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DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity

Nature Microbiology (2024)Cite this article

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Abstract

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR–Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

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Fig. 1: Phylogeny and structure of short pAgo systems.
Fig. 2: Collateral nuclease activity of SPARDA.
Fig. 3: SPARDA is activated by plasmids.
Fig. 4: SPARDA is active against phages.
Fig. 5: Repurposing of SPARDA for DNA detection and the proposed mechanism of SPARDA.

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Data availability

The data generated during this study are included in the published article, Extended Data and Supplementary Information. The gRNA and total RNA sequencing datasets generated in this study are available from the Sequence Read Archive (SRA) database (BioProject ID PRJNA975910). Source data are provided with this paper.

Code availability

The code used for data analysis is available via GitHub at https://github.com/prostovna/short-argonaute-guides.git (ref. 66).

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Acknowledgements

We thank A. Aravin for the inspiration for this work and helpful discussions, A. Olina for help in the analysis of SPARDA-associated small RNAs, A. Zavgorodniaia and D. Atroshenko for advice on protein purification, A. Isaev for phages (Skolkovo Institute of Science and Technology, Moscow), S. Pitelyak for help with phage assays and N. Mikhaylov for invaluable programming clues. This study was supported by the Russian Science Foundation (grant 22-14-00182 to A. Kulbachinskiy). N.N.S. and K.V.P.T. were partially supported by the Ministry of Science of Higher Education of the Russian Federation.

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Author notes

  1. These authors contributed equally: Maria Prostova, Anna Kanevskaya.

Authors and Affiliations

  1. Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

    Maria Prostova, Anna Kanevskaya, Vladimir Panteleev, Lidia Lisitskaya, Daria Esyunina & Andrey Kulbachinskiy

  2. A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia

    Kristina V. Perfilova Tugaeva & Nikolai N. Sluchanko

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  1. Maria Prostova
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Contributions

Conceptualization and supervision: M.P., D.E. and A. Kulbachinskiy. Experimental design: M.P. and A. Kanevskaya. Phylogenetic analysis: M.P. Structural modelling: M.P. and A. Kanevskaya. Cloning and purification of SPARDA: A. Kanevskaya and M.P. Experiments in vitro: A. Kanevskaya under the supervision of M.P. Analysis of plasmid interference and phage infections: A. Kanevskaya, V.P. and M.P. Degradation of plasmid and genomic DNA and RNA: A. Kanevskaya. CFU measurements: A. Kanevskaya and M.P. EOP assays: A. Kanevskaya. PFU measurements and microscopy: A. Kanevskaya and V.P. SEC–MALS analysis: N.N.S., K.V.P.T. and A. Kanevskaya. Preparation and sequencing of RNA libraries: A. Kanevskaya, L.L. and M.P. Analysis of sequencing data: M.P. Writing—original draft: A. Kulbachinskiy. Writing—review and editing: A. Kulbachinskiy and M.P. with contribution from all of the authors.

Corresponding authors

Correspondence to Maria Prostova, Anna Kanevskaya or Andrey Kulbachinskiy.

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Competing interests

M.P., A. Kanevskaya, D.E. and A. Kulbachinskiy have filed a patent application based on the findings of this study. The other authors declare no competing interests.

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Nature Microbiology thanks Malcolm White, Phillip Zamore and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Conservation, structural modeling and purification of SPARDA.

a, Alignment of the MID and PIWI domains in short pAgos from the SPARDA (NbaAgo, CmeAgo), SPARTA (CrtAgo), SPARSA (CcAgo) systems and in long pAgos (RsAgo, TtAgo, CbAgo, MjAgo, PfAgo). Residues involved in interactions with the guide 5’-end in the MID pocket are shown with green dots (YKQK in RsAgo, RKQK in NbaAgo and CmeAgo, see Fig. 1e). The active site residues in the PIWI domain in active long pAgo nucleases (TtAgo, CbAgo, MjAgo, PfAgo) are shown with blue dots. The source of pAgo proteins: NbaAgo – N. baekryungensis (WP_022673743.1), CmeAgo – C. metallidurans (WP_011516870.1), CrtAgo – Thermoflavifilum thermophilum (former Crenotalea thermophila) (WP_092459742.1), CcAgo - Caballeronia cordobensis (WP_053571899.1), RsAgo - Rhodobacter sphaeroides (A4WYU7.1), TtAgo - Thermus thermophilus (WP_011174533.1), CbAgo - Clostridium butyricum (WP_058142162.1), MjAgo - Methanocaldococcus jannaschii (WP_010870838.1), PfAgo - Pyrococcus furiosus (WP_011011654.1). The amino acid numbering is shown for NbaAgo. b, Residues of the nuclease active site in DREN in SPARDA systems from various species: Nba – N. baekryungensis (WP_033317603.1), Cme – C. metallidurans (WP_011516871.1), Rhod - Rhodoplanes elegans (WP_111355734.1), Rph - Rhizobium phaseoli (WP_126906501.1), Mma - Mycobacterium marinum (WP_117407168.1), M.leaf - Methylobacterium sp. Leaf113 (WP_056186121.1). The amino acid numbering is shown for Nba DREN. Predicted active site residues are indicated with blue dots. Predicted elements of the secondary structure are indicated (α-helices, π-helices and β-strands; strict β-turns are shown as TT letters). The alignment is generated with ESPript 3.0. c-d, Confidence of the AlphaFold prediction for NbaSPARDA. c, pLDDT (local-distance difference test) – the per-residue estimate of prediction confidence measure for the five best rank models of the NbaAgo and DREN-APAZ complex. Positions 1–485 correspond to NbaAgo, while the rest correspond to DREN-APAZ (separated by a vertical line). The lDDT value > 80 reflects high confidence of the backbone prediction. d, PAE (Predicted Aligned Error) – the expected position error at residue x if the predicted and true structures were aligned on residue y, shown for the best ranked model. Positions 1–485 (top on the y-axis) correspond to NbaAgo, the rest (bottom on the y-axis) correspond to DREN-APAZ. The color gradient from blue to red indicates the error value, ranging from low to high. The graph shows that the relative positions of NbaAgo and the APAZ domain are confident, while the position of the DREN domain is flexible. Both graphs were created in ColabFold v1.5.3. e-g, Purification of SPARDA complexes. e, Co-elution of NbaAgo and DREN-APAZ during Ni-chelating, heparin and anion-exchange (MonoQ column) chromatography steps. Individual fractions and the final protein sample are shown for the wild-type complex. f, Final purified samples of wild-type (WT) NbaSPARDA and its mutants with substitutions in the active site of DREN (CD, catalytically dead) and in the MID pocket of NbaAgo (MID). g, Purification of CmeSPARDA. The elution fractions from the final chromatography step (MonoQ) are shown.

Source data

Extended Data Fig. 2 Analysis of the optimal reaction conditions for NbaSPARDA in vitro.

a, Structure of gRNA and tDNAs (single-stranded or double-stranded) used in the assays. b, Activity of SPARDA with single-stranded tDNAs of various lengths (20, 30, and 50 nt), in the absence and in the presence of gRNA. c, Activity of SPARDA with double-stranded (ds) tDNA (20 nt, lanes 3–5, or 50 nt, lanes 6–8), tested for 60 minutes at 30 °C or 40 °C. Control reactions were performed with single-stranded (ss) tDNA. The non-target DNA strand was annealed with the complementary target strand at the 1:1 or 3:2 ratio. While low level of SPARDA activity was observed at the 1:1 ratio (lanes 3 and 8), it was suppressed at the 3:2 ratio (lanes 4–5 and 6–7), indicating that this activation resulted from the presence of non-annealed single-stranded tDNA. The position of non-denatured 50 nt duplex DNA is indicated with an asterisk. d, Activity of SPARDA in buffers containing various divalent cations (5 mM each), with either 5 mM or 100 mM KCl. d, Activity of SPARDA with different concentrations of Mg2+ (left, with 5 mM KCl) or Mn2+ (right, with 100 mM KCl). f, Activity of SPARDA with different concentrations of Mn2+ in the absence and in the presence of 5 mM Mg2+ (with 100 mM KCl). g, Activity of SPARDA at various pH. h, Activity of SPARDA at various temperatures. i, Sequences of gRNAs and tDNAs used for analysis of the optimal gRNA structure. For gRNAs with substitutions of the first or second nucleotides, tDNA contained complementary substitutions at corresponding positions (indicated with ‘N’). j, Activity of SPARDA with gRNAs of different lengths. k, Kinetics of substrate DNA cleavage with 5’-phosphorylated and 5’-OH gRNAs. l, Effects of substitutions of the 5’-nucleotide in gRNA (lanes 2–6) or in gDNA (lanes 7–10) on the activity of SPARDA. SPARDA is activated only by gRNAs with 5’-A (lanes 2 and 3). m, Effects of substitutions of the second nucleotide in gRNA on the activity of SPARDA. The 5’-end dinucleotides in each gRNA are shown above the gel. All reactions were performed at 30 °C in reaction buffers containing 5 mM Mn2+ (with 100 mM KCl) or 5 mM Mg2+ (with 5 mM KCl), unless otherwise indicated. The reactions in panels b, c, k, j, l were performed in the presence of Mn2+. All reactions except for panels c and h were performed at 30 °C. In each panel, representative gels from several independent experiments are shown, which produced similar results: 4 replicates in panel b, 2 replicates in panels c-m. Positions of gRNA, tDNA, single-stranded collateral substrate DNA (71 nt) and the cleavage products are indicated. The lengths of guides, targets and substrates are shown in nucleotides (see Supplementary Table 1 for oligonucleotide sequences).

Source data

Extended Data Fig. 3 Multiple-round DNA cleavage by NbaSPARDA.

a-c, Cleavage of ssDNA substrates. a, Cleavage of an excess of 5’-P32-labelled 71 nt tDNA (5 μM) with increasing amounts of the activated SPARDA complex (from 10 to 500 nM, SPARDA:gRNA:tDNA = 1:1:1). b, Multiple-round cleavage of the 71 nt tDNA substrate (5 μM) by activated NbaSPARDA (500 nM SPARDA, 500 nM gRNA, 500 nM tDNA). c, Multiple-round cleavage of 5’-P32-labeled 55 nt tDNA (5 μM) under the same conditions. d-e, Cleavage of a ssRNA substrate. d, Cleavage of 5’-FAM-labeled 55 nt ssRNA (5 μM) with increasing amounts of the activated SPARDA complex (from 10 to 500 nM, SPARDA:gRNA:tDNA=1:1:1). e, Multiple-round cleavage of the 55 nt RNA substrate (5 μM) by activated NbaSPARDA (500 nM SPARDA, 500 nM gRNA, 500 nM tDNA). All reactions were performed in three replicates at 30 °C in the buffer containing 5 mM Mn2+ and 100 mM KCl. The reaction products were visualized by phosphorimaging (panels a-c) and fluorescence scanning (panels d-e). In each case, representative gels from three independent experiments are shown. f, Kinetics of ssDNA and ssRNA cleavage by NbaSPARDA. Means and standard deviations from three replicates. See Supplementary Table 1 for oligonucleotide sequences.

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Extended Data Fig. 4 Activities of NbaSPARDA and CmeSPARDA with various collateral substrates.

a, Kinetics of cleavage of supercoiled plasmid DNA (pBAD) by activated NbaSPARDA. Control reactions were performed for 40 minutes without SPARDA (lane 1) or without gRNA (lane 2). b, Cleavage of linear dsDNA fragments of indicated lengths (obtained after treatment of pBAD by restriction endonucleases with gel purification). c-e, Analysis of collateral cleavage of 55 nt ssRNA, RNA/DNA duplex and dsRNA. The reactions were performed with 5’-FAM and 5’-HEX labeled substrate RNA and DNA oligonucleotides, as indicated below the gels. f, Analysis of gRNA and tDNA cleavage by NbaSPARDA in the absence of collateral substrates. g,h, Comparison of the activities of wild-type (WT) NbaSPARDA and its MID mutant (g) or CD (h) mutants with the collateral ssDNA substrate (71 nt) in the presence of Mg2+ and Mn2+. i, Cleavage of the ssDNA substrate (71 nt) by CmeSPARDA. j, Cleavage of FAM-labeled 55 nt ssRNA substrate by CmeSPARDA. k, Cleavage of plasmid DNA (pBAD) and its linear dsDNA fragment (~3000 bp) in the presence of gRNA and tDNA. Linear plasmid (lane 3) was obtained by treatment with HindIII. Control reactions in lanes 4 and 7 were performed in the absence of gRNA. M, length marker. Linear DNA fragments in panels b and k have lower gel mobility in comparison with molecular weight markers (lane 1) than expected, likely due to differences in the loading buffer composition (see Materials and Methods). The lengths of guides, targets and substrates in panels c-j are shown in nucleotides (see Supplementary Table 1 for oligonucleotide sequences). The reactions were performed for 30 min (1 hour in panel i) at 30 °C with standard 20 nt gRNA (20 nt 5’-A-RNA) and 50 nt single-stranded tDNA (200 nM each; 100 nM in panels a,b,g,h). The samples contained 200 nM NbaSPARDA (panels a,b), 500 nM NbaSPARDA (panels c-h) or 350 nM CmeSPARDA (panels i-k). The experiments in panels a,b,g and h were performed with WT SPARDA samples containing admixture of endogenous gRNAs, so that the relative activity of guide-free WT SPARDA would be even higher in these assays (see below Extended Data Fig. 8a, c). The reaction buffers contained 5 mM Mg2+ (with 5 mM KCl) or 5 mM Mn2+ (100 mM KCl), as indicated. The reactions in panels a,b and k were performed in buffers containing 5 mM Mg2+ (with 5 mM KCl). In all cases except panel j, nucleic acids were visualized by SYBR Gold staining; 5’-FAM-labeled RNA in panel j was visualized by fluorescence scanning. In each panel, representative gels from several independent experiments are shown, which produced similar results: 3 replicates in panels a,c,g,h,i,j,k; 2 replicates in panels b,d,e,f. Positions of gRNA, tDNA, collateral substrates and the cleavage products are indicated. See Supplementary Table 1 for oligonucleotide sequences.

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Extended Data Fig. 5 Analysis of the oligomeric state of NbaSPARDA by size-exclusion chromatography and multi-angle light scattering.

a, Analysis of the WT SPARDA complex by SEC-MALS. The absolute molar weight of the NbaAgo/DREN-APAZ heterodimer is indicated. The Mw/Mn polydispersity index was 1.006, suggesting particles with the same Mw across the peak. b, Analysis of the MID mutant of NbaSPARDA by SEC-MALS, with the absolute molecular weight indicated. The Mw/Mn polydispersity index was 1.000. c, Size-exclusion chromatography of the purified WT NbaSPARDA complex in the absence of nucleic acids (top), in the presence of 20 nt gRNA (middle) and in the presence of 20 nt gRNA and 20 nt tDNA (bottom). For each sample, OD280 and OD260 absorbance profiles are shown. OD500 profiles are also shown for samples containing gRNA (which was labeled with a FAM residue at its 3’-end, see Supplementary Table 1). In each panel, positions of monomeric (apparent Mw of 83 or 96 kDa) and dimeric (apparent Mw of ~150 kDa) SPARDA peaks are indicated. Note that gRNA binding slightly increases the apparent size of SPARDA, as suggested by an increase of the apparent Mw from 83 to 96 kDa and hydrodynamic radius from 3.88 to 4.12 nm, but does not involve any distinct oligomeric state transition. d, Analysis of the apparent hydrodynamic radii and masses of monomeric (heterodimers of NbaAgo and DREN-APAZ) and dimeric (dimers of heterodimers) SPARDA complexes, based on calibration with standard albumin samples. e, Analysis of nucleic acids from various fractions after size-exclusion chromatography of the SPARDA sample pre-incubated with gRNA (20 nt) and tDNA (20 nt) (from the bottom profile in panel C). In each case, the results of a single experiment are shown. Based on the OD260/OD280 ratio (panel c) and the electrophoretic analysis of gRNA and tDNA in each fraction (panel e) it can be concluded that dimers of the heterodimeric SPARDA complex contain only gRNA, while monomeric heterodimers contain both gRNA and tDNA. Thus, the dimeric peak of SPARDA observed in the presence of gRNA (middle profile in panel c) or gRNA and tDNA (bottom profile in panel c) likely corresponds to gRNA-bound SPARDA. The presence of a minor dimeric fraction in WT SPARDA (top profile in panel c and bottom profile in panel d) likely results from binding of gRNAs co-purified with SPARDA, while the main peak corresponds to gRNA-free monomeric SPARDA.

Source data

Extended Data Fig. 6 Plasmid interference induced by SPARDA.

a, Measurements of CFU numbers for E. coli strains containing empty pBAD (AmpR) or pBAD encoding NbaSPARDA (WT or CD and MID mutants), and transformed with pACYC (CmR, left) or pCDF (SmR, right) interfering plasmids. The cells were grown for 2.5 hours at 30 °C (see Fig. 3b and panel d in this figure) in the presence of Amp and 0.1% Ara (to induce expression of SPARDA), serial dilutions were plated on LB agar containing Amp or Cm or Sm and 0.5% Glc (to repress SPARDA expression), and CFU numbers were counted after overnight growth. The calculations of the CFU numbers for the pCDF and pACYC experiments are shown in Fig. 3d and in panel e in this figure, respectively. b, Measurements of CFU numbers for E. coli strains containing empty pBAD (AmpR) or pBAD encoding CmeSPARDA in the absence and in the presence of pCDF (SmR). The cells were grown for 6 hours at 30 °C (see Fig. 3c) in the presence of Amp and 0.1% Ara (to induce expression of SPARDA), serial dilutions were plated on LB agar containing Amp (left) or Sm (right) and 0.5% Glc (to repress expression of SPARDA), and CFU were counted as described above. c, Numbers of viable cells in cell cultures expressing wild-type CmeSPARDA (WT) or containing empty pBAD, depending on the presence of the pCDF plasmid, measured in the presence of Amp or Sm (calculation of data from panel b). Means and standard deviations from three independent experiments. Statistically significant differences are indicated (from left to right: **p = 0.002, **p = 0.005, ****p = 0.00005). d, Growth of E. coli strains expressing WT or mutant NbaSPARDA from pBAD, or containing control empty pBAD, depending on the presence of the second interfering plasmid pACYC184. Means and standard deviations from 6 independent experiments. The growth curves obtained in the absence of pACYC184 (left panel) correspond to the left panel in Fig. 3b. e, Numbers of viable cells (CFU) in cell cultures (shown in panel d) expressing NbaSPARDA or its mutants, depending on the presence of the pACYC184 plasmid (CmR), measured in the presence of Amp or Cm. Means and standard deviations from 3 independent experiments. Statistically significant differences are indicated (from left to right: *p = 0.026, **p = 0.004, ****p = 0.00004). f, Growth of E. coli strains expressing CmeSPARDA or containing empty pBAD, in the absence and in the presence of pACYC184. Means and standard deviations from 6 independent replicate experiments. g, CFU numbers in cell cultures grown for 7 hours (shown in panel f), expressing CmeSPARDA and containing pACYC184, measured in the presence of Amp or Cm. Means and standard deviations from 3 independent experiments. Statistically significant differences are indicated (from left to right: **p = 0.008, **p = 0.002, ****p = 0.000006). h, Numbers of viable cells in cell cultures expressing NbaSPARDA or containing empty pBAD, in the absence and in the presence of RSF1010 or pMB9. The cells were grown for 2.5 hours at 30 °C hours in the presence of 0.1% Ara to induce SPARDA expression and CFU numbers were determined as described above. Means and standard deviations from 3 independent experiments. Statistically significant differences are indicated (from left to right: *p = 0.044, **p = 0.002, *p = 0.02). In all cases, statistics was calculated using the two-sided t-test for independent samples with Bonferroni correction.

Extended Data Fig. 7 Analysis of the effects of SPARDA on cell viability and nucleic acid degradation in vivo.

a, SYTO 9 and propidium iodide (PI) staining of E. coli cultures expressing SPARDA or containing control empty pBAD. Cell cultures were grown for 5 and 7 hours after induction in experiments with NbaSPARDA (left column) and CmeSPARDA (right column), respectively. Strains containing the second interfering plasmid pCDF were grown under identical conditions. The cells were collected by centrifugation, stained with SYTO 9 and PI, and analyzed by microscopy. The scale bar is 10 μm. The experiment was independently repeated 3 times, producing similar results. Representative fields of view (from 5–7 analyzed in each experiment) are shown. b, Analysis of the integrity of plasmid DNA in E. coli strains expressing CmeSPARDA (lanes 6–7, 11–12) or containing a control empty pBAD plasmid (‘E’, lanes 3–4, 9–10), in the absence (lanes 3–4, 6–7) and in the presence (lanes 9–12) of pCDF. Plasmid DNA was purified at 7 h of growth after induction of CmeSPARDA. For each condition, the results of two replicate experiments are shown. Lanes 2, 5 and 8 contain control samples of intact pBAD, pBAD-CmeSPARDA and pCDF. c, Purification of plasmid DNA from E. coli strains containing empty pBAD (‘E’, lanes 3–4), or pBAD encoding NbaSPARDA (WT or CD, lanes 6–9), or pBAD together with pCDF or pACYC (lanes 11–14 and 16–17, respectively) at 6 h of growth after induction of SPARDA expression (0.1% Ara). The results of two replicate experiments are shown. Lane 1 contains length marker, lanes 2, 5, 10 and 15 contain control samples of intact pBAD, pBAD-WT NbaSPARDA, pCDF and pACYC184. d, Analysis of total RNA purified from E. coli cultures containing pBAD (empty ‘E’, or encoding WT or CD NbaSPARDA), or pBAD together with pCDF, at 7 h of growth after induction of NbaSPARDA. Positions of 23 S and 16 S rRNA are indicated. A representative gel from two independent experiments, which produced similar results. Lane 1 in panels b-d contains length marker.

Source data

Extended Data Fig. 8 Purification and sequence analysis of small guide RNAs associated with NbaSPARDA in E. coli.

a, Small RNAs associated with WT SPARDA at different steps of the standard purification protocol (Ni-chelating, heparin and MonoQ columns). Most gRNAs are removed after heparin chromatography. The OD260/280 ratio of the final sample is 0.55. b, Small RNAs associated with WT and mutant SPARDA variants at different steps of purification using a modified protocol, in which EDTA was omitted from the buffers during heparin and anion-exchange chromatography. Significant amounts of gRNAs remain associated within the WT and CD complexes (but not the MID mutant) through all purification steps, indicative of stable gRNA binding. The OD260/280 ratios of the final WT, MID and CD samples are 0.98, 0.53 and 0.82, respectively. Panels a,b show representative gels from two independent experiments, which produced similar results. c, Comparison of the activities of WT SPARDA purified by different protocols. Representative gels from 3 independent experiments. SPARDA samples containing endogenous gRNAs (OD260/280 = 0.98, right) have a lower nuclease activity in comparison with guide-free SPARDA (OD260/280 = 0.55, left), when activated by specific gRNA (20 nt) and tDNA (50 nt). Results d, Correlation between the abundance of gRNAs and the RNA transcriptome of E. coli strains expressing NbaSPARDA from pBAD and containing pACYC184 (three individual replicate experiments for Fig. 3h). Plasmid genes are shown in violet and blue (pBAD and pACYC, respectively). Regulatory antisense RNAs, prophage genes, and IS elements are shown in orange, pink and turquoise, respectively. e, Analysis of the distribution NbaSPARDA-associated gRNAs and long RNAs along plasmid sequences, pBAD (top) and pACYC184 (bottom). Short gRNAs and total RNA were isolated from E. coli containing pBAD_NbaSPARDA and pACYC184 at 5 h after induction of SPARDA, sequenced and mapped to the plasmids. The amounts of short gRNAs and long RNA transcripts are shown in RPKM (reads per kilobase per million aligned reads in the library) for each DNA strand and plotted for each plasmid region (left and middle panels, respectively). The ratio of short to long RNAs is shown on the right panels. Means and standard deviations from three replicate experiments. The map of each plasmid is shown above the plots. f, Distribution of various types of transcripts among the genes enriched or depleted for gRNAs. For all E. coli genes, the ratios of small gRNAs to long RNA transcripts were calculated, and the genes were sorted by this ratio in descending order (from left to right). The distributions of coding, noncoding and regulatory antisense transcripts among the top and bottom 100 genes are shown in circular diagrams. The list of top enriched genes (with the highest ratio of short to long RNAs) is shown below the diagram. g, Distribution of gRNAs and long RNAs along the E. coli chromosome. Short gRNAs and total RNA isolated from E. coli cells containing pBAD_NbaSPARDA and pACYC184 were mapped to the MG1655 reference genome (GenBank ID GCA_000005845.2), the amounts of short gRNAs and long RNA transcripts were expressed in RPKM for each E. coli gene and their ratios were calculated. Only reads corresponding to the sense strand of each gene were counted. The sequences corresponding to rRNA were omitted from analysis. Means from three replicate experiments.

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Extended Data Fig. 9 Effects of SPARDA expression on cell growth during phage infection at different MOI.

a, Growth of E. coli cultures (DH5α Turbo for Qβ and N15, MG1655 for other phages) expressing NbaSPARDA or containing control empty pBAD and infected with indicated phages at low MOI (~3 × 10-6 for Qβ, 8 × 10-3 for N15, 1 × 10-5 for T6, 4 × 10-3 for 121Q, 4 × 10-6 for T7, T4, T2). The phages were added in the starting liquid cultures simultaneously with the induction of SPARDA. The results of a single experiment for each phage are shown. b, Growth of E. coli cultures (MG1655) expressing WT or mutant NbaSPARDA and infected with phages P1, λ, T5 and P2 at low MOI (MOI 3.6 × 10-4 for P1 and T5, 3.6 × 10-3 for λ, 3.6 × 10-6 for P2). Means and standard deviations from 5 replicate experiments. The phages were added in the starting liquid cultures simultaneously with the induction of SPARDA (indicated with gray asterisks). c, Growth of E. coli cultures (BL21(DE3)) expressing CmeSPARDA and infected with phages P1 and λ at low MOI (MOI = 3.6 × 10-4 for P1, MOI = 3.6 × 10-3 for λ). The phages were inoculated in the starting liquid cultures simultaneously with the induction of SPARDA. Means and standard deviations from 4 replicates. d,e, Growth of E. coli cultures (BL21(DE3)) containing empty pBAD or expressing NbaSPARDA or CmeSPARDA and infected with phages P1, λ, T5 and P2 at MOI 1.0 (d) and 0.1 (e). The cells were grown for 3 hours 20 min after induction of SPARDA expression before phage inoculation (indicated with gray asterisks). Means and standard deviations from 4 replicate experiments. The results for phages P1 and λ correspond to Fig. 4b, c. For measurements of CFU and PFU numbers (Fig. 4f, g), the samples were collected at 8 h 20 m of growth after SPARDA induction (indicated with blue asterisks).

Extended Data Fig. 10 DNA detection with SPARDA.

a, Scheme of the fluorescent ssDNA and dsDNA substrates. b, Kinetics of fluorescence increase after guide-dependent recognition of target DNA by NbaSPARDA, using dsDNA (left) and ssDNA (right) beacon substrates. The concentrations of target DNA in the samples are shown on the right. Means and standard deviations from 3 measurements. The reactions were performed with 1 µM NbaSPARDA, 1.4 µM gRNA and serial dilutions of tDNA20 at 30 °C in a reaction buffer containing 20 mM HEPES pH 7.5, 5 mM MnCl2, 5 µg/ml BSA, 100 mM NaCl, 2 mM DTT. c, Scheme of the assay with PCR amplification followed by exonuclease digestion of the non-target DNA strand. d, Sensitivity of the assay without amplification (top, molar concentrations of the ssDNA target) and with PCR amplification (bottom, number of molecules in the sample before amplification). Means and standard deviations from 3 replicates. The data correspond to the 150 and 100 min time points from Fig. 4b and 4e, respectively.

Supplementary information

Supplementary Information

Suppplementary text containing (1) a detailed description of the in vitro activity and specificity of NbaSPARDA and (2) a description of the analysis of the oligomeric state of NbaSPARDA, and Supplementary Tables 1–3.

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Supplementary Data 1

Source data for plots from Figs. 3–5 and Extended Data Figs. 3, 6, 8 and 9.

Supplementary Data 2

AlphaFold2 model of the NbaSPARDA complex.

Source data

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Unprocessed photo of agar plates for the plaque assay.

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Prostova, M., Kanevskaya, A., Panteleev, V. et al. DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01654-5

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