Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact on RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR Cas systems. We report a cryo-EM (cryo-electron microscopy) structure of the active Cas7-11 from  Ribonucleic acid or RNA is an important molecule involved in making proteins, transmitting diseases, offering immunity in form of vaccines, and also degrading itself. Programmed RNA degradation is a common method used by bacteria to protect themselves from invading viruses.Bacteria acquire viral genetic materials during infections, which are then converted into RNA fragments, or guide RNA. The guide RNAs both locate and recruit enzymes to help destroy the infectious RNA. These programmable RNA degradation machineries can be repurposed for biotechnology applications to help regulate gene expression or to minimize the effect of viral infections.Similar machineries, like the CRISPR/Cas9 gene editing tool, act like genetic scissors, allowing researchers to make precise modifications to DNA to study and alter the role of genes in the cell. Like in bacteria, the CRISPR system uses fragments of RNA from viruses as a guide to identify matching targets and create breakages in the genetic material. Recently, researchers discovered Cas7-11, which is used to break sections of RNA in viruses.To better understand how Cas7-11 works, Goswami et al. studied its three-dimensional structure. Detailed views of each segment of the protein, together with biochemical studies of the protein’s activity, helped to identify their respective roles. The structural information also highlighted three regions involved in snipping RNA and revealed how they drive this process. This analysis showed that a short segment of Cas7-11 alone is sufficient to prepare and bind the guide RNA fragments.These findings add to the understanding of how Cas7-11 prepares its guide and creates breakages in RNA. It has a similar structure to a previously known assembly of proteins that also breaks down RNA, providing insight into its evolution. The detailed analysis of how Cas7-11 works also demonstrates the possibility of engineering it as a laboratory tool to remove specific RNA sequences in cells. Desulfonema ishimotonii Cas7-11 (DiCas7-11) at an overall resolution of 2.82 Å (The CRISPR Cas systems confer adaptive immunity to prokaryotic hosts against invading viruses by encoding a range of different CRISPR Cas efforts that interfere with the invader nucleic acids . Three tf 2.82 Å . DiCas7-f 2.82 Å . Our strLactococcus lactis Csm (LlCsm) complex (The wild-type DiCas7-11 was incubated with its precursor crRNA (pre-crRNA) and a complementary target RNA under a reactive condition before being made frozen specimen . Under t complex   pre-crRNA are successfully processed by DiCas7-11  linked to the programmed spacer . To confiCas7-11 . A well-iCas7-11 . The reqiCas7-11 , althougiCas7-11 , suggest10 helix in proteins. Interestingly, the non-conserved 5’-tag nucleotides, A(–3), C(–6), and C(–8) form minimal contacts with the protein, suggesting their minor roles in maintaining protein-RNA interactions  domain may be sufficient for binding and processing pre-crRNA. We thus created and purified a truncated mutant comprised of residues 1–238 (hereafter fC7L.1 for free C7L.1) . Followi2+ in target cleavage  due to weak density, focused classification and refinement led to a low-resolution map that matches the AlphaFold-predicted model of the insertion domain . This moA structure of catalytically inactive DiCas7-11  bound with a slightly modified crRNA (U(–15) is replaced by G(–15)) recently became available (PDB ID: 7WAH) . We supeThe structure and complementary biochemical assays show that Cas7-11 has a minimal architecture required for programmable RNA cleavage. The covalent linkage of the homologous units suggests an evolutionary advantage in dedicating Cas7-11 to RNA cleavage. Considering the known collateral RNase activity of Cas13 and the complicated Csm/Cmr systems, Cas7-11 offers a desirable alternative in developing gene regulation tools. While DiCas7-11 has been successfully demonstrated to function in mammalian cells, the efficiency and accuracy remain to be improved. With now the available structures and the accurately mapped processing and target cleavage sites, protein engineering may assist the efforts in designing improved Cas7-11-derived RNA interference platforms. Both fC7L.1 and DiCas7-11-Δint1 lend a proof-of-concept for such an effort.Escherichia coli NiCo21 (DE3) competent cells were transformed with either plasmid used in protein production. A single colony was picked and transferred to 100 mL LB media containing 50 μg/mL ampicillin and grown for 12 hr at 37°C before inoculation into 1 L LB culture. The cells were induced at mid-log phase with the addition of 0.5 mM IPTG  and grown overnight at 16°C and harvested. Cells were lysed and centrifuged at 4000 rpm for 30 min in buffer A  sonicated 10 times on pulse for 20 s with 40 s rest between the pulses. The cell lysate was centrifuged at 16,000 rpm for 1 hr at 4°C and the resulting supernatant was passed through the pre-equilibrated Ni-NTA resin column. The protein bound resins were washed by 100 mL buffer B  and eluted with buffer C . For the sumo-tagged DiCas7-11, the ULP1 protease was added to the elutant to remove the sumo-tag from DiCas7-11 while dialyzing at 4°C overnight. The digested protein solution was diluted twofold before being loaded onto a heparin column pre-equilibrated with buffer D . The bound protein was eluted with a salt gradient. Pooled fractions were further purified on a gel filtration column in buffer E . The protein containing fractions were pooled, concentrated to 21 mg/mL, aliquoted, and stored at –80°C for future use. We found that the quality of the sumo-tag purified protein is superior to that of the his-tag purified protein and used it in subsequent experiments. The DiCas7-11 mutants, DiCas7-11-Δint1 and fC7L.1, were prepared by Q5 ×2 master mix mutagenesis kit (New England Biolabs) using primers listed in Two DiCas7-11 expression constructs were tested for protein expression. The first one comprises pACYC-duet-1 plasmid inserted with the codon-optimized sequence encoding for DiCas7-11 with an N-terminal his-tag. The second plasmid encodes sumo-tag fused DiCas7-11 (Addgene: 172503). 2, 0.5 mM NTPs, and 48  μg/mL T7 RNA polymerase in a 50 μL reaction. The in vitro transcription reaction was kept overnight at 37°C and treated with 2 U of Turbo DNase (Invitrogen) for 1 hr at 37°C. The final product was purified by Monarch RNA Cleanup kit (New England Biolabs), eluted in water, flash-frozen using liquid nitrogen and stored at −80°C.For synthesis of 59-nt pre-crRNA, DNA oligonucleotides appended with T7 promoter sequence were ordered from Eurofins . The comFive hundred nM of DiCas7-11 of fC7L.1 was incubated with 500 nM of pre-crRNA at 37°C for 30 min in a 15 μL reactions containing ×1 processing buffer . The reactions were stopped by using ×2 formamide dye . The samples were heated at 95°C for 5 min and run on 15% TBE-UREA gels  at RT at 180 V in ×1 Tris Borate EDTA (TBE) running buffer. The gels were stained with SYBR Gold II (Invitrogen) stain and scanned by BIO-RAD ChemiDoc MP scanner.2. A binary complex was first prepared by incubating 400 nM Cas7-11 with 500 nM crRNA for 30 min at 37°C. The resulting samples were then incubated with 500 nM target RNA (5Cy3 labeled or non-labeled) for 1 hr at 37°C. To test the metal dependence cleavage activity, reaction was supplemented with 16 mM EDTA. The reactions were stopped by using ×2 formamide dye . The samples were heated at 95°C for 5 min and run on 15% TBE-UREA gels  at RT at 180 V in ×1 TBE running buffer. The gels were stained with SYBR Gold II (Invitrogen) stain and scanned by Bio-Rad ChemiDoc MP scanner. When Cy3-labeled RNA was used, gels were directly scanned by Bio-Rad ChemiDoc MP scanner.The in vitro target RNA cleavage assays were performed in a cleavage buffer containing 40 mM Tris pH 8.0, 70 mM sodium chloride, 10 mM MgClFor electrophoretic mobility shift assay, 1 µM fC7L.1 was separately added with 150 nM of polyA pre-crRNA (pA3) and non-specific RNA in the buffer containing 20 mM Tris pH 7.5, 70 mM NaCl, 5% glycerol, and 25 µg/mL heparin. The reaction was incubated for 30 min at 37°C. After the reaction was complete, the samples were mixed with ×6 loading dye  and run on a 10% TBE gel  at 4°C at 150 V. Gels were stained with SYBR Gold II (Invitrogen) and scanned by Bio-Rad ChemiDoc MP scanner.2, and 2 mM TCEP). The peak fraction at 0.3 mg/mL determined by UV 280 nm absorbance was collected and further incubated with 2 molar excess target RNA at 37°C for 10 min before grid preparation. The Cas7-11-crRNA-target RNA sample in 5 μL volume was added onto glow-discharged 300 mesh Cu R1.2/1.3 holey carbon grids (Quantifoil) with extra layer of carbon (2 nm), blotted for 3 s at 100% humidity using FEI Vitrobot Mark IV. After flash-freezing in liquid ethane, the grids were transferred to liquid nitrogen for storage until cryo-EM imaging.To reconstitute the ternary complex, 400 μg wild-type DiCas7-11 protein was incubated with 1.5 molar excess of a pre-crRNA in the buffer  at 37°C for 45 min followed by separation on a Superdex 200 increase 10/300 gel filtration column in the buffer  equipped with Gatan Bioquantum K3 direct electron detector (Gatan) with 15 eV energy filter in a counted super-resolution mode. All 4177 images were collected at a dose rate of 60 eonCorr 2  in a wraonCorr 2  and contonCorr 2  implemenonCorr 2 . The staThe DiCas7-11 protein was built from an AlphaFold-  predicte This manuscript is a timely contribution to the CRISPR/Cas field: the mode of function of the type III-E Cas7-11 CRISPR-Cas system. This is an RNA-guided RNA targeting system only characterized last year. In contrast to Cas13 systems, Cas7-11 does not possess collateral damaging activity, hence does not show cytotoxicity when introduced into human cells. These are highly desirable traits in practical applications. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Timothy Nilsen as Reviewing Editor and James Manley as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Molecular Mechanism of Active Cas7-11 in Processing CRISPR RNA and Interfering Target RNA" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:eLife. Nevertheless, each raised a significant number of concerns regarding the presentation of the work. Please address these concerns as thoroughly as possible. We hope that the concerns can be handled through textual revisions and revision of the figures.The reviewers thought that the work was interesting and in principle appropriate for Reviewer #1 (Recommendations for the authors):I have the following points for the authors to address.1. Comparison with Type III Csm complex – In page 4, the authors claim "the similarity in the bound RNA trajectory at the core between the two complex[es] is striking ". However, Figure S4 only showed side-by-side comparison of the complex. There was no superpositioning nor r.m.s.d. value for the readers to evaluate the claims. These need to be provided.2. "The first 18 nucleotides of the target RNA (+19* ~ +2*) remain base paired with the spacer region of the crRNA ." – What happens to the +1* nucleotide? Is it base paired with the guide? If not, why?3. Along the same line, the role of the PFS motif in DiCas7-11 should be discussed. While most of the 5´ handle residues are sequestered, the last two nucleotides are displayed by DiCas7-11. The authors should discuss whether their structural observations in this region agree the biochemical data by Ozcan et al. For example, can the authors define the A-1*, G-2*, and the preceding nucleotides in their density map? Is there any interaction between this region and C-1A-2 in the 5´ handle?4. Along the same line, the authors should include a zoom-in view of each guide RNA-target segment. It is hard to tell from viewing Figure 1C whether the guide and target form perfect, distorted, or shearing base pairing in each segment. Suggesting including a supplementary figure showing the zoomed base pairing densities.5. I suggest the authors to improve the rendering of the EM maps in supplementary figure 3. For the three enzymatic centers, the important H-bonds should be marked with dashed lines, and distances labeled.6. Page 7, 5´ handle recognition: as the authors pointed out, not all 15 handle residues are highly conserved among Cas7-11 guide RNAs. Can the authors rationalize the handle nucleotide conservation based on variations in the recognition residues in Cas7-11?7. Enzymatic mechanism of crRNA biogenesis – Typically besides His, additional polar residues are involved in assisting the proton shuttering. Are there any such residues conserved among Cas7-11s?8. Cleavage at site 1 – I did not quite follow the author's conclusion that because Y360 is not required for site 1 cleavage, D429 is sufficient for mediating the metal-dependent cleavage. Is Y360 the only alternative catalytic residue in this site? Given that site 1 cleavage is metal independent, D429 is unlikely the general acid/base. Did I misunderstand something here?9. Figure 3c – No target band, nor cleavage product bands in lanes 4 and 5. Why is that? Are they not labeled? I could not make sense of this panel from reading the text and legend.10. Figure 3e – Why isn't substrate 5 cleaved but substrate 7 cleaved. Both results seem surprising.11. Page 5, first sentence of the 2nd paragraph: how does supplementary Figure 5 support the claim of structural similarity?Reviewer #2 (Recommendations for the authors):1. I found Figure 2 and 3  difficult to follow due to the lack of description of gel visualization methods and the overuse of "asterisk" in the figure legends. The following suggestions may help to reduce such confusion for future readers of this paper:a. In the biochemical experiments, gels were visualized using either SybrGold staining or fluorescence labeling of the target RNA. The visualization method should be clearly stated in the figure legend for each panel containing a gel, to make it easier for the reader to understand the gel.b. In some cases, the "target" label on the left side of the gel is not aligned correctly with all targets shown on the gel , because targets of multiple sizes were used on the same gel. Please adjust these labels.c. Please describe which substrates were Cy5 labeled in the figure legend for Figure 3b, and/or describe what the yellow circle at the 5' end of target RNA 1 indicates.d. Please list the target RNAs in Figure 3b in the order they are shown on gels in panels 3c-f.e. Please describe the blue stars depicting deoxy positions for RNA target 2 in Figure 3b as "blue stars" rather than as asterisks.f. The use of an asterisk to indicate the labeled target in panel 3d and the spurious RNA in panel 3e is confusing. Could one of these be indicated using a different label? For example, the labeled target RNA could be indicating using the same yellow circle that was used in the cartoon of target RNA 1.2. Kato et al. described Zinc-finger domains in the Cas7 domains. Is evidence for these domains present in the cryo-EM map obtained in this study? These were not built into the structural model that was provided to reviewers.3. p. 4 line 15: The authors state that the density map revealed a "partially cleaved target RNA" but do not explain their interpretation of the map that led them to conclude that the RNA is partially cleaved. What was this determination based on? As mentioned in the public review, there appears to be little density for the site 1 product. Further discussion of this interpretation should be included in the manuscript.4. p. 3 line 22: The authors suggest that Cas7-11 may be an "evolutionary intermediate" between Class 1 and 2 effectors. This terminology is misleading, as it suggests that Class 1 and 2 effectors are homologous. Please reword this sentence.5. p. 6 line 6: The authors refer to the catalytic mechanism employed for crRNA processing as an "RNase A-like mechanism". A more generic term like "general acid-base mechanism" would be more appropriate, as there are likely several differences between RNase A and the Cas7-11 catalytic activity .6. p. 7 line 2: The authors compare a structure in the crRNA 5' handle to a 3-10 helix of a protein. It is unclear how these are similar, as this region of the RNA has the base of residue -5 stacked between the bases of residues -7 and -8, which is quite a distinct architecture than what would be observed for the side chains of a 3-10 helix. The phrase about the 3-10 helix could be removed.7. Please use consistent nomenclature for the Csm complex subunits. It would be helpful to a broad audience to describe Csm3 as the Cas7-like subunit and Csm2 as the Cas11-like subunit in the text (p. 4 lines 20-21). Cas10 is referred to as Csm1 at the top of p. 5. Please use the updated nomenclature of Cas10 only. Csm1 could be indicated in parenthesis when first mentioning Cas10.8. Reference 1 could be updated to the more recent Makarova review (PMID: 31857715).9. p. 3 line 4: "efforts" should be "effectors"10. p. 3 line 11: "Higher Prokaryotic and Eukaryotic binding domain" should be "Higher Eukaryotic and Prokaryotic Nucleotide binding domain"11. p. 5 line 8: The reference to Supplementary Figure 5 seems extraneous here.12. p. 6 line 21: Please cite Figure 2a at the end of this sentence.13. p. 8 line 18: The authors state that Asp429 is sufficient for mediating metal-dependent cleavage, but the paragraph presents data that target cleavage in this active site is metal independent. This sentence is incongruous with the rest of the paragraph and requires clarification.14. A general comment regarding the structure figures: The figures are very attractive, but it would be helpful if the side chains of proteins were colored by heteroatom to more readily observe the identity of the residue and potential polar contacts.15. In Supplementary Figure 1, please indicate which peak was collected for the bottom elution profile.16. In Supplementary Figure 2, does "pcts" stand for "particles"? It may be helpful to spell out the whole word, as this is not a standard abbreviation as far as I know.17. In Supplementary Figure 4, it would be helpful to show Cas7-11 in this figure so that the reader does not need to compare with a separate figure. Reviewer #1 (Recommendations for the authors):I have the following points for the authors to address.1. Comparison with Type III Csm complex – In page 4, the authors claim "the similarity in the bound RNA trajectory at the core between the two complex[es] is striking ". However, Figure S4 only showed side-by-side comparison of the complex. There was no superpositioning nor r.m.s.d. value for the readers to evaluate the claims. These need to be provided.We revised Figure S4 to include three views of superimposed structure images as the new panel b and the associated r.m.s.d in the figure legend.2. "The first 18 nucleotides of the target RNA (+19* ~ +2*) remain base paired with the spacer region of the crRNA ." – What happens to the +1* nucleotide? Is it base paired with the guide? If not, why?We should have clearly labeled the nucleotides that are modeled based on density, which does not include +1*. We now include in Figure 1 caption to indicate that the gray colored nucleotides, though included in the RNA constructs, are not modeled due to weak or no density.3. Along the same line, the role of the PFS motif in DiCas7-11 should be discussed. While most of the 5´ handle residues are sequestered, the last two nucleotides are displayed by DiCas7-11. The authors should discuss whether their structural observations in this region agree the biochemical data by Ozcan et al. For example, can the authors define the A-1*, G-2*, and the preceding nucleotides in their density map? Is there any interaction between this region and C-1A-2 in the 5´ handle?Unfortunately, our model does not include any of the PFS nucleotides. However, we did not do a good job in indicating which nucleotides are modeled (see above). We now include in Figure 1 caption to indicate that the gray colored nucleotides, though included in the RNA constructs, are not modeled due to weak or no density.Though we did not observe any of the PFS nucleotides mentioned by the reviewers, we do have an opportunity to speculate the roles of PFS given the observed 5’-tag structure. We added a statement on page 8 speculating that the two unpaired (C-1 and A-2) could pair with PFS to influence Cas7-11 structure and its downstream activity such as activation of TPR-CHAT by Hu et al.20.”“The different mode of 5’ tag binding to Cas7-11 from that of Csm explains the lack of a Cas10-like effector as in Csm. Rather, it allosterically activates a different effector, the TPRCHAT protease, in a 3’ PFS-dependent manner. Though 13 of the 15 5’-tag nucleotides are buried by the protein, the last two, C(-1) and A(-2), are partially solvent-accessible and can potentially participate in PFS-mediated activation of the TRP-CHAT activity 4. Along the same line, the authors should include a zoom-in view of each guide RNA-target segment. It is hard to tell from viewing Figure 1C whether the guide and target form perfect, distorted, or shearing base pairing in each segment. Suggesting including a supplementary figure showing the zoomed base pairing densities.We added additional close-up views of density of the guide RNA-target pairings in Figure S3. In addition, we added an image of superimposed guide RNA-target helix on a standard A-form helix.5. I suggest the authors to improve the rendering of the EM maps in supplementary figure 3. For the three enzymatic centers, the important H-bonds should be marked with dashed lines, and distances labeled.Thanks for the suggestion. The EM maps and the close contacts are now updated for Figure S3.6. Page 7, 5´ handle recognition: as the authors pointed out, not all 15 handle residues are highly conserved among Cas7-11 guide RNAs. Can the authors rationalize the handle nucleotide conservation based on variations in the recognition residues in Cas7-11?We revised the paragraph in page 7 extensively and with careful citations to supplementary figure 6  to reflect the fact that “In general, well-conserved residues form base-specific interactions while non-conserved residues maintain interactions with RNA sugar phosphate backbone.”. We clearly state if an amino acid is conserved or not when describing its mode of interactions. We explicitly point out that the non-conserved RNA nucleotides, A(-3), C(-6), and C(-8), form minimal contacts with proteins.7. Enzymatic mechanism of crRNA biogenesis – Typically besides His, additional polar residues are involved in assisting the proton shuttering. Are there any such residues conserved among Cas7-11s?We appreciate this suggestion. We now extend our discussion to include discussion of the surrounding residues that can potentially influence processing activity.8. Cleavage at site 1 – I did not quite follow the author's conclusion that because Y360 is not required for site 1 cleavage, D429 is sufficient for mediating the metal-dependent cleavage. Is Y360 the only alternative catalytic residue in this site? Given that site 1 cleavage is metal independent, D429 is unlikely the general acid/base. Did I misunderstand something here?In the original manuscript, we meant to use the phrase “metal-independent” instead of “metal-dependent” because at the time, we were convinced this site uses a metalindependent mechanism, which prompted us to look for possible general acid/base residues such as the well-conserved Tyr360.Nature 597.7878 (2021): 720-725.). Though both encode an identical protein with affinity tag differences, the in-house clone produced samples with noticeably more degraded or prematurely terminated proteins (see figure in and response to item 3 of Reviewer 2). Though the cleavage gel patterns from the in-house clone-derived protein resemble those by Ozcan et al. that seem to suggest metal independence for site 1, the cleaner protein prep from the Addgene clone clearly showed metal dependent cleavage for both sites (see figure in and response to item 3 of Reviewer 2). We thus updated our target cleavage results and discussions throughout the manuscript to reflect this new result. The exact reason for the different cleavage patterns with different proteins is not clear at this time (see figure in and response to item 3 of Reviewer 2).This conclusion had since been revised given the new data described below (see response to item 3 of Reviewer 2). In an effort of repeating the target cleavage assay, we discovered that two different batches of DiCas7-11 resulted in different metal-dependence. Whereas the older batch used for biochemistry studies such as those in Figure 3 reproducibly produces metalindependent cleavage of site 1, the batch later purified showed a complete dependence on metal . The two batches were from two different clones, one was constructed in-house and another described by Ozcan et al. and purchased from Addgene , though flanking both cleavage sites, has only 12-base pairs with crRNA, which could be insufficient for substrate binding and is thus not cleaved. In contrast, substrate 7 (now 5), though flanks only site 2, can base pair with 18 crRNA nucleotides and is thus cleaved.Given the fact that substrate 6 is also cleaved (16-base pairs), we suggest that target RNA pairing with either end of the crRNA for at least 16 bases are sufficiently cleaved.11. Page 5, first sentence of the 2nd paragraph: how does supplementary Figure 5 support the claim of structural similarity?We mistakenly cited Supplementary Figure 5 here. It is now deleted.Reviewer #2 (Recommendations for the authors):1. I found Figure 2 and 3  difficult to follow due to the lack of description of gel visualization methods and the overuse of "asterisk" in the figure legends. The following suggestions may help to reduce such confusion for future readers of this paper:a. In the biochemical experiments, gels were visualized using either SybrGold staining or fluorescence labeling of the target RNA. The visualization method should be clearly stated in the figure legend for each panel containing a gel, to make it easier for the reader to understand the gel.Thank you for the suggestion. We have revised all figures to contain consistent symbols indicating either processing or target cleavage sites, various types of RNA used and the cleavage products. In addition, we also found a way to distinguish the two different types of staining used for various target RNA .b. In some cases, the "target" label on the left side of the gel is not aligned correctly with all targets shown on the gel , because targets of multiple sizes were used on the same gel. Please adjust these labels.See item a.c. Please describe which substrates were Cy5 labeled in the figure legend for Figure 3b, and/or describe what the yellow circle at the 5' end of target RNA 1 indicates.See item a.d. Please list the target RNAs in Figure 3b in the order they are shown on gels in panels 3c-f.See item a.e. Please describe the blue stars depicting deoxy positions for RNA target 2 in Figure 3b as "blue stars" rather than as asterisks.See item a.f. The use of an asterisk to indicate the labeled target in panel 3d and the spurious RNA in panel 3e is confusing. Could one of these be indicated using a different label? For example, the labeled target RNA could be indicating using the same yellow circle that was used in the cartoon of target RNA 1.See item a.2. Kato et al. described Zinc-finger domains in the Cas7 domains. Is evidence for these domains present in the cryo-EM map obtained in this study? These were not built into the structural model that was provided to reviewers.Thanks for pointing this out. We did neglect the zinc atoms at these four Zinc fingers that are now added to the revised coordinate and the revised Figure 1.3. p. 4 line 15: The authors state that the density map revealed a "partially cleaved target RNA" but do not explain their interpretation of the map that led them to conclude that the RNA is partially cleaved. What was this determination based on? As mentioned in the public review, there appears to be little density for the site 1 product. Further discussion of this interpretation should be included in the manuscript.The “partially cleaved target RNA” phrase is a result of weak density for nucleotides beyond site 1 (+2* and +3*) but clear density flanking site 2, despite the presence of the active enzyme. This feature indicates that cleavage likely had taken place at site 1 but not site 2 in most of the particles went into the reconstruction. To further support this phrase, we added “The PFS region plus the first base paired nucleotide (+1*) are not observed.”4. p. 3 line 22: The authors suggest that Cas7-11 may be an "evolutionary intermediate" between Class 1 and 2 effectors. This terminology is misleading, as it suggests that Class 1 and 2 effectors are homologous. Please reword this sentence.We revised the phrase to “Cas7-11 is therefore believed to be a unique Type III CRIPSR-Cas system.” to avoid the confusion.5. p. 6 line 6: The authors refer to the catalytic mechanism employed for crRNA processing as an "RNase A-like mechanism". A more generic term like "general acid-base mechanism" would be more appropriate, as there are likely several differences between RNase A and the Cas7-11 catalytic activity .16,17, although detailed roles of His43 and other possible catalytic residues remain to be characterized.”This statement is indeed speculative, and we agree there are likely differences between RNase A and Cas7-11. Without thorough enzyme kinetic studies, the catalytic mechanism of Cas7-11 remains at large. We do not know if His43 acts as general acid or base, nor do we know if there are other amino acids nearby that participate in rate enhancement. We revised the statement to “The requirement for His43 without requiring divalent metals in processing suggests that the C7L.1 employs a general acid-base catalysis mechanism similarly suggested for Cas6 6. p. 7 line 2: The authors compare a structure in the crRNA 5' handle to a 3-10 helix of a protein. It is unclear how these are similar, as this region of the RNA has the base of residue -5 stacked between the bases of residues -7 and -8, which is quite a distinct architecture than what would be observed for the side chains of a 3-10 helix. The phrase about the 3-10 helix could be removed.The reviewer is correct that we need further clarify the reference to 3-10 helix. We should have emphasized that the comparison is between the backbone of the two kinds of polymers, especially the intra-chain interactions established among the backbone atoms.10 helix in proteins.”. This statement is also better positioned in the text after a series of descriptions of the unusual tight turn interactions.We revised the statement to “The tight and the N+3 interactions involving the C(-8)-A(-7)C(-6)-G(-5) backbone atoms is reminiscent of those within a 37. Please use consistent nomenclature for the Csm complex subunits. It would be helpful to a broad audience to describe Csm3 as the Cas7-like subunit and Csm2 as the Cas11-like subunit in the text (p. 4 lines 20-21). Cas10 is referred to as Csm1 at the top of p. 5. Please use the updated nomenclature of Cas10 only. Csm1 could be indicated in parenthesis when first mentioning Cas10.Lactococcus lactis Csm (LlCsm) complex 13 with the C7L.1-C7L.4 ridge matching that formed by four Cas7-like (Csm3) subunits and C11L matching the Cas11-like (Csm2) subunit adjacent to Cas10 (Csm1) .”Thanks for the suggestion. We revised the statement to “The entire DiCas7-11 complex can be superimposed onto the closely matched homologous 8. Reference 1 could be updated to the more recent Makarova review (PMID: 31857715).We have updated reference 1.9. p. 3 line 4: "efforts" should be "effectors"Corrected.10. p. 3 line 11: "Higher Prokaryotic and Eukaryotic binding domain" should be "Higher Eukaryotic and Prokaryotic Nucleotide binding domain"Corrected.11. p. 5 line 8: The reference to Supplementary Figure 5 seems extraneous here.Corrected.12. p. 6 line 21: Please cite Figure 2a at the end of this sentence.Corrected.13. p. 8 line 18: The authors state that Asp429 is sufficient for mediating metal-dependent cleavage, but the paragraph presents data that target cleavage in this active site is metal independent. This sentence is incongruous with the rest of the paragraph and requires clarification.Corrected, and please also see the response to Reviewer 1’s #8 comment.14. A general comment regarding the structure figures: The figures are very attractive, but it would be helpful if the side chains of proteins were colored by heteroatom to more readily observe the identity of the residue and potential polar contacts.We have replaced the figures with heteroatom colored side chains of proteins.15. In Supplementary Figure 1, please indicate which peak was collected for the bottom elution profile.We labeled the peak now.16. In Supplementary Figure 2, does "pcts" stand for "particles"? It may be helpful to spell out the whole word, as this is not a standard abbreviation as far as I know.We replaced “pcts” by “particles”.17. In Supplementary Figure 4, it would be helpful to show Cas7-11 in this figure so that the reader does not need to compare with a separate figure.A new panel (b) showing superimposed Cas7-11 and LlCsm is now added to Figure S4.


More specifically,the effect of “linear offset-induced image drag” ispresented, which refers to a spatial distortion in SESORS images causedby the magnitude and direction of the linear offset and highlightthe need for an annular SORS collection geometry during imaging toneutralize these asymmetric effects. Additionally, building on theseprinciples, the concept of “ratiometric SESORS imaging”is introduced for the location of buried inclusions in three dimensions.Together these principles are vital in developing a methodology forthe location of surface-enhanced Raman scattering-active inclusionsin three dimensions. This approach utilizes the relationship betweenthe magnitude of the spatial offset, the probed depth, and ratiometricanalysis of the NP and tissue Raman intensities to ultimately imageand spatially discriminate between two distinct NP flavors buriedat different depths within a 3D model for the first time. This researchdemonstrates how to accurately identify multiple objects at depthin tissue and their location using SESORS which addresses a key capabilityin moving SESORS closer to use in biomedical applications.A fundamental question crucial to surface-enhanced spatiallyoffsetRaman spectroscopy (SESORS) imaging and implementing it in a clinicalsetting for in vivo diagnostic purposes is whether a SESORS imagecan be used to determine the exact location of an object within tissue?To address this question, multiple experimental factors pertainingto the optical setup in imaging experiments using an in-house-builtpoint-collection-based spatially offset Raman spectroscopy (SORS)system were investigated to determine those critical to the three-dimensional(3D) positioning capability of SESORS. Here, we report the effectsof the spatial offset magnitude and geometry on locating nanoparticles(NPs) mixed with silica powder as an imaging target through tissueand outline experimental techniques to allow for the correct interpretationof SESORS images to ascertain the correct location of NPs in the two-dimensional  Imaging is traditionally undertaken by tracking the intensityof a particular SERS band, pertaining to the targeted NPs of interest,across a grid on the surface of a biological sample to ascertain thelocation of the NPs at depth in the two-dimensional (2D) imaging plane.Using a handheld SORS spectrometer with a point-based collection geometry,that is, a linear offset vector, Nicolson et al. reported the throughtissue imaging of ex vivo MCF7 multicellular tumor spheroid (MTS) models incubated with SERS-activegold NPs. In this study, the MTS models were buried beneath the porcinetissue and mapped in a grid across the tissue surface using a handheldspectrometer.21For SESORS imagingto be considered as a viable optical medicalimaging method, it is crucial to not only apply the technique to differentassays and disease states but also to cultivate theoretical principlesfor the correct interpretation of such images and, hence, the accuratelocation of targeted NPs in vivo at depth in three dimensions. Wesought to address a fundamental question relevant to all the SESORSimaging studies alluded to here, namely, how does a SESORS image,collected at points offset from the excitation points on an imaginggrid by a constant spatial offset, relate to the physical locationof a SERS-active inclusion through tissue? We have conducted a studythat investigates multiple factors pertaining to the optical settingswithin imaging experiments on an in-house-built SORS system to addressthis question.x, y-imaging planeat depth. More specifically, we present the effect of “linearoffset-induced image drag” which refers to a spatial distortionin SESORS images caused by the magnitude and direction of the linearoffset vector and highlight the need for a radial SORS collectiongeometry during imaging to neutralize effects such as asymmetric imageartifacts that would otherwise lead to misinterpretation of data andincorrect identification of the inclusion location. Additionally,building on these principles, we simultaneously introduce the conceptof “ratiometric SESORS imaging” for the location ofburied inclusions in three dimensions. This technique differs fromthe more traditional form of SESORS imaging, in which the intensityof a specific SERS band from the subsurface NPs is used to quantifyeach pixel, in that the relative contribution of the NP SERS bandto the through tissue spectrum, INP/ITis, is used to quantify the value of each pixelto build the image. This ratiometric technique was first reportedby Asiala et al. in a study that utilized spin-coated SERS-activeNP samples to monitor INP/ITis as a function of tissue barrier thickness and magnitudeof a linear offset vector and indicated a positive relationship betweenthe thickness of the tissue barrier and the offset magnitude requiredto access inclusions behind those barriers.21 Mosca et al. reached a similar conclusion using Monte Carlo simulationsand stipulated that when analyzing a turbid sample with a point-basedcollection SORS geometry, it is possible to relate the magnitude ofthe linear offset vector to the depths that are being probed by thelaser.22 These principles are vital indeveloping a methodology for the location of SERS-active inclusionsin three dimensions, and we test the robustness of our approach, whichutilizes the relationship between the magnitude of the spatial offset,the probed depth, and the ratiometric analysis, INP/ITis, to ultimately imageand spatially discriminate between two distinct NP flavors buriedat different depths in three dimensions for the first time.Herein, we reportthe effects of the magnitude of the spatial offsetand the nature of the geometry in locating SERS-active NPs in a novelSESORS imaging template through tissue and outline experimental techniquesto allow for the correct interpretation of SESORS images and the locationof NPs in the 2D H-pyrazol-4-yl)pyridine(PPY), 1,2-bis(4-pyridyl)ethylene (BPE), methanol, and (3-aminopropyl)triethoxysilane(APTES) were purchased from Sigma-Aldrich. Milli-Q water (MQ H2O) was prepared in-house. Sodium silicate in aqueous solutionand a silica gel (40–63 μm) were purchased from VWR chemicals.The Raman reporters, BPE and PPY, were prepared in stock solutionsby dissolving the solid in methanol. Subsequent dilutions were thencarried out in MQ H2O. Lean pork back tissue was used asa mammalian tissue analogue. Samples were purchased from a local supermarketand cut into sections (roughly 50 mm × 50 mm with a 3 mm thickness).Sodium tetrachloroaurate(III)dihydrate, trisodium citrate, 4-(123 A 785 nm laser (Innovative Photonics Solutions)with an attenuable output was coupled to one of the two fiber opticRaman probes (Wasatch Photonics Ltd) with built-in filtering optics.The probes were mounted on independent x, y, z-translational stages with rotationalmounts (ThorLabs) for accurate positioning and as a convenient meansof directly measuring the spatial offset magnitude. The collectionprobe was coupled to a f/1.3 Raman spectrometer , and the excitation and collection probeswere angled at approximately 60° from each other. Spectra werecollected for 1 s using a laser power of 400 mW. It is worth commentingthat the maximum permitted exposure for the skin is exceeded withthe laser settings used and that the settings were selected primarilyto improve the through tissue signal of the NPs for demonstrationpurposes. Since the intended application of this work is in vivo biomedicalsensing, any future clinical studies would have to account for thisfactor.A Cary 60 UV–Visible spectrophotometer with a wavelengthrange of 300–800 nm and a scan rate of 24,000 nm/min was usedto acquire extinction spectra, which were used to characterize theoptical properties of the NPs. A Malvern Zetasizer nano ZS systemwas used to conduct dynamic light scattering (DLS) and zeta potentialmeasurements. Samples were added to a poly(methyl methacrylate) cuvettewith 1 cm path length for all measurements. For zeta potential measurements,a dip cell was placed in the cuvette. SESORS experiments were conductedon an in-house-built SORS system with a point-based collection geometry,developed by Asiala et al. based on the design of Shand and co-workersand is shown in 24 Sodium tetrachloroaurate(III) dihydrate(70 mg) was added to MQ H2O (500 mL) and heated to boiling.Trisodium citrate (42 mg) was added to MQ H2O (7.5 mL),and the two solutions were combined. The resulting solution was boiledfor 15 min with continuous stirring and then allowed to cool. SERS-activesilica-coated NP aggregates were synthesized using a method modifiedfrom the study by Li et al.25 Raman reporters were added separately to solutions ofAuNPs (100 mL) under stirring. The solution was stirred until aggregationoccurred, and the SERS response was optimized. APTES  was then added, followed immediately by sodium silicate. The mixture was stirred vigorously at 90°C for 30 min, and it was then removed from heat and stirredovernight. The NPs were then centrifuged  and resuspended in MQ H2O (30 mL). Powder-basedSERS-active imaging templates, shown in Gold NP cores with an average sizebetween 50 and 60 nm were synthesized using a modified Turkevich methodwhere citrate ions act as the reducing and capping agents for theNPs.x–y stage with a range of 2.54 cm was used to maneuver the tissue samplesin the x, y-imaging plane, z-translational stage below the imagingstage. The samples were focused by raising the stage vertically withthe laser and spectrometer operational until the tissue contributionto the resulting SORS spectrum was maximized, that is, by trackingthe intensity of the 1425 cm–1 CH2 scissoringband characteristic of lipids in mammalian tissues. This indicatedthat the excitation and collection probes were focused on the samespot on the tissue surface because the relative contribution of thesurface was maximized in the resulting spectra. To mimic a ring-collectionoffset, a rotational mount, shown in x, y, z-translationalstage to allow for rotation of the samples four times through 360°in 90° steps and simulation of a rotating linear offset vector.Images were recorded between each rotation in the manner shown in SESORS imaging was performed on an in-house-builtSORS system with a point-based collection geometry. Approximately30 mg of the powder-based SERS-active NP imaging templates was transferredand placed on top of a section of a porcine tissue, –1 for PPY and BPE, respectively,based on Raman shift calibrations) was plotted at each pixel as afalse-color 2D heat map. For ratiometric images, the SERS intensityof the NPs was divided by the maximum Raman intensity of the tissue(1425 cm–1) across all pixels in the image (INP/ITis max). These values were then plotted at each pixel as false-color 2Dheat maps. Where stated, the color bars across each image in a setof ratiometric images were scaled for clarity, for example, in a fixeddepth and variable offset experiment to allow for three-dimensional(3D) sectioning of the tissue block. To mimic a ring-collection offset,ratiometric images were collected as the sample was rotated through360° in 90° steps to simulate rotation of the linear offsetvector. Since the samples were being physically rotated anticlockwise,the images were rotated clockwise during processing to counteractthis and to simulate the linear offset vector being rotated in 90°steps; then, the images containing the four different linear offsetvectors were averaged at each pixel. The averaged ratiometric valuesat each pixel were then plotted as false-color 2D heat maps.All spectra were processed using MATLABsoftware . Preprocessinginvolved truncating and baselining the spectra which correspondedto image pixels. For intensity images, the SERS intensity of the NPs biological window for through tissue SESORSimaging experiments. Previous SESORS studies utilized NP-spin-coatedglass slides with uniform SERS signals across the sample or NP liquidsspotted and dried directly on the tissue.INP/ITis, is useful for providing information aboutthe depth of NPs within tissue in SESORS measurements.28 When the depth of the NPs is varied and the offset is fixed, thisanalysis technique can be used to calibrate the Raman intensity ofthe NPs against the depth using log-linear regression, and when theoffset is varied and the depth is fixed, it allows for different depthsto be probed within the NP/tissue system. Therefore, it would be highlyadvantageous to apply ratiometric analysis to SESORS imaging to notonly allow for determination of the location of a SERS-active inclusion,and hence a disease state, in the x, y-imaging plane but also gain information about the inclusion locationin the z-axis, that is, the depth. A comparison betweenintensity and ratiometric SESORS imaging is provided in Figure S2.It has previously been establishedthat ratiometric analysis of the subsurface NPs versus the surfacetissue Raman intensities, INP/ITis can be modeled as a function of the tissue barrier thickness andthe spatial offset magnitude, it follows logically that the intensityratio should be maximized at an offset proportional to the depth atwhich the NPs are buried behind the tissue barrier. For example, theintensity ratio would be maximized at a small offset magnitude whenthe NPs are buried close to the surface–air interface and viceversa. This result is in accordance with previous work that noteda strong correlation, the nature of which varies with the scatteringproperties of the turbid medium, between the SORS-probed depth andthe spatial offset applied.22 This phenomenonwas observed, and the intensity ratio of the high-intensity areaswithin the images was maximized at the larger spatial offset magnitudes.The ability of ratiometric SESORSimaging to probe different depths through tissue using NPs at a fixeddepth and incrementally varying spatial offsets was investigated usingthe in-house-built SORS spectrometer. The silica powder mixed withsilica-coated PPY-functionalized gold NPs (PPY powder) was buried15 mm behind the tissue barrier, and ratiometric images were collectedacross a 25 × 25 mm grid in 5 mm steps with linear offset magnitudesof 0, 2, 6, and 8 mm, x, y-imaging plane since the excitation and collection probes were focusedon the same position on the tissue surface at each pixel, and anyRaman-scattered photons from the subsurface NPs would only be collectedat a pixel if the SERS-active inclusion was present in that location.It is crucial to note throughout this study that the Raman intensityat any given pixel is related to where the sample was excited andnot collected because when a spatial offset is applied, these twopoints become separated. The directionality of this effect was comparedto the direction of the linear offset vectors on the set of images,that is, the path from the excitation point, E, to the collectionpoint, C. The direction of the offset vector was calculated by mappingeach pixel from the physical experiment onto the final image and mappingthe physical position of the excitation and collection probes in thesame way. Inclusion of the linear offset vector in the set of ratiometricimages revealed the position of the maximum intensity areas withinthe images. This was noted previously by Botteon et al. in a micro-SORSimaging study of art samples, and the effect can also be observedin variable offset SORS images of a plastic inclusion through bonein the work by Nicolson et al., although in both cases the implicationof the effect for the through barrier x, y-plane localization of subsurface targets was not discussed.29 We have termed this phenomenon of movement in the image againstthe offset vector as “linear offset-induced image drag”.This is observed in SESORS imaging when a linear offset is appliedbecause the excitation and collection probes are focused on differentareas on the tissue surface, and the photons follow a linear paththrough the tissue medium between the two spots.31 Therefore, if a SERS-active NP inclusion were to lie within theaxis of the linear offset vector, it would likely be excited, andhence, the resulting Raman-scattered photons would be detected. Linearoffset-induced image drag occurs in the direction opposite to thelinear offset vector because an inclusion may be detected when itis buried beneath the collection point, that is, it will be detectedeven when the excitation point is offset from the real location ofthe inclusion. Linear offset-induced image drag is shown and describedfurther in Figure S3. The discrepancy betweenthe two probes used in SESORS imaging is shown in x, y-plane is not an objective.It was also observed that as the spatial offsetmagnitude was increased,the maximum intensity area within the images moved relative to theimages collected with no spatial offset. This can be observed moreclearly in the corresponding intensity SESORS images recorded at linearoffset magnitudes of 0 mm and 8 mm, High-resolution ratiometric SESORSimaging was performed to ascertainwhether the magnitude of the linear offset vector was proportionalto the magnitude of linear offset-induced image drag between images.The PPY powder was buried beneath 3 mm of tissue, and images werecollected on the in-house-built SORS system across a 25 × 25mm grid in 1.25 mm steps with linear offset magnitudes of 0 and 2mm. This was repeated, but the image step size was altered to 5 mm,meaning that the resolution was lower (36 pixels vs 441 pixels). Theseresults can be observed in 32 Initial numerical studies of SORS specified that when using a ring-collectionSORS geometry, spectral data could be improved significantly overa point collection SORS geometry in terms of the signal-to-noise ratio.33 Additionally, since this optical setup is symmetricaland involves the collection of photons from 360° around a centralexcitation point, it was hypothesized that it could be adopted toremove asymmetrical linear offset-induced image drag. This sectioninvestigates the effect that a mimicked ring-collection spatial offsethas on the location of NP inclusions in ratiometric images with avariable offset magnitude. We sought to ascertain if it would be abetter indicator of the location of NPs through the tissue in the x, y-imaging plane by introducing a rotationalvariable in imaging experiments. A ring-collection SORS geometry wasimitated by mounting a rotational platform with a range of 360°beneath the optical setup of the in-house-built SORS spectrometerand on top of the previously used x, y, z-translational stage. Ratiometric images werecollected of the PPY powder buried behind 3 mm of tissue across a25 × 25 mm grid in 2.5 mm step sizes with linear offset magnitudesof 0 and 8 mm. This was repeated three times with the sample beingrotated through 360° in 90° steps to mimic the rotationof the linear offset vector. Since the samples were being physicallyrotated anticlockwise, the images were rotated clockwise during processingto counteract this and to simulate the linear offset vector beingrotated in 90° steps, as can be observed in x, y-imaging plane than a linear point-basedcollection SORS geometry.From the previous sections, itis evident that steps must be taken to eradicate linear offset-inducedimage drag for the correct location of SERS-active NP inclusions throughtissue. Linear offset-induced image drag occurs because of asymmetryin the imaging process, and to remove this effect, it is necessaryto either correct for it numerically, which is possible because previouswork stipulates that the maximum signal in a linear SORS measurementis typically placed halfway between the excitation and collectionpoints, or by adopting an alternative optical geometry.31 Schematics in To explain why a ring-collection SORS geometryremoves linear offset-inducedimage drag and is a better indicator of the inclusion location throughthe tissue than point-based collection, a visual aid showing fourhypothetical scenarios is provided in 22 At the larger offsets used, the contribution of the NPs in the spectraand images is lower, indicating that these offsets are too large,and the excitation photons are focused on depths beneath the NP inclusions.This has the potential to be a new methodology for detecting the locationof a NP inclusion in the 2D x, y-imaging plane using the ring-collection offset to remove image dragand in the z-depth plane by altering the magnitudeof the ring-collection offset. Unlike an alternative method developedby the authors that requires internal calibration of the NP SERS intensity,this technique would not require prior knowledge of the NP locationbecause it is primarily affected by the parameters of the opticalsetup.28 However, associating a specificdepth in a quantitative manner with experimental observables wouldstill require knowledge of the optical properties of the tissue andparallel photon propagation simulations because the relationship betweenthe spatial offset magnitude and the penetration depth of the laserhas been previously found to be dependent on the scattering propertiesof the sample matrix.22It followsfrom the previous sections that ratiometric SESORS imaging with anincreasing ring-collection offset magnitude should allow 3D trackingof NP inclusions through the depth axis in a fixed NP/tissue system.Here, this hypothesis was tested through the ratiometric imaging ofthe PPY powder buried at a depth of 6 mm beneath the tissue usingthe in-house-built SORS system across a 20 × 20 mm grid in 5mm step sizes with mimicked ring-collection offset magnitudes of 0,2, 6, and 8 mm. Again, to do this, ratiometric images of the powderwere collected with the four linear offset magnitudes and a constantlinear offset vector direction. This was repeated three times withthe sample being rotated through 360° in 90° steps to mimicthe rotation of the linear offset vector; then, to imitate ring-collectionSORS images, the images containing the four different linear offsetvector directions and the same offset magnitude were averaged at eachpixel. The results of this are shown in z-plane,but at an equivalent position in the x, y-imaging plane, within a single tissue model. Since the spatial offsetmagnitude can be related to the depth probed by the laser, it followsthat ratiometric analysis of two different NP inclusions buried atdifferent depths should yield maximum NP contributions at separateoffset magnitudes, thereby separating the two inclusions in threedimensions. To achieve this spatial discrimination, samples consistingof the silica powder mixed with silica-coated BPE-functionalized goldNPs (BPE powder) and the PPY powder were buried separately beneath3 mm (BPE) and 9 mm (PPY) of tissue and imaged using the in-house-builtSORS system across a 20 × 20 mm grid in 5 mm step sizes withmimicked ring-collection offset magnitudes of 0, 2, 6, and 8 mm. Carewas taken to ensure that the PPY and BPE powders were placed at thesame position in the x, y-imagingplane, so they were only separated in the depth axis. Ratiometricimages of the two flavors were collected with the four linear offsetmagnitudes and a constant linear offset vector direction. This wasrepeated three times with the sample being rotated through 360°in 90° steps to mimic the rotation of the linear offset vector.For ring-collection SESORS images, the images containing the fourdifferent linear offset vector directions for the same offset magnitudewere averaged at each pixel. The results of this section are shownin Ratiometric SESORS imaging with an increasing ring-collection offsetmagnitude has been presented for 3D tracking of NP inclusions througha depth axis in a fixed NP/tissue system. To test the robustness ofthis method for 3D ratiometric imaging and inclusion location, wesought to achieve spatial discrimination between two “flavors”of SERS NPs, that is, the silica powder mixed separately with differentRaman reporters, placed at different depths, 34 The maximum contribution fromthe PPY powder occurred at the larger 6 mm offset since it was placedat a depth of 9 mm. These results and indeed all of the results hereinthat utilize ratiometric analysis for depth determination are broadlycomparable to the theoretical findings and experimental studies onplastic models in the study by Mosca et al., in which the median,10, and 90% quantiles of SORS-probed depths are determined as a functionof linear spatial offset magnitude in samples with different scatteringproperties. Deviations from these models can be explained by the morelimited number of spatial offset magnitudes investigated, the differentoptical arrangement used, and heterogeneity in the optical propertiesof the ex vivo porcine tissue.22 It islikely that with a larger number of measurements, this ratiometricanalysis technique would demonstrate a maximum contribution of BPEat a spatial offset magnitude between 0 and 2 mm. However, it is clearthat these two observations together suggest spatial discriminationof the two samples in three dimensions. This result reinforces thepotential that SESORS imaging holds for multiplexed in vivo detectionbecause the experiments conducted not only detect and classify multiplevibrational fingerprints at depth but also provide a means to locatemultiple targets simultaneously in three dimensions.19 This has implications for a wide range of clinical applications,including cancer imaging, infection diagnostics, and drug deliverymonitoring.38Since the two flavors of nanotags were buried atdifferent depthswithin the single tissue model, the combination of techniques alloweddiscrimination between the two samples in a 3D space using ratiometricanalysis and the mimicked ring-collection SORS geometry. The resultingratiometric images for the BPE powder show a maximum contributionof SERS NPs at the 0 mm offset, in line with previous experimentsin this study, since the BPE powder was placed at a small tissue depthof 3 mm.INP/ITis), and it was thisratiometric intensity value that was used for the quantification ofpixels in SESORS imaging experiments. Combining the principles ofratiometric analysis and linear offset-induced image drag minimization,we ultimately imaged and spatially discriminated between two distinctNP flavors buried at different depths in three dimensions for thefirst time. This is a significant step forward in the field of SESORSand through tissue detection, and to the best of our knowledge, itis the first time that theoretical principles have been defined forthe universal interpretation of SESORS images, which have the potentialto be applied to in vivo research and diagnostics in a clinical settingin the future.In summary, we have conducted a study examiningexperimental techniquesfor the correct interpretation of SESORS images and outlined a methodologyfor the accurate location of targeted NPs at depth in three dimensions,which could be applied to the detection of any relevant disease state.The work investigated multiple factors pertaining to the optical settingswithin through tissue imaging experiments on an in-house-built point-collection-basedSORS system and reported the effects of the spatial offset magnitudeand geometry in locating SERS-active NPs through the tissue. We presentedthe effect of linear offset-induced image drag which refers to a spatialdistortion in SESORS images caused by the magnitude and directionof the linear offset vector and then utilized a ring-collection-basedSORS geometry to remove asymmetric drag effects in images to locatea NP inclusion more accurately in two dimensions. Additionally, toallow for the location of an inclusion through tissue in three dimensions,we introduced the concept of ratiometric SESORS imaging which utilizesthe widely reported relationship between the magnitude of the appliedoffset and the depth probed by the laser. This relationship can beobserved experimentally by conducting ratiometric analysis of thesubsurface NP and tissue barrier Raman intensities (


The activitiesof both polymerases are monitored simultaneously by using Broccoliand malachite green light-up aptamer systems. In our toggle switch,a T7 promoter drives the expression of SP6 inhibitory aptamers, andan SP6 promoter expresses T7 inhibitory aptamers. We show that thetwo distinct states originating from the mutual inhibition of aptamerscan be toggled by adding DNA sequences to sequester the RNA inhibitoryaptamers. Finally, we assessed our RNA-based toggle switch in degradingconditions by introducing controlled degradation of RNAs using a mixof RNases. Our results demonstrate that the RNA-based toggle switchcould be used as a control element for nucleic acid networks in syntheticbiology applications.SyntheticRNA systems offer unique advantages such as faster response,increased specificity, and programmability compared to conventionalprotein-based networks. Here, we demonstrate an  Consequently, a great deal of attentionhas recently been devoted to the development of various RNA-basedcore elements such as riboswitches,8 ribozymes,9 short interfering sRNAs,10 RNA aptamers,12 and riboregulators.14 Among these, RNA aptamers—nucleic acid sequences capableof recognizing and binding to their target molecule with high affinityand specificity—have been used to create RNA-based circuitsby interfacing RNA with proteins and other small molecules.19RNA synthetic biologyuses RNA-based components to control geneexpression and engineer biological systems at the molecular level.in vitro setups. In vitro transcription systems have becomean attractive alternative not only to construct and characterize biologicalcircuits without involving the complexities of living systems, butalso for other applications such as diagnosis.21 With full control over the concentrations and stoichiometries ofeach component, these platforms are ideal for the rapid prototypingof genetic circuit designs as well as for developing mathematicalmodels to characterize these systems and help fine-tune their design.23RNA-based networks lend themselves to in vitro transcriptionsystems are bacteriophage RNA polymerases due to their high processivityfor catalyzing the formation of RNA from DNA templates. Among them,T7 and SP6 RNA polymerases (RNAP) are DNA-dependent RNA polymerasesthat are specific for the T7 and SP6 promoters, respectively. Overthe last years, several RNA aptamers have been described as specificallyinhibiting the activity of the T7 RNA polymerase24 and SP6 RNA polymerase.25 Theseinhibitory RNA aptamers have been used as regulators for syntheticbiological circuits and metabolic engineering applications.27 For instance, Lloyd et al. demonstrated dynamic control of bothRNAPs in in vitro reactions by applying the principlesof strand displacement reactions.28 Theyproposed the use of DNA kleptamers—single-stranded DNA oligonucleotideswith complementary sequences to the RNA inhibitory aptamers—tocharacterize minimal circuits with dynamic control over the activationof the RNA polymerases. Transcription-based RNA circuits have beensuccessfully implemented with other networks such as CRISPR-Cas9 systems.31 Here, we take RNA-based circuits to the next level in complexityand bring them closer to a future application in biological systems.In this work we described an in vitro RNA-based toggleswitch in the presence of both RNA synthesis and degradation. A toggleswitch is a bistable network that can be set to either one of twostable states creating a memory unit with an “on” and“off” position. Our circuit is an experimental realizationof a strategy proposed by Mardanlou et al.32The most commonly used enzymes for 33 Among them, spinach and Broccoliaptamers emit a green fluorescence signal in the presence of the fluorophoremolecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI).36 Mango aptamer can bind to thiazole orange molecules to increasefluorophore fluorescence.37 The malachitegreen aptamer binds to a triphenylmethane dye and emits red (650 nm),39 and the corn aptamer can dimerize activating the fluorescence ofa modified fluorophore from DsRed.40 Tominimize the bleed-through signal between different channels, we usedtBroccoli and malachite green aptamers.To visualize the transcriptional process in real-time, light-upRNA aptamers are commonly used as transcriptional reporters. Light-upaptamers are short RNA sequences capable of mimicking the activityof fluorescence proteins. These probes have specific stem-loops thatserve as binding pockets for small fluorophores. The quantum yieldof the fluorophore significantly increases when it binds to the RNAaptamer.The architecture ofthe RNA-based toggle switch here presentedis composed of two DNA templates: a first template where a T7 promoterexpresses a SP6 inhibitory aptamer and tBroccoli aptamer, and anothertemplate where a SP6 promoter produces a T7 inhibitory aptamer anda malachite green aptamer 1. The prIn this work, we first show thatthe transcriptional activity ofboth RNAPs can be suppressed when the inhibitory RNA aptamers areexpressed in either cis- and trans-acting circuits. We then demonstratethe recovery of function of RNAPs from cis- and trans-acting circuitsby adding DNA kleptamers. Next, we show that the RNA-based toggleswitch works at the transcriptional level and can be “toggled”by the addition of DNA kleptamers. Finally, the RNA-based toggle switchproposed is studied in the presence of external RNases to build afoundation for future biological technologies.in vitro transcriptionreactions. In the presence of the specific dye, the fluorescence signalof each reporter is detected without significant bleed-through . In this work, we decided to separate both molecules by intercalatingthe hammerhead ribozyme (CChMVd-U10) between the light-up and inhibitoryRNA aptamers to improve their folding efficiency and performance.41Wefirst tested the ability of the inhibitory RNA aptamers to bind toand repress the activity of their RNAP targets. For this purpose,we built two cis-acting circuits: one with the SP6 promoter expressingmalachite green and SP6 inhibitory aptamers, and another circuit whereT7 promoter expresses tBroccoli and T7 inhibitory aptamers. In eachDNA template, the inhibitory aptamers are coexpressed along with thefluorescence reporters to monitor the Both cis-acting circuits showed a significantdecrease in the fluorescence signal when the inhibitory aptamers wereexpressed compared to the expression of only the fluorescent aptamers2a,b. TheSI Figure 1). Whileprevious works characterized DNA kleptamers that partially cover thesequence of the inhibitory aptamers,28 wedesigned and tested DNA kleptamers that cover the full sequence ofthe inhibitory aptamer. Longer kleptamers performed slightly betterthan the previous reported sequences. We also tested different architecturesof the synthetic circuits to observe a significantly better recoverywhen the fluorescent RNA aptamer was placed 5′ upstream ofthe ribozyme in the DNA template .After confirming repression of the RNAPs by their cognateinhibitoryRNA aptamers, we tested the recovery of transcriptional activity byusing DNA kleptamers. We used the malachite green aptamer as the reporterfor both T7 and SP6 systems and tested the efficiency of DNA kleptamersthat covered the sequence of RNA inhibitory aptamers to varying degrees. As shownin SI Figure 4).Motivated by these results, we decided to test more designsforDNA kleptamers covering different regions of the sequences. We alsoincorporated additional binding domains on the kleptamers to testif the dissociation efficiency could be improved by introducing additionalbinding energy  to obtain better recovery rates than with tBroccoli reporter.In addition, we observed a similar performance of tBroccoli when itwas expressed within the SP6 circuit . We observed a less significant increase with the tBroccolifluorescence signal using the SP6 kleptamer. Nonetheless, the recoveryrates, normalized with their negative controls, were similar for T7and SP6 kleptamers, and different versions of kleptamers showed onlya marginal difference in recovering the fluorescence signal. On thebasis of these results, the recovery of SP6 and T7 RNAP’s activitywas deemed sufficient for the purpose of constructing an RNA-basedtoggle switchThe activity of T7 RNAP was also recovered after adding 2 μMof the kleptamer 2d. AlthoSI Figure 8). We observed that the addition of the incorrect DNAkleptamer had no significant effect in the fluorescence signal ofthe reporter. This orthogonality is particularly important to ensurethe correct performance of the RNA-based toggle switch.The next step was to assess the performance of bothtrans-actingcircuits that, together, will generate the RNA-based toggle switch.First, we built a circuit in which the T7 promoter expresses tBroccoliand SP6 inhibitory aptamer that, in turn, represses the expressionof malachite green aptamer in another DNA template 3a. With We also tested the other trans-acting circuit inwhich the T7 inhibitoryaptamer is produced via an SP6 promoter. The T7 inhibitory aptamerrepresses the expression of tBroccoli aptamer in another DNA template3c. When We createdthe RNA-based toggle switch by combining both trans-acting circuitsthat we characterized 4a. When SI Figure 9). This is an essential feature fora correct operation of the toggle switch as it pushes down the signalfrom the suppressed aptamer when its transcription is being repressed. SI Figures 10–12.Finally, we introduced a mix of purified RNases in definedamountsto continuously degrade the RNA components in our toggle switch SI 9. This iin vitro RNA-based toggle switch and observedits performance in degrading conditions. The advantages of RNA moleculesincluding fast dynamics, high turnover, increased specificity, andprogrammability make them ideal for building complex networks formetabolic engineering and synthetic biology. On the basis of previousworks on minimalist RNA-based circuits28 and theoretical modeling of an RNA-based toggle switch,32 we demonstrated the behavior of this specificnetwork in vitro and highlighted the potential forimplementing this network in living cells and other in vitro applications.21 Degrading conditions are closerto how the circuit would behave in a cellular environment and canhelp to understand its performance in that context. The next stepwould be testing the RNA-based toggle switch in more cell-like conditionssuch as cell lysates.We have presented the experimental implementationof an 42 We observed better recovery of RNAP activities via DNA kleptamerswhen the light-up RNA aptamers were placed before the ribozyme . We hypothesize thatthe residual ribozyme sequence left at the 3′-end (self-cleavingoccurs near its 5′-end) could have more effects in the secondarystructure of light-up aptamers than the inhibitory aptamers. Thisfactor seems to particularly affect the tBroccoli aptamer, for whichrecovery of the fluorescence signal is less significant than for themalachite reporter after adding the kleptamers. We found that bothT7 aptamer and T7 kleptamers performed better when used with malachitegreen aptamer . We also foundthat tBroccoli expressed in the SP6 cis-acting circuit produces evenless significant recovery than with the T7 system . For these reasons, we believe that RNA secondarystructures play an important role in the performance of RNA-basednetworks in vitro. However, this effect could potentiallybe further improved by the use of other ribozymes,44 catalytic tRNAs,45 or through engineeringthe sequence of the HHRs9 and also, testingthe performance of other RNA fluorescence aptamers such as Spinach,35 mango,37 or corn.40 Combining our in vitro systemwith high-throughput measurements would allow the characterizationof the system over a wider area of operating conditions and parametervalues.The successful performance of the RNA-basedcircuits involving RNA inhibitory aptamers and light-up aptamers dependson the secondary structure of the RNA elements. Considering this possiblelimitation, we decided to separate both RNA aptamers using a self-cleavinghammerhead ribozyme between both structures.in vitro transcription slows down as rNTPs areconsumed. To maintain the activity of RNAPs for longer, we used higherconcentrations of rNTPs than standard in vitro conditions.With these conditions, RNAPs remain active for 10 h and we could demonstratetransitions between states by adding kleptamers. However, even a 10h-long window was not enough to reach the steady states within eachbistable regime in these in vitro experiments. Byusing techniques such as microfluidics, it should be possible to maintainthe activity of RNAPs for longer and characterize the behavior ofour circuit more completely. Taking into account this degrading environment,we used DNA-based kleptamers for building the RNA-based toggle switchrather than RNA-kleptamers. However, RNA kleptamers could be usedfor this circuit as well as single-stranded DNA expression systems, although more theoreticaland experimental work is required to ensure its bistability since,in the presence of RNases, RNA kleptamers would also be susceptibleto degradation.To prove the functionality of the RNA-based system indegradingconditions, we added a mix of RNases in our system. However, the rateof Supporting Information.DNA oligonucleotides and double-strandedDNA (gBlocks) were synthesized by Integrated DNA Technologies  and resuspended in nuclease-free water. NUPACK software was usedto computationally check the secondary structures for cis- and trans-circuits.gBlocks were amplified by PCR using Q5 High-Fidelity DNA Polymerasefrom NEB (NEB #M0492L), and the NEB Tm calculator tool was used toestimate the annealing temperature. PCR products were purified usingMonarch PCR & DNA Cleanup Kit (#T1030). All nucleic acid sequencesare reported in the H-imidazol-5(4H)-one) (DFHBI-1T) was purchased from Bio-TechneLtd. (Cat. 5610/10), resuspended in DMSO and stored at −20°C. RNase Cocktail Enzyme Mix was added with a 1/320.000 dilutionfactor (Life technologies #AM2286) when relevant. Replicates shownin SI Figures 1 and 2 were performed bycreating a reaction master mix with the corresponding DNA templates,enzymes, and dyes and then, splitting it into individual reactions.Technical replicates were performed for the main figures in whichreplicates were individually composed . Each technical replicate is shown in a different graphwhen the experiments were carried out on different days.These reactionswere performed in 50 μL in a transcription mix prepared with200 ng of each DNA template, 1X RNA polymerase Buffer (NEB #B9012S),8 mM rNTPs (NEB #N0466S), 250 U T7 RNA Polymerase (NEB #M0251L), and/or100 U SP6 RNA Polymerase (NEB #M0207L), 15 μM of DFHBI-1T and/ormalachite green dye, and RNase-Free water (Thermo Fisher #AM9932).Malachite green oxalate salt was purchased from Sigma-Aldrich (Cat.No. M6880-25G) and was dissolved in nuclease-free water and storeat −20 °C. For tBroccoli aptamer, (Z)-4--2-methyl-1--1Samples were transferredto flat μClear bottom 96-well plates(Greiner). Fluorescent measurements were obtained using a CLARIOstarPlus microplate reader (BMG LABTECH) and reading from the bottom.The temperature of the plate reader was set at 37 °C. Fluorescencesignals were measured throughout the duration of the experiment. Onlyat the time of manual addition of kleptamer at a concentration of2 μM, was the plate reader stopped, and the plate was outsideof the plate reader for less than 5 min. The sample was mixed to ensurethe distribution of the DNA kleptamer inside the well. Excitationand emission wavelengths for the dyes were as follows: ex/em = 600/650nm for malachite green light-up aptamer and ex/em = 466.5/512.2 nmfor DFHB-1T.In vitro reactionswere performed in 50 μL for 2 h at 37 °C in a transcriptionmix prepared with 200 ng of each DNA template, 1X RNA polymerase buffer(NEB #B9012S), 8 mM rNTPs (NEB #N0466S), 250U T7 RNA polymerase (NEB#M0251L) and/or 100U SP6 RNA polymerase (NEB #M0207L), and RNase-Freewater (Thermo Fisher #AM9932). GeneJET RNA Cleanup and ConcentrationMicro Kit (Thermo Fisher #K0841) was used to purify the RNA molecules.


RNA-dependent RNA polymerases (RdRPs) represent a distinctive yet versatile class of nucleic acid polymerases encoded by RNA viruses for the replication and transcription of their genome. The structure of the RdRP is comparable to that of a cupped right hand consisting of fingers, palm, and thumb subdomains. Despite the presence of a common structural core, the RdRPs differ significantly in the mechanistic details of RNA binding and polymerization. The present review aims at exploring these incongruities in light of recent structural studies of RdRP complexes with diverse cofactors, RNA moieties, analogs, and inhibitors. RdRPs EC 2.7.7.48.) are ancient enzymes that catalyze the formation of phosphodiester bonds between ribonucleotides in an RNA template-dependent fashion using divalent metal ions. The reaction involves the nucleophilic attack of the 3’-hydroxyl group of the nascent RNA on the α-phosphate of the incoming nucleoside triphosphate (NTP), resulting in the formation of a new phosphodiester bond and the release of a pyrophosphate moiety A. Togeth.7.7.48. RdRPs are classified as alpha and beta proteins and fold majorly into three subdomains, namely the fingers, thumb, and palm B 3,5,6,,6,7,8. TReoviridae and phage φ6 are 148 and 76 kDa, respectively, and possess extended N- and C-terminal domains in addition to the core . T. TInflued A/H3N2 , where td A/H3N2 ,79. The d A/H3N2 . The stud A/H3N2 .Tick-borne encephalitis virus (TBEV) (PDB 7D6N) [Yellow fever virus (YFV) (PDB 6QSN)   and the DB 6QSN) F 82] in inTick-biviridae . RdRP has emerged as the most versatile protein for developing drugs against RNA viruses ,14,83. TPicornaviridae inhibitor complexes were shown to target antivirals at the interface of the fingers and the palm subdomains ..50 of ~6The derivatives of the NIT29 inhibitor (Ligand ID KY3) that showed preferential binding to an allosteric pocket between the thumb and palm subdomains  were used to screen for potential non-nucleoside inhibitors of Zika viral RdRP . FluoresPoliovirus , and EV71 . After protein preparation and removal of water molecules, the ligand-based docking was carried out with default parameters using a genetic algorithm. The NADPH showed a striking similarity in the binding poses that depended solely on the occupancy of the template channel of the RdRP. In the apo structures, the NADPH was bound inside the template channel closer to the pinky of the fingers subdomain, whereas in the template-bound elongation complexes, the ligand was displaced by more than 5 Å away from its original position and towards the thumb subdomain owing to the binding of the RNA (The in vitro titration of the polymerases of EVD68, EV71, and Poliovirus with NADPH resulted in decreased activity where the ligand acted as an allosteric inhibitor rather than a chain terminator during replication . Further the RNA . The int the RNA . Intrigureoviral RdRPs demonstrated that the disruption of capsid protein interactions with RdRP during disassembly provided the essential room and energy for transcription to start [picornaviruses [caliciviruses [mononegaviruses [arenaviruses [pestiviral RdRPs promoted the unwinding of the RNA duplex [Recent investigations, largely cryo-EM-based, have revealed structural details of the RdRPs bound with cofactors, nucleic acid moieties, analogs, and inhibitors, enhancing our understanding of their mechanism of action. Although the fundamental structure of RdRP has stayed constant over time, there are variations at the individual level that remain unexplored. The association of RdRPs with cofactors that are either self-encoded or derived from the host facilitates the timing of genome replication and transcription. For instance, the in situ structures of to start ,70. Likeaviruses  and the iviruses  aided inaviruses ,47, and aviruses ,97, promaviruses  inhibiteaviruses , and theA duplex . The varA duplex ,98. For A duplex . CuriousA duplex . Thus, t


High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3′ termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) – a novel RNA-seq library preparation method directed towards mRNA 3′ termini. We demonstrate the specific enrichment for 3′-terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates. Most isogenic cells of multicellular organisms produce transcripts with a complex spatial, temporal and structural variety. RNA abundance regulation and alternative processing play a central role in shaping phenotypic complexity, with more than 90% of human genes undergoing alternative splicing and nearly 70% having multiple polyadenylation sites .Advances in sequencing technologies empower to study quantitative and structural aspects of RNA biology down to a single-nucleotide level resolution from inputs as low as the contents of individual cells ,5. WholeDifferent types of noise which are usually classified as either technical or biological by origin influence quantitative results in RNA-seq . TechnicWe have previously shown that introduction of sequencing adapters via enzymatic incorporation of base-modified dideoxynucleotides into nascent DNA can substantially improve the informativeness of 16S rRNA sequencing for the characterization of microbial communities . Here, wWe developed MTAS-seq , which lThe use of ‘click’ chemistry as a mechanism of adapter addition that follows terminated polymerization reaction was previously reported ,20. HereTo validate the technique, we sequenced MTAS-seq libraries prepared from well-characterized HeLa and UHRR total RNA samples spiked with ERCC transcript mixes, with three technical replicates per RNA input, which ranged from 0.5 ng to 500 ng. The obtained libraries were of similar size indicating that OTDDN incorporation rate is robust across different RNA inputs given the same OTDDN ratio to corresponding dNTPs . 99.4–99.8% of sequencing reads from each sample mapped to the human genome and ERCCs after UMI trimming, with strand specificity of >99%. Importantly, on average 94.5% of reads were uniquely mapped in HeLa samples, and 93.4% were in UHRR samples. The high percentage of uniquely aligned reads might be attributed to rather long insert sizes of MTAS-seq libraries. We obtained sequences for more than 19,000 genes in UHRR samples and nearly 15,000 genes in HeLa samples with only 2 M reads. Reverse transcription conditions demonstrated high specificity for mRNAs even though starting material was total RNA: there were virtually no traces of rRNA reads indicating no mispriming events and read coverage, as expected, concentrated at the 3′ terminal region of RNA transcripts .2) with the expected distribution is 0.91–0.94. We next evaluated the discriminatory power of differential expression detection by assessing ERCC ratio detection performance with receiver operating characteristic (ROC) curves and area under the curve (AUC) statistics. With at least 13 ERCC spikes detected per abundance ratio, AUC analysis indicated good diagnostic power of MTAS-seq assay, with AUC values >0.96 for all ratios  and, in parallel, from purified bulk RNA which amount corresponded to the cellular RNA contents used in the crude lysate experiment. Libraries for each input were prepared in quadruplicates.On average, 99.1% (98.6–99.3%) of sequencing reads aligned to the human genome in crude lysate samples, and 99.2% (96.7–99.5%) aligned to the human genome in purified RNA samples. The percentages of uniquely mapped reads on average were 92.8% for crude lysates and 94.5% for purified RNA. We observed a good agreement of gene detection capacity between corresponding lysate and RNA samples  A and B Despite the decreasing cost of next-generation sequencing, sample preparation remains expensive and can be prohibitive for large-scale experimental studies; thus, newly proposed library generation methods are expected to be both reliable and cost-effective. Here, we have demonstrated that the use of OTDDNs enables the simple and quick generation of cDNA libraries enriched for mRNA 3′-terminal sequences and reduces the number of workflow steps. Moreover, randomized sequence within the oligonucleotide modification tags each fragment with a unique barcode which in turn contributes to highly accurate estimation of transcript abundance in sequencing data.MTAS-seq approach can be viewed as a simplified version of chemoenzymatic library preparation, such as Poly(A)-ClickSeq . AlthougGene expression analysis strives for minimal perturbation of the original cellular RNA content during sample preparation. Previous studies revealed the feasibility of reverse transcription directly on cell lysates , and theOverall, MTAS-seq provides an accurate and efficient method for high-throughput gene expression profiling with the following highlights: (i) simple single-tube library preparation protocol exhibits solid performance in the range of 0.12–500 ng of total RNA; (ii) easy UMI labelling ensures bias correction for highly accurate estimation of transcript abundance and differential expression; (iii) protocol does not require RNA pre-processing, i.e. enrichment/depletion and fragmentation steps, before library generation and (iv) workflow is compatible with reverse transcription on cell lysates. This approach might greatly facilitate gene expression profiling studies to unravel molecular signatures of complex diseases. Further development of MTAS-seq could include the investigation of its broader applicability both in terms of compatible sample types, e.g. including degraded RNA samples and single-cell analysis protocols, and in terms of analytical capabilities, such as providing information on alternative polyadenylation.All reaction components were added to the reaction mixture as solutions in water unless specified otherwise. Modified oligonucleotide 5′-hexynyl-NNNNNNNNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG-phosphate-3′ (ON) was used for coupling to dideoxynucleotides. All oligonucleotides used in this work were synthesized by Metabion GmbH requesting HPLC purification.4  and THPTA  was then added to the reaction mixture, followed by the addition of sodium ascorbate . The reaction mixture was stirred for 20 min at 42°C, quenched with 0.5 M EDTA-Na2 solution . The products were purified by C18 reversed-phase chromatography using 100 mM TEAAc/ACN (11–18%) as eluent and desalted using water/ACN (0–100%) as eluent.5-(3-(2-Azidoacetamido)prop-1-ynyl)-2’,3’-dideoxycytidine-5′-triphosphate or 5-(3-(2-azidoacetamido)prop-1-ynyl)-2’,3’-dideoxyuridine-5′-triphosphate (3 eq.) solution was added to 5′-hexynyl-modified oligonucleotide (200–210 nmol) solution in sodium phosphate buffer . A premixed solution of CuSOONTP was obtained with 37% (78 nmol) yield. HRMS (ESI−): calculated 39 nt 8 N random oligonucleotide mean mass [M]: 12,743.080; found: 12,743.088. The oligo-modified ddUONTP product was obtained with 20% (40 nmol) yield. HRMS (ESI−): calculated 39 nt 8 N random oligonucleotide mean mass [M]: 12,744.064; found: 12,744.063.The oligo-modified ddCThe general scheme of OTDDN synthesis is depicted in Fig. S5.Samples. Universal Human Reference RNA and HeLa total RNA were used for proof-of-principle experiments. Invitrogen™ ERCC ExFold RNA Spike-In Mixes (Thermo Fisher Scientific) were used as external controls. Library preparation from cell lysates was performed using HEK-293 (ATCC CRL-1573) and BALB/3T3 (ATCC CCL-163) cells. To compare the library preparation performance using cell lysates and purified RNA, total RNA was extracted from 1 million HEK-293 cells using the Invitrogen™ PureLink™ RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer′s instructions. RNA quality was assessed by Agilent 2100 Bioanalyzer™ using the RNA 6000 Pico Kit (Agilent Technologies). RNA concentration was measured by NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific).Cell cultivation. Cells were cultured according to the standard mammalian tissue culture protocols and sterile technique. HEK-293 cell line was maintained in Dulbecco′s Modified Eagle's Medium (DMEM) supplemented with 2 mM L-glutamine, 10% foetal bovine serum, 1% gentamicin and 0.00028% β-mercaptoethanol (Thermo Fisher Scientific). BALB/3T3 cells were cultured in DMEM supplemented with 2 mM L-glutamine, 10% donor-sourced bovine serum and 1% gentamicin (Thermo Fisher Scientific). Cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C.MTAS-seq library preparation. Library preparation does not require rRNA depletion or mRNA enrichment as the RT primer effectively selects for polyadenylated transcripts. 0.1–500 ng of total RNA or 10–10,000 cells were used to generate libraries. Reverse transcription was performed in 20 µl reaction mixture containing 200 U of SuperScript™ IV reverse transcriptase (Thermo Fisher Scientific), 50 pmol of RT primer of sequence 5′-CTGGAGTTCAGACGTGTGCTCTTCCGATCT(T)30–3′, 20 pmol of dNTP mix, 40 U of RiboLock™ RNase Inhibitor (Thermo Fisher Scientific), 5 mM DTT, 2 pmol of ddUONTP, 0.4 pmol of ddCONTP in 1× of SuperScript IV RT buffer (Thermo Fisher Scientific). For library preparation from cell lysates, reverse transcription reaction was supplemented with 0.3% IGEPAL™ CA-630 (Sigma-Aldrich) to ensure cell lysis. The reaction was performed for 30 min at 50°C followed by termination at 80°C for 10 min. After reverse transcription, the reaction mixture was used directly for cDNA amplification. Reverse transcription reaction was supplemented with 25 µl of Invitrogen™ Collibri™ Library Amplification Master Mix (Thermo Fisher Scientific), 20 U of 3′-5′ exonuclease-deficient Phusion polymerase (Thermo Fisher Scientific) and 50 pmol of each of the unique dual indexing primers:i5 primer: 5′-AATGATACGGCGACCACCGAGATCTACAC[index]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′i7 primer: 5′-CAAGCAGAAGACGGCATACGAGAT[index]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′Cycling was performed as follows: denaturation at 98°C for 30 s, followed by 10–25 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 1 min and final extension at 72°C for 1 min. Each PCR reaction was then purified using Dynabeads™ Cleanup Beads (Thermo Fisher Scientific). DNA binding to the beads was performed by mixing 45 µl of bead suspension with 50 µl of sample and subsequent incubation at room temperature for 5 min. The sample was then placed on a magnet, the supernatant was removed and beads were resuspended in 50 µl of elution buffer containing 10 mM Tris-HCl (pH 8.0). 50 µl of fresh beads was added again to the sample and binding was repeated. After room temperature incubation, the sample was placed on a magnet, the supernatant was removed and beads were washed twice with 85% ethanol. To elute libraries, beads were resuspended in 15 µl of elution buffer and incubated for 1 min at room temperature.To generate enough material for sequencing, low RNA inputs (0.1–0.5 ng) required an additional amplification step. Reamplification was performed in a 50 µl reaction with Invitrogen™ Collibri™ Library Amplification Master Mix with Primer Mix (Thermo Fisher Scientific) for 6–12 cycles according to the recommended temperature conditions. Final libraries were purified using Dynabeads™ Cleanup Beads (Thermo Fisher Scientific) as described above. Fragment size distribution was then assessed by Agilent Fragment Analyzer™ system with HS NGS Fragment kit or Agilent 2100 Bioanalyzer™ with High Sensitivity DNA kit (Agilent Technologies). Quantification of sequenceable molecules was performed with Invitrogen™ Collibri™ Library Quantification Kit (Thermo Fisher Scientific).For library quality control, 1 × 150 bp SE sequencing was performed on the Illumina MiSeq™ instrument using MiSeq Reagent Kit v3 (150-cycle). Deep sequencing was performed on the Illumina NovaSeq™ 6000 System , aiming for ≥2 M reads per sample.All NGS data analysis workflows were implemented using the Snakemake workflow manager v6.1.0 .Quality control for the PE raw reads obtained upon sequencing with the NovaSeq system was performed with BBDuk tool from BBMap suit v37.90  to trim Click here for additional data file.