A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia

Abstract

Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from Verrucomicrobia bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.

Keywords: RNA target specificity; bacterium; programmable nucleases; prokaryotic argonaute.

Figure 1

VbAgo exhibits DNA-guided RNA cleavage activity at 37°C (A) Maximum likelihood phylogenetic tree analysis of VbAgo based on amino acid sequences. The numbers at the nodes indicate the bootstrap values for maximum likelihood analysis of 1000 resampled data sets. (B) The sequence of the synthetic guide and target. gDNAs and RNA targets (T-RNA) were used in most experiments. The black triangle indicates the cleavage site. (C) VbAgo exhibits DNA-guided RNA cleavage activity. (D) VbAgo exhibits no DNA cleavage activity. Positions of T-RNA and products (P) are indicated on the left of the gels. VbAgo, guide and target were mixed at a 4:2:1 molar ratio (800 nM VbAgo preloaded with 400 nM guide, plus 200 nM target) and incubated at 37°C for 60 min. Catalytic mutant VbAgo_DM was used as a control. Lanes M1 and M2 contain chemically synthesized 5′-end, FAM-labelled, 34 nt RNA and DNA corresponding to the cleavage products of target RNA and target DNA (T-DNA), respectively.

Figure 2

Effects of the reaction system on VbAgo cleavage activity (A) Effects of NaCl concentration on RNA target cleavage activity. (B) Effects of pH value on RNA target cleavage activity. (C) Effects of temperature on RNA target cleavage. (D) Effects of different divalent metal ions on target cleavage. Data are presented as the mean±standard deviation (SD) from three independent experiments. In all experiments, VbAgo, guide and target were mixed at a 4:2:1 molar ratio (800 nM VbAgo preloaded with 400 nM guide, plus 200 nM target). Experiments in (A) and (C) were incubated at 37°C for 60 min, experiments in (B) were incubated at 37°C for 15 min and experiments in (D) were incubated at 50°C for 15 min.

Figure 3

Effects of the guide length on RNA target cleavage (A) Cleavage assays with 5′P-gDNA (upper panel) and 5′OH-gDNA (lower panel) of varying lengths. The positions of RNA targets (T) and products (P) are indicated on the left of the gels. (B) Quantification of cleavage efficiencies by VbAgo (the percentage of target cleavage). The results from three independent experiments were quantified. (C–E) Kinetics analyses of RNA cleavage by VbAgo with 15, 16 and 18 nt gDNAs. The kobs values were analysed from the single-exponential fits of the data. Data are presented as the mean±standard deviation (SD) from three independent experiments. In all experiments, VbAgo, guide and target were mixed at a 4:2:1 molar ratio (800 nM VbAgo preloaded with 400 nM guide, plus 200 nM target) and incubated at 50°C for 15 min.

Figure 4

Effects of the guide-target mismatches on target cleavage (A) Cleavage assays with single-nucleotide or dinucleotide mismatches at a certain position. The positions of RNA targets (T) and products (P) are indicated on the left of the gels. (B) Quantification of cleavage efficiencies by VbAgo (the percentage of target cleavage). Data are presented as the mean±standard deviation (SD) from three independent experiments. In all experiments, VbAgo, guide and target were mixed at a 4:2:1 molar ratio (800 nM VbAgo preloaded with 400 nM guide, plus 200 nM target) and incubated at 50°C for 15 min.

Figure 5

Effects of the 5′-nucleotide of the guide on target cleavage and VbAgo is a multiple-turnover enzyme (A) Effects of the 5′-nucleotide of the guide on target cleavage. The assays were performed at 50°C for 15 min. The kobs values were analysed from the single-exponential fits of the data. Data are presented as the mean±standard deviation (SD) from three independent experiments. (B) P values for all comparisons of kobs values from (A). nsP>0.05, compared to the 5′-T guide using Student’s t-test. (C) VbAgo was loaded with 16 nt 5′P-gDNA at 37°C for 60 min. In the turnover experiments, 200 nM RNA target was mixed with increasing concentrations of VbAgo–gDNA (50-800 nM). (D) VbAgo was loaded with 16 nt 5′P-gDNA at 50°C for 60 min. In the turnover experiments, 200 nM RNA target was mixed with increasing concentrations of VbAgo–gDNA (50-800 nM). The numbers in panels C and D are the ratios of VbAgo to RNA targets. Data were fitted to a single-exponential equation if the [VbAgo–gDNA]/RNA target ratio was ≥ 1. The linear parts of the reaction kinetics were used for the calculation of the steady-state velocities of RNA cleavage if the [VbAgo–gDNA]/RNA target ratio was <1. Data are presented as the mean±standard deviation (SD) from three independent experiments.

Figure 6

Cleavage of highly structured HIV-1 ΔDIS 5′-UTR RNA by VbAgo (A) Schematic overview of the HIV-1 ΔDIS 5′-UTR. Twelve regions (shown in different colors) were selected from the RNA target sequence, with each region targeted by a 16 nt 5′P-gDNA. (B) VbAgo cleaved HIV-1 ΔDIS 5′-UTR RNA with 16 nt 5′P-gDNA at 37°C and 50°C for 15 min, and products (indicated by red triangle) were observed at predicted positions. The positions of the targets (T), gDNAs (G) and products (P) are indicated on the left of the gels. M, RNA marker.