme exhibits\nup to ~7.4-fold amplification at 16-fold more ribozyme than trigger (p < 0.05)(Figure 2G).”\nResponse Figure 22 and updated Figure 2G:\nCleavage assays of T-E-gene_Cl-29nt-clvRNA ribozyme show that cleavage product release\nincreases with increasing concentration of ribozyme, when keeping trigger RNA concentration\nconstant at 50nM. This demonstrates that the original trigger amount has been transduced into\namplified amount of cleavage product. Chart plots the ratio of dual-cleaved product over the\nconstant trigger amount. Data represents three biological replicates with error bars showing\nmean and SEM; statistical significance was assessed by non-parametric one-way ANOVA\n(Friedman test) with Dunn’s multiple comparisons post-hoc test (* = p < 0.05).\n17.5. Replication and statistical analyses for Figure 2H, which describes specificity for the E-\ngene ribozyme when triggered by triggers with single, or dual mutations, or an unrelated RNA:\nLines 294-298:\n“An unrelated RNA (SARS-CoV-2 S-gene fragment) was unable to trigger cleavage product\nrelease by T-E-gene_Cl-29nt-clvRNA (p < 0.01). The effects of single-nucleotide mismatches\ndepended on their locations, with position 4 (from the 5’ end) reducing cleavage product release\nby ~90% (p < 0.05)(Figure 2H).”\nResponse Figure 23 and updated Figure 2H:\nTesting mutant variants of the E-gene trigger RNA against the T-E-gene_Cl-29nt-clvRNA\nribozyme in cleavage assays shows that UNBAR ribozymes can distinguish between unrelated\nand closely related trigger RNAs with 1-3nt differences. Sequences of test triggers are shown in\nFigure S2D. Data represents three biological replicates with error bars showing mean and SEM;\nstatistical significance was assessed by non-parametric one-way ANOVA (Friedman test) (* = p\n< 0.05, ** = p < 0.01).\n17.6. Replication and statistical analyses for Figure 4D, which shows the T-let-7f-Cl-sgRNA\nribozyme:\nLines 404-409:\n“We assayed T-let-7f_Cl-sgRNA, T-let-7f_Cl-shRNA and T-let-7f_Cl-Red-Broccoli for trigger-\nactivated cleavage in cell-free assays, and observed substantially more cleavage product\nreleased in the presence of trigger than without (Figures 4D-4F). Quantitative analyses showed\nthat no cleavage was observed with non-specific triggers for the sgRNA and shRNA riboyzmes\n(Figures S4Bi-ii; p < 0.05) or with their respective non-cleaving catalytic mutants (Figures S4Bi-\nii).”\nResponse Figure 24 and new Figure S4Bi.\nCleavage assays for wild-type and A38C mutant of T-let-7f_Cl-sgRNAGFP ribozyme tested\nribozymes with specific cognate let-7f or non-specific triggers. Sp = Specific trigger; NS = Non\nspecific trigger. Data represents three biological replicates with error bars showing mean and\nSEM. Statistical significance was assessed by non-parametric one-way ANOVA (Friedman test)\nwith Dunn’s multiple comparisons post-hoc test (* = p < 0.05, ns = p > 0.05).\n17.7. Replication and statistical analyses for Figure 4E, which shows the T-let-7f-Cl-shRNA\nribozyme:\nLines 404-409:\n“We assayed T-let-7f_Cl-sgRNA, T-let-7f_Cl-shRNA and T-let-7f_Cl-Red-Broccoli for trigger-\nactivated cleavage in cell-free assays, and observed substantially more cleavage product\nreleased in the presence of trigger than without (Figures 4D-4F). Quantitative analyses showed\nthat no cleavage was observed with non-specific triggers for the sgRNA and shRNA riboyzmes\n(Figures S4Bi-ii; p < 0.05) or with their respective non-cleaving catalytic mutants (Figures S4Bi-\nii).”\nResponse Figure 25 and new Figure S4Bii:\nCleavage assays for wild-type and A38C mutant of T-let-7f_Cl-shRNAGFP ribozymes with\nspecific cognate let-7f or non-specific triggers. Sp = Specific trigger; NS = Non specific trigger.\nData represents three biological replicates with error bars showing mean and SEM. Statistical\nsignificance was assessed by non-parametric one-way ANOVA (Friedman test) with Dunn’s\nmultiple comparisons post-hoc test (* = p < 0.05).\n17.8. Replication and statistical analyses for Old Figure 4F, which shows the T-let-7f-Cl-Red-\nBroccoli ribozyme. This was not statistically significant due to the small sample size but the\neffect size was medium and the result was highly replicable.\nResponse Figure 26 and new Figure S4Biii:\nCleavage assays for wild-type and A38C mutant of T-let-7f_Cl-Red Broccoli wild-type ribozymes\nwith specific cognate let-7f or non-specific triggers. Sp = Specific trigger; NS = Non specific\ntrigger . Data represents three biological replicates with error bars showing mean and SEM.\nStatistical significance was assessed by non-parametric one-way ANOVA (Friedman test) with\nDunn’s multiple comparisons post-hoc test (ns = p > 0.05).\n17.9. Replication and statistical analyses for updated Figure 4I, demonstrating that with\nincreasing trigger concentration, there is increased dual cleavage and release of Broccoli\nLines 437-440:\n“In-gel staining of cleavage assay products showed strong fluorescence only of the dual-cleaved\nBroccoli cleavage product (Figure 4Ii), with increased dual cleavage and release of Broccoli as\ntrigger concentration was increased (Figure 4Iii).”\nResponse Figure 27 and new Figure 4Iii:\nFraction of ribozyme dual-cleaved at increasing trigger concentration fit a hyperbolic (least\nsquares fit) model (R² = 0.96). Data represents three biological replicates with error bars\nshowing mean and SEM.\n17.10. Replication and statistical analyses for Figure 4J showing fluorescence fold change\ndirectly detectable on a plate reader with increasing trigger concentration.\nLines 440-443:\n“Increasing fluorescence with increasing trigger concentration could be detected on a plate\nreader , with turn-on signals of up to ~10-fold (up to 20-fold could potentially be reached with a\nhigher concentration of ribozyme; Figure S4G) and sensitivity of at least 25 nM (Figure 4J).”\nResponse Figure 28 and updated Figure 4J:\nFluorescent microplate readings of purified cleavage assays of T-E-gene_Cl-Broccoli cleaved in\nvarying concentration of trigger allows direct readout of cleaved Broccoli as a function of trigger\nconcentration. Data represents three biological replicates with error bars showing mean and\nSEM and fit a hyperbolic (least squares fit) model (R² = 0.969).\n17.11. Replication and statistical analyses for the T-E-gene_Cl-sgRNA-U39C ribozyme used for\nflow cytometry experiments in Figure 5, is now an additional panel in Figure S5Giii, and\ndescribed in Lines 511-514:\n“This ribozyme exhibited robust trigger-dependent cleavage in cell-free assays (Figure S5Gi)\nonly with cognate and not control trigger (Figure S5Gii, iii; p < 0.05), and its corresponding\nA38C catalytic mutant did not cleave (Figure S5Gii).”\nResponse Figure 29 and new Figure S5Giii:\nDensitometry analysis of the fraction dual-cleaved by the wildtype T-E-gene_Cl-sgRNASTOPLIGHT-\nU39C ribozyme shows significant release of cleavage product with specific but not non-specific\ntrigger. Data represent three biological replicates with error bars showing mean and SEM.\nStatistical significance was assessed by non-parametric one-way ANOVA (Friedman test) with\nDunn's multiple comparisons test (* = p < 0.05).\nComment 18:\nSignal-to-noise ratio optimization (Lines 231–239; Figure 2D):\nOptimization was carried out using the T-Egene_Cl-29nt-clvRNA construct. Is this enhancement\ngeneralizable to other UNBAR variants with different trigger RNAs? Demonstrating improved\nsignal-to-noise ratios across various triggers would better support claims of modularity.\nResponse 18:\nThank you for pointing out that our engineering efforts would be more useful if generalisable\nacross various triggers. Indeed, we had tested the same panel of CMs for two other different\ntrigger RNAs, mir-451a and SARS-CoV-2 S-gene, which are shown in Fig S2C, found that 3bp-v1 and 3bp-v2 also improve ribozymes triggered by these different RNAs, albeit to different\nextents. To highlight this, we added the following elaboration in Lines 248-250:\n“We tested the same panel of CMs for two other different trigger RNAs, mir-451a and SARS-\nCoV-2 S-gene, and found that 3bp-v1 and 3bp-v2 CMs also improve ribozymes triggered by\nthese different RNAs (Figure S2C).”\nComment 19:\nSignal amplification (Lines 255–272; Figures 2F–G):\nThe observed linear relationship between trigger RNA concentration and ribozyme activity does\nnot demonstrate true signal amplification (i.e., one trigger molecule catalyzing the cleavage of\nmultiple ribozymes).\nResponse 19:\nIndeed, the reviewer has made a sharp observation and is correct. In Fig. 2F, we do not\ndemonstrate amplification. The linear relationship shows trigger-dependent turn-on with\nincrease in trigger amount (cleavage product released in presence vs absence of the trigger).\nThis panel shows that the ribozyme can be trigger-dependent across a large dynamic range. To\nmeasure this turn on signal, the ribozyme amount was held constant while trigger amount was\nvaried.\nIn Fig. 2G is where we are looking at signal amplification. Here, the trigger amount is held\nconstant while the ribozyme concentration is gradually increased, and we do compare cleavage\nproduct and trigger amount, as suggested by the reviewer in Comment 20. Please refer to\nResponse 20 where we address this in detail.\nComment 20:\nTo support this claim, quantify both trigger RNA and cleaved products and show that product\namounts exceed the trigger RNA concentration.\nResponse 20:\nThank you for highlighting that a conclusion of ribozyme signal amplification requires\ndemonstration that one trigger molecule can catalyse cleavage product release from multiple\nribozymes. As the reviewer suggests, this is best demonstrated when trigger concentration is\nfixed, and examining the amount of cleavage product released when increasing ribozyme\namount is provided,\nWe have now made it clearer that we have quantified both the trigger RNA and dual-cleaved\nproduct. In Figure 2G and reproduced below in Response Figure 30, you can see in the bottom\nboxed region in the gel that when Trigger amount is kept constant (50nM), the amount of\ncleaved 29-nt cleavage product increases as more ribozyme is added. At the lowest\nconcentration (200nM) of ribozyme and 50nM of trigger, the amount of cleavage product\nreleased is about 1:1. As more ribozyme is added, the amount of cleavage product released\nfrom the self-cleaving ribozyme visibly exceeds the fixed amount of trigger, demonstrating that\nthe original trigger signal has been amplified into released cleavage product that exceeds the\noriginal trigger amount. Quantification of the amount of cleavage product vs trigger shows that\nthe ribozyme exhibits up to 7.4-fold amplification under these parameters.\nResponse Figure 30 and updated Figure 2G. With a fixed amount of trigger (50nM), by adding\nincreasing amounts of ribozyme (200 nM to 1600 nM), increasing amount of cleavage product is\nreleased from the ribozyme, so that the cleavage product amount visibly exceeds trigger\namount by up to 7-fold, when ribozyme concentration reaches more than 800nM. Data\nrepresents three biological replicates with error bars showing mean and SEM; statistical\nsignificance was assessed by non-parametric one-way ANOVA (Friedman test) with Dunn's\nmultiple comparisons test (* = p < 0.05).\nTo improve the clarity of the description of this experiment, we now add the following in Lines\n280-289:\n“We observed that when trigger amount is kept constant (50nM), the amount of cleaved 29-nt\ncleavage product increases as more ribozyme is added (boxed region in Figure 2G). At the\nlowest concentration (200nM) of ribozyme, the amount of cleavage product released relative to\nthe fixed amount of trigger was about 1:1. As more ribozyme is added, the amount of cleavage\nproduct released from the self-cleaving ribozyme gradually exceeds the fixed amount of trigger,\ndemonstrating that the original trigger signal has been amplified into released cleavage product.\nQuantification of the amount of cleavage product vs trigger showed that the ribozyme exhibits\nup to ~7.4-fold amplification at 16-fold more ribozyme than trigger (p < 0.05)(Figure 2G).”\nComment 21:\nLines 297, 412–423, 533, 538:\nGiven the lack of evidence for signal amplification, this claim should be removed or rephrased to\nreflect current data.\nResponse 21:\nWe hope that our clarifications and additional explanations in Response 20 provide clear\nevidence for signal amplification. At a constant trigger concentration, increase in Rz conc leads\nto amplified cleavage product release, which is visible on the gel because there is more\ncleavage product than trigger.\nComment 22:\nModeling of ribozymes:\nWhile structurally interesting, the modeling did not contribute meaningfully to the subsequent\nexperimental design or analysis. The in silico predictions would have been more valuable if they\nhad informed the rational design that guided in vitro optimization. Moreover, the modeling\nresults are not referenced in the discussion.\nResponse 22:\nWe thank the reviewer for their thoughtful comments and the opportunity to clarify the role of\nmodeling in our study. While we acknowledge that the in silico predictions were not explicitly\nused to guide the initial stages of experimental design, the computational modeling presented\nhelped us to understand why the best ribozymes performed well experimentally, by providing\nmechanistic insight into the conformational dynamics that govern the system’s performance.\n[Redacted]\nWe agree with the reviewer that integrating predictive modeling into the initial rational design\nphase can be highly impactful, and we are now doing this as a follow-up study, to further\nexplore the interplay between the trigger-binding site and H4 structural features. This will allow\nus to design and validate the functional model presented in this work, which will inform next-\ngeneration designs with improved performance.\nWe also acknowledge the oversight in not referencing the modeling results more explicitly in the\ndiscussion. In response to the reviewer’s feedback, we have revised the discussion section to\nemphasize the interpretative value of the models, particularly in contextualizing the observed\nfunctional outcomes and in shaping our current and future experimental directions. We therefore\nexpanded our discussion of these findings as follows, in Lines 632-639:\n“Computational modelling identified principles that underlie optimal ribozyme activation by\ntrigger-binding, including an auto-inhibitory effect by unbound trigger arms that results in low\nleakiness and a large turn-on signal for the best-performing ribozymes. The models we created\naccurately reproduce the behaviour observed experimentally, particularly the sensitivity of\nsignal-to-noise ratio on H4 stability.\n[Redacted]\nComment 23:\nLines 437–438:\nThe statement \"embedding a sgRNA within the ribozyme can reduce its editing functionality\"\nshould also consider that the amount of sgRNA released may be insufficient to achieve an\nextent of editing comparable to that obtained through direct sgRNA delivery. An appropriate\npositive control for this experiment would be to inject exactly the same RNA molecule that is\nexpected to be released from the ribozyme.\nResponse 23:\nThe reviewer makes a good point that the amount of sgRNA released by the ribozyme in this\nexperiment likely does not reach the level of the free sgRNA alone. In mammalian cell\nexperiments, we have directly compared a mutant ribozyme to free sgRNA and saw that\nembedding the sgRNA within a mutant ribozyme, that cannot be cleaved, led to a 13-fold\nrepression of editing compared to the sgRNA alone (Fig. 5C).\nThe reviewer also makes a good suggestion to express the exact amount of RNA that is\nexpected to be released. We did this in mammalian cells, by extracting ribozymes pre-cleaved\nin cleavage buffer that resembles cellular ionic conditions, which showed the ribozyme was not\nfully cleaved in these conditions, and that the amount of cleavage product released led to an\nincrease in editing compared to mutant ribozyme, but yet much lower editing than the free\nsgRNA.\nWe hope that the above data demonstrate that embedding a sgRNA within the ribozyme can\nreduce its editing functionality. However, we agree with the reviewer that this statement was\nconfusing within its context, and hence, to avoid confusion, we have now removed it, and\nreplaced it with the reviewer’s hypothesis, in the new Lines 468-471 as follows:\n“Injection of equimolar amounts of ribozyme T-let-7f_Cl-sgRNAGFP with Cas9 also led to robust\nediting, although ~50% lower than the sgRNA alone (Figure S5D), which could be due to\nincomplete dual-cleavage of the ribozyme within the embryo.”\nRegarding the suggested addition of the above pre-cleaved ribozyme control into the fish\nexperiments, unfortunately, we seek the reviewer’s understanding that we are unable repeat\nsuch extensive fish experiments due to a lack of funding and personnel, as these experiments\nwere done in a different lab by a student/co-author who has since graduated.\nComment 24:\nLines 426–445; Figure 5A:\nThe results show a trend without an statistical analysis that endorses the conclusion. The\ndispersion of the data and the unclear differences might likely be due to the complexity of the\nexperimental system: triggering cleaving activity in vivo with a highly-expressed miRNA.\nResponse 24:\nThe reviewer is right that this is a complex system, which may underlie the noise, nonetheless,\nthe result is statistically significant (p < 0.0001), and we have now more strongly emphasised\nthe statistics in the text, as follows:\nLines 472-474:\n“Compared to co-injection with a control inhibitor, co-injection of the let-7f inhibitor significantly\nreduced ribozyme-induced editing in the fish (Figure 5A; p < 0.0001)”\nComment 25:\nA clearer, although less physiological, approach might involve using a let-7 knockout\nbackground and testing cleavage upon introduction of let-7f versus an unrelated miRNA.\nAlternatively, ectopic expression of let-7f under an inducible promoter in mammalian cells would\nprovide a more controlled setting.\nResponse 25:\nThank you for this suggestion. Unfortunately there are 11 members of the let-7 microRNA\nfamily, and hence a knockout is not possible to carry out. At the same time, the high levels of\nendogenous let-7 expression in mammalian cells means that over-expression of let-7 would\nlikely have little effect or cause signals that are difficult to differentiate from noise. This type of\nmore complex experimental setup in cells can be explored in future studies with a more\ncontrollable trigger RNA.\nComment 26:\nLogically, cellular experiments should precede more complex systems in the manuscript.\nResponse 26:\nWe appreciate the reviewer’s comment. In our manuscript, we chose to present the zebrafish\nexperiment first, as it was performed with the sgRNA-GFP construct that was already described\nin Figures 4A and 4D. This is followed by data in mammalian cells using optimized sgRNA-\nStoplight UNBAR constructs, which allowed a more systematic interrogation of intracellular\nUNBAR activity. We chose this order of presentation to provide logical progression from Figure\n4 to Figure 5 and to guide the reader clearly through the discussion.\nComment 27:\nFigure 5C–D:\nThe co-transfection setup raises concerns about extracellular ribozyme activation.\nResponse 27:\nThank you for highlighting that it is important to rule out extracellular ribozyme activation. To\ndo this, we first carried out an additional experiment showing that RNA extracted from transfection\nmix containing ribozyme and trigger showed no cleavage of the ribozyme (updated Figure S5J).\nThis is described in Lines 563-564:\n“Northern blotting of the transfection mix sample at 24 hours showed no ribozyme cleavage\n(Figure S5J; lanes 1, 2).”\nPlease also refer to Response 28 to see the referenced Figure S5J, and additional experiments\nand explanations that definitively show that cleavage is occurring strictly intracellularly.\nComment 28:\nCellular experiments should ensure cleavage occurs strictly intracellularly. Consider using\nstable cell lines expressing both T-E-gene and a catalytically inactive mutant (e.g., A38) and\ntransfecting them either with plasmids expressing the E-gene or the let-7f (as control)\ntriggers under an inducible promoter. This allows to assess the sgRNA-mediated gene editing\nbefore and after induction.\nResponse 28:\nThank you for highlighting that it is indeed critical to show that the ribozyme is able to cleave\nwithin the cellular environment. To clarify this, we have carried out an additional time-course\nexperiment in HEK293T cells, which is shown in Response Figure 33, updated Figure S5J and\ndescribed in Lines 570-584 as follows:.\n“HEK293T cells were transfected with T-E-gene_Cl-sgRNASTOPLIGHT ribozyme and either control\nor cognate trigger. After 24 hours, all media and transfection mix was removed from the culture\nwells, so that any ribozyme remaining MUST have entered cells to be protected from the media\nremoval. At this point, we incubated the cells with either normal or spermine-supplemented\nreplacement media. Ribozymes from cells replaced with normal media exhibited limited\ncleavage by 72 hours (Figure S5J, lane 9). In contrast, ribozymes from cells replaced with\nspermine-supplemented media exhibited robust cleavage, with a marked increase in cleavage\nproduct released just two hours after spermine addition, at 26 hours (lane 11), with a further\nincrease at the 48 hour mark (lane 13). Notably, RNA in the transfection mix alone at 24h (lane\n2) did not show detectable cleavage product, in contrast to RNA from spermine-treated cells\ntested just 2 hours later (lane 11). Because spermine was only added after the removal of\ntransfection mix from the culture media, all ribozymes in the well were inside cells before\nspermine was added, and this rules out the possibility of extracellular cleavage from residual\ncomponents of the transfection mix. Hence, this additional cleavage observed post-24 hours\nhad to have occurred within cells.”\nThese results demonstrate that substantial cleavage occurred in cells after media with\ntransfection mix was removed at 24 hours, and did not occur in the transfection mix prior to cell\nentry, and that ribozyme cleavage occurred in cells only with cognate trigger and not control\ntrigger.”\nWith the improved explanation in the text and new experiments showing increase in ribozyme\ncleavage over time that can only be happening within cells, we hope that we have now\ndemonstrated that cleavage unequivocally occurred within cells.\nResponse Figure 31 and updated Figure S5J: Northern blot of total RNA from HEK293T cells\ntransfected with T-E-gene_Cl-sgRNASTOPLIGHT , with control or cognate trigger, where\ntransfection media was removed at 24 hours and replaced with either normal media or\nspermine-supplemented media. Cleavage and release of the embedded sgRNA is not visible in\nthe transfection mix at 24 hours, and increases visibly only with cognate trigger after\ntransfection mix in the media is removed and replaced with spermine-supplemented media\n(lanes 10-15), demonstrating that cleavage occurred within cells.\nThank you also for suggesting an experiment using stable cell lines expressing both T-E-gene\nand a catalytically inactive mutant (e.g., A38 mutant). We tried to express the sgRNA-embedded\nUNBAR ribozymes under a U6 promoter, but observed that the ribozymes exhibited ~5x lower\nexpression compared to sgRNA expressed from the same construct (Response Figure 33),\nsuggesting that it was unstable or being degraded. This would be interesting to follow up upon\nin a future study; however, because of this large difference in expression between the sgRNA\nand sgRNA-embedded Rz, it would be very difficult to interpret functional flow results.\nResponse Figure 32: Expression of ribozyme is ~5x lower under U6 promoter compared to\nsgRNA alone.\nWe have now added the following sentence in Discussion in Lines 672-675:\n“UNBAR ribozyme could also potentially be genetically encoded in a transgene to enable a\nfurther range of applications; this would require study of ribozyme integrity, trigger-dependent\ncleavage and cleavage-dependent function when expressed in this manner.”\nComment 29:\nInclude additional control ribozymes, with embedded mutant sgRNAs incapable of guiding\nediting.\nResponse 29:\nThank you for suggesting this important control to ensure that the observed fluorescent-turn on\nis sgRNASTOPLIGHT-mediated. In response, we have carried out experiments with a non-targeting\nguide and its embedding within wildtype and mutant ribozymes, as detailed in Response 12.\nComment 30:\nMoreover, to demonstrate modularity, use a different UNBAR, with distinct sensing capability\nand functional output.\nResponse 30:\nThank you for suggesting that we use a different UNBAR, with distinct sensing capability and\nfunctional output, to demonstrate modularity of UNBAR.\nIn our manuscript, we have already shown potential applications in three different systems: a\ncell-free system, an in vivo zebrafish system and in vitro human cell culture system:\n1) Cell-free diagnostic sensor with a direct fluorescent readout (Fig. 4H-J);\n2) Trigger-activated sgRNA editing in zebrafish embryos (Fig. 5A)\n3) Trigger-activated sgRNA editing in human cultured cells (Fig. 5C-E).\nWe believe that this strongly demonstrates its modularity and broad applicability and hope that\nthe reviewer agrees.\nComment 31:\nIntroduction, Line 60:\nThe list of processes—circular RNA, riboswitches, suppressor tRNAs—might be all ascribed to\npost-transcriptional regulation and could be presented under this unified category for clarity.\nResponse 31:\nThank you for this helpful suggestion. We have now amended the sentence in Lines 58-63 to\nemphasise that these are all post-transcriptional modes of regulation.\nLines 58-63:\n“RNA is a particularly attractive material for engineering due to its diverse biological functions,\nwhich include post-transcriptional regulation resulting in gene repression (RNAi), gene\nexpression (mRNA, circular RNA), translation activation (riboswitches) and reprogramming of\ntranslation (suppressor tRNA), as well as functioning as programmable guides for RNA and\nDNA effector complexes (CRISPR, ADAR).”\nComment 32:\nSection Title: Trigger-activated self-cleaving tandem ribozymes (Line 107):\nThis section focuses on construct optimization rather than inducible activity via trigger RNA\nbinding. The title should reflect that.\nResponse 32:\nThank you for this suggestion to improve the readability of our manuscript. We now amend this\nsection title in Line 109 to:\n“Engineering trigger-activated tandem ribozymes”\nComment 33:\nLine 258:\n\"In RT-qPCR, RNA is first amplified and copied into complementary DNA\" is incorrect. RNA is\nreverse transcribed to cDNA without amplification. A single RNA molecule cannot participate in\nmultiple cDNA synthesis rounds as the RNA and cDNA remain hybridized.\nResponse 33:\nThank you for highlighting this error in our description of RT-qPCR. We now amend the\nsentence in Line 271-272 to:\n“...in RT-qPCR, RNA is first copied into complementary DNA (cDNA) by enzyme-mediated\nreverse transcription, and the cDNA is subsequently amplified by gene-specific primers.”\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe revised manuscript presents a strong and original study introducing UNBAR, a dual-\ncleavage ribozyme platform that senses RNA triggers and releases functional RNA effectors.\nThe authors have convincingly addressed the major scientific issues raised previously,\nadding new experiments on partial cleavage, zebrafish and cell-based assays with\nappropriate controls, and clearer methodological details. The work is scientifically sound and\nof broad interest to RNA engineering and synthetic biology.\nWe thank the reviewer for their suggestions that have improved the manuscript substantially,\nand are glad that they find the work of broad interest to RNA engineering and synthetic\nbiology\nComment 1: However, several figures (Fig 2) remain visually crowded and would benefit\nfrom re-shuffling to improve readability.\nResponse 1: We thank the reviewer for their helpful feedback. We agree that parts of Fig. 2\nremained visually crowded. In response, we have reorganized the figure layout to reduce\ndensity and improve visual clarity.\nComment 2: Moreover, a few statements in the Abstract and Discussion should be slightly\ntempered to reflect the efficiencies compared with conventional delivery. The manuscript is\nwell suited for publication in Nature Communications in my opinion, albeit with the correction\nof these minor changes.\nResponse 2: We thank the reviewer for this helpful suggestion. We agree that our findings\ncan be better contextualized to reflect the relative efficiencies of UNBAR-based sgRNA\nactivation compared with conventional direct sgRNA delivery. In response, we have added\nthe following to the Discussion:\n“In addition, sgRNA-embedded UNBAR does not currently attain the gene editing levels\nobserved with direct sgRNA delivery (Figure 5C), likely due to incomplete cleavage.\nEngineering of UNBAR to optimize robust trigger-dependent dual-site cleavage will be\nrequired to improve UNBAR function on these fronts.” (Lines 682 - 685).\nReviewer #2 (Remarks to the Author):\nThe authors have made a substantial experimental effort to revise the paper according to\nmost of the reviewers’ suggestions. They have included essential controls, repeated\nexperiments, and incorporated quantification and statistical analyses, all of which collectively\nstrengthen the conclusions. The storyline, although still a bit overstuffed with results, has\nbeen streamlined and is now easier to follow. Furthermore, the authors have successfully\nintegrated and discussed MD results within the storyline. With the addition of further controls,\nthe experiments in zebrafish (ZF) and mammalian cells are now more consistent and\ncompelling. Overall, I believe the authors have addressed the major concerns raised in my\ninitial review. Therefore, I consider the current version of the manuscript acceptable for\npublication in Nature Communications, provided the authors address some minor concerns\noutlined below. These issues do not require additional experiments but are important for\nimproving the manuscript's clarity.\nWe thank the reviewer for the review and suggestions, which have greatly improved the\nmanuscript.\nMain concerns.\nConcern 1.\nThe authors have demonstrated that cleavage can occur within mammalian cells, although\nspermine supplementation in growth media is required. However, the experiments in both\ncells and ZF were conducted using RNA devices that were pre-folded in vitro (via\ntransfection or microinjection). This raises questions about the functionality of the ribozyme\nwhen expressed from a DNA construct in cells. In fact, I had previously suggested\nexperiments along these lines (comment 28: “stable cell lines expressing the T-E-gene and\nthe A38 mutant as a control, transfected with either a plasmid expressing the E-gene or let-\n7f triggers under an inducible promoter”), and Reviewer #1 specifically questioned whether\n“the ribozyme could also be genetically encoded in a transgene.”\nIn the authors’ response to comment 3 from R#1 and my comment 28, they provided an\nimage that I assume is from a northern blot hybridized with a probe against the sgRNA.\nHowever, it is unclear whether the trigger RNA was co-transfected. This is critical, as it\nwould allow the authors to assess whether the ribozyme properly folds and functions in the\nintracellular environment when expressed from a DNA construct. The authors state that “the\nribozymes exhibited ~5x lower expression compared to sgRNA expressed from the same\nconstruct (under a U6 promoter), suggesting that it was unstable or being degraded.” This\nresult, coupled with the necessity for spermine supplementation to achieve substantial\nintracellular cleavage (Fig. S5J), suggests that the ribozyme is not functional when\nexpressed in cells.\nWhile I believe the manuscript could still be acceptable for publication even if the construct is\nnot functional when expressed from a DNA construct, it is essential that the authors address\nthis point explicitly. The discussion should be expanded (lines 643–646) to include these two\nresults (low expression and spermine dependency) and a statement acknowledging that the\nresults suggest that the construct is not functional when expressed from a transgene.\nResponse 1:\nWe thank the reviewer for their thoughtful assessment and for highlighting the importance of\nevaluating whether UNBAR can properly fold and function in cells when expressed from a\nDNA construct.\nWe clarify that the northern blot previously provided in the Response to Reviewers in\nRevision 1 (reproduced below), showed a catalytically inactive sgRNA-embedded\nribozyme or sgRNA cleavage product alone expressed under U6 promoter, hybridized with a\nprobe against the sgRNA. The cognate trigger RNA (modified at the terminal three\nnucleotides at the 5’ and 3’ ends with 2’MOE) was co-transfected with the ribozyme DNA\nconstruct. As the mutant does not cleave, this experiment was carried out to investigate\nstability of the ribozyme when expressed under U6 promoter without being confounded by\ncleavage. When we observed that its expression was very low when expressed as a\ntransgene, we decided to focus on delivery of UNBAR as RNA. The effect of spermine\nsupplementation on improving UNBAR cleavage was discovered after we decided to focus\non RNA delivery, and hence spermine efficacy on transgene expression or function was not\ntested.\nResponse Figure 1 and new Figure S5L: Northern blot of a catalytically inactive (mutant) sgRNA-embedded\nribozyme expressed under a U6 promoter, co-transfected with cognate trigger RNA, vs the sgRNA cleavage\nproduct (CsgRNA) alone expressed under a U6 promoter. Hybridization with an sgRNA-specific probe shows that\nribozyme expression is ~5x lower than the CsgRNA alone.\nIn response to the reviewer’s suggestion, we have included the above Figure in\nSupplementary Figure S5L, and added the following text to the Discussion:\nLines 661-664:\n“UNBAR cleavage in some cell types may benefit from supplementation of spermine, a\nnaturally occurring polyamine found in many foods67; understanding the underlying\nmechanism will enable further engineering for improved future iterations of UNBAR.”\nLines 693-699:\n“UNBAR ribozyme could also potentially be genetically encoded in a transgene to enable a\nfurther range of applications; however, we observed that the expression levels of a\ncatalytically inactive UNBAR was ~5-fold lower than the sgRNA alone when either was\nexpressed under a U6 promoter (Figure S5L). Hence, UNBAR integrity, trigger-dependent\ncleavage and cleavage-dependent function remains to be studied and optimised when\nexpressed from a transgene.”\nConcern 2.\nMy second concern is about modularity. Lines 325-339: If structural predictions revealed a\nhigh probability base-pairing between trigger-binding arms and catalytically critical and highly\nconserved nucleotides A38 and C25 in the absence of trigger. Does this imply that choosing\na different trigger could prevent the autoinhibitory state from being reproduced? Could these\nobservation suggest sequence constraints that limit the choice of triggers? In contrast, the\nauthors demonstrate minimal trigger-independent cleavage for UNBARs activated by T-ban-\n5p (Fig. 2), mir-451a (Fig. S2), and, to a lesser extent, the SARS-CoV-2 S-gene. This\napparent discrepancy should be clarified/discussed, as it could challenge the claimed\nmodularity of UNBARs (lines 610–612).\nResponse 2:\nWe thank the reviewer for this important point regarding modularity. The reviewer is correct\nthat our structural predictions indicated a higher-probability autoinhibitory interaction\nbetween the trigger-binding arms and the catalytically essential nucleotides A38 and C25 for\none specific, high-functioning trigger sequence (T-E-gene). This interaction arises from\nsequence-specific complementarity and may not arise for all triggers/trigger-arm sequences.\nThe structural data provides a hypothesis for why T-E-gene exhibits strong trigger-\ndependent activity. However, this does not imply that all triggers will rely on the same\nstructural interactions to achieve trigger-dependent cleavage. Different triggers may activate\nUNBAR through distinct mechanisms that have not yet been fully explored or characterized.\nAs the reviewer correctly pointed out, we have demonstrated in this work comparable\ntrigger-dependent activity for UNBAR with a variety of trigger sequences such as by T-let-7f\n(Figure 2 ), T-mir-451a (Figure S2), and T-S-gene (Figure S2).\nWe agree with the reviewer that the current iteration of UNBAR is not yet a fully plug-and-\nplay platform. Different choices of triggers, and consequently the trigger arm sequences, will\nlikely affect the efficacy of trigger-dependent and trigger-independent cleavage. That said,\none of the outcomes of this work is the development of engineering strategies that can be\nexplored to achieve trigger-dependent behaviour across diverse triggers. For example, we\nfound that modulating helix 4 enables tuning of the energetic balance between inactive and\nactive conformations, and may be required to ensure robust trigger dependency for different\ntrigger sequences.\nIn response, we modified the Discussion to clarify the following:\n“Computational modelling of an E-gene-triggered ribozyme identified an auto-inhibitory effect\nby unbound trigger arms, which results in low leakiness and a large turn-on signal for this\nribozyme” (Lines 645-647).\nWe also add in Lines 654-655:\n“Computational and structural analyses can help us to understand why some triggers and\ndesigns perform better.”.\nFinally, we clarify in Lines 655-658 that:\n“These studies show that UNBAR is modular in concept, but careful sequence-aware design\nis necessary for maximal performance. Engineering strategies described in this study can be\nexplored to optimise trigger-dependency for a specific trigger sequence”.\nConcern 3\nRegarding the potential orthogonality of UNBAR ribozymes, the authors mention in lines\n615–616 that, due to trans-cleaving activity, UNBAR cannot be multiplexed (i.e., assembled\ninto a sequential array of multiple devices). This trans-cleaving activity would also render\nUNBAR unsuitable for orthogonal integration of multiple devices expressed from different\nconstructs within the same cellular system. This point should be further discussed.\nResponse 3:\nWe thank the reviewer for this important point. We agree that, due to trans-cleaving activity,\nthe current iteration of UNBAR cannot be multiplexed or integrated orthogonally across\nmultiple constructs within the same cell. We have added text to the Discussion to clarify this\nas follows:\nLines 628-630:\n“Due to the ribozymes’ trans-cleaving characteristics, they cannot be multiplexed to\ndifferentiate between different triggers to release different cleavage products, including\nwithin the same cellular system”\nMinor concerns:\n• Line 43: Remove \"and.\"\nFixed.\n• Line 166: Replace \"In order for the strategy of trigger-activated cleavage to work\" with \"For\nthe trigger-activated cleavage strategy to work.\"\nFixed as suggested.\n• Lines 233–234: \"However, dual cleavage appeared to be trigger-independent (Figures 2Cii,\np > 0.05; and S2Bii).\" Consider moving this sentence to the beginning of the next paragraph\n(line 241) to improve the logical flow of the argument.\nEdited as suggested.\n• Lines 235–237: This sentence sounds redundant. I suggest replacing it with: \"This is the\nfirst example of a single hairpin ribozyme capable of cleaving at two different sites, a\nphenomenon not previously described.\"\nEdited as suggested.\n• Lines 241–243: This sentence is difficult to read. Consider revising to: \"To minimize trigger-\nindependent cleavage, we aimed to identify the optimal length and sequence for the H4\nstem, which we predicted would function as a trigger-responsive communication module\n(CM).\"\nEdited as suggested.\n• Lines 269–273: I would suggest removing these sentences for clarity.\nResponse 4:\nWe thank the reviewer for this suggestion, but we think that a key feature of the ribozyme is\nits ability to amplify the target/trigger RNA signal into multiple copies of a sequence-\nindependent RNA cleavage product, and hence have retained this sentence, but edited it to\nimprove clarity.\n• Figure 2J: Replace \"T-EgeneCL_29nt A38WT\" with \"T-Egene_Cl-29nt.\" Only include the\ncleaved products in boxes.\nFixed. Indeed, only cleaved products are boxed.\n• Lines 414-422 and-Fig. S4C and D: Why is there no reduction of C+D fragment parallel to\nthat of the P fragment in constructs with longer H2 and H3 stems when the trigger was not\nadded?\nResponse 5:\nWe thank the reviewer for their thoughtful examination of the data. Indeed, this is puzzling as\none would expect the C+D fragment to reduce when P is reduced, or be accounted for by\nthe C and/or D fragments. We apologise for the confusion as we realised that this gel was\nmislabelled. The shRNA-ribozyme fragments comprise extensive complementarity, and\nhence it can be more difficult to resolve the higher molecular weight bands that comprise the\nshRNA stem-loop, even in a denaturing gel. In Figure S4D, the full-length ribozyme for the\n5H3 ribozyme, which is 256 nt (79 + 106 + 71) in length, appears to migrate at ~500 nt, while\nthe C+D fragment, at 185 nt (79 + 106), appears to migrate at ~300 nt. We initially identified\nthe 300 nt as the full-length based on corroboration with the ladder, but your comment\nprompted us to re-examine the original gel. We hypothesise that these are double-stranded\ncomplexes of the respective bands, which hence run larger. With the correct assignment of\nthe fragments, we can clearly see a concomitant decrease in the C+D fragment as the H3 is\nextended.\nWe have now revised the gel image in Fig. S4D, re-annotated the bands and boxed the C+D\nand P fragments, respectively, and added the following sentence to describe this\nphenomenon:\nLines 436-438:\n“we note that the larger shRNA ribozyme fragments occasionally migrate at larger sizes,\nlikely due to dsRNA formation of the highly self-complementary fragments even in a\ndenaturing gel”\nWe also checked all the other shRNA gels and found that this was the only gel to be\nincorrectly annotated. We would like to thank the reviewer again for highlighting this.\n• Lines 481–485: Remove these lines and adjust line 486 accordingly.\nRemoved as suggested\n• Line 511: Check the format of the colon.\nChecked, and the colon appears to be suitable, as it refers to the systematic steps that we\nexplored\n• Line 569: Remove the capitalized “MUST” for tone consistency.\nFixed.\n• Lines 575–588: This section is repetitive. Please condense it.\nCondensed as suggested.\n• Lines 618–620: What about potential innate responses via cytoplasmic RNA sensors such\nas TLRs (TLR3, TLR7, TLR8) or RLRs (RIG-I, MDA-5)? While this may not be an issue in\n293T cells, how might this affect other cell types or animal models?\nResponse 6: We thank the reviewer for this important consideration. Indeed, RNA-based\ntechnologies like UNBAR may elicit innate immune responses depending its length,\nmodifications, as well as on the cell type or animal model used. While our current studies in\nHEK293T cells did not investigate immune-related effects, future investigations of UNBAR in\nother cellular contexts or in vivo models could provide insight into potential activation of TLR\nor RLR pathways. To clarify this, we have expanded the discussios as follows:\nLines 632-635:\n“For cellular applications, this may avoid undesired adaptive immune responses to\nextraneous foreign proteins (although exogenous RNA and some delivery systems can still\nactivate innate immune sensing) and dependence on varying host-cell protein expression\nmachinery.”\n• Lines 643–646: Consider citing the following reference: Xiang, J.S.; Kaplan, M.; Dykstra,\nP.; Hinks, M.; McKeague, M.; Smolke, C.D. \"Massively Parallel RNA Device Engineering in\nMammalian Cells with RNA-Seq.\" Nat. Commun. 2019, 10, 4327, doi:10.1038/s41467-019-\n12334-y.\nAdded citation"}]

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