Biology subjects

Kulbachiskiy, A.

Publications and source records attributed to Kulbachiskiy, A..

7 recordsLinked to original sources

bioRxiv2025

Targeting of human mitochondrial DNA with programmable pAgo nuclease

Manipulating the mitochondrial genome remains a major challenge in genetic engineering, largely due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mtDNA has remained elusive. Here we report the first successful mitochondrial import of a functional RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By directing AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings lay the groundwork for further development of programmable systems for mitochondrial genome manipulation. HighlightsAmAgo targeted to mitochondria significantly reduces human mtDNA copy number Mitochondrial localization of AmAgo is essential for changes in the mtDNA content AmAgo effects on mtDNA are modulated by exogenous guide RNAs Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/689640v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@ddd069org.highwire.dtl.DTLVardef@9db9a3org.highwire.dtl.DTLVardef@1068b02org.highwire.dtl.DTLVardef@904c87_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Target-induced Argonaute-HNH filaments confer bacterial immunity

Argonaute proteins provide innate immunity in all domains of life through guide-dependent recognition of invader nucleic acids. While eukaryotic Argonautes (eAgos) act on RNA during RNA interference, prokaryotic Argonautes (pAgos) mainly recognize DNA targets. Many eAgos and some pAgos are active nucleases that directly cleave their targets. In contrast, short pAgos lack the nuclease activity and are co-encoded with additional effectors. Diverse effector domains in short pAgo systems include NADases and tentative nucleases, but their mechanisms of activation remain largely unknown. Here, we characterize SPARHA systems (short prokaryotic argonautes, HNH-associated) encoding HNH nuclease effectors. We show that short pAgo activates the HNH effector after RNA-guided DNA recognition. Target recognition induces formation of filaments of SPARHA with double active sites formed at the interfaces of repetitive HNH domains, which results in indiscriminate collateral degradation of DNA and protects bacterial population from invaders. The results show that pAgos and associated effectors act as modular two-component systems that translate recognition of specific DNA into immune response through assembly of supramolecular complexes, deleterious for invaders and potentially useful for biotechnology.

molecular biology↗

bioRxiv2023

DNA-targeting short Argonaute triggers effector nuclease to protect bacteria from invaders

Two prokaryotic defence systems, Argonautes (pAgos) and CRISPR-Cas, detect invader nucleic acids using complementary guides. Upon recognition, the target is cleaved through nuclease activities of pAgo or Cas proteins thus protecting the cell from invasion. However, not all pAgos are active nucleases. Members of a large clade of short pAgos bind nucleic acid guides but lack nuclease activity suggesting a different mechanism of action. Here, we have investigated short pAgo from Novosphingopyxis baekryungensis (NbaAgo). We have shown that NbaAgo forms a heterodimeric complex, SPARDA, with a co-encoded effector nuclease. RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA results in cell death during plasmid transformation or phage infection, thus protecting bacterial population from invaders. The collateral activity of SPARDA allows highly sensitive detection of specific DNA targets. SPARDA expands the list of prokaryotic immune systems that elicit suicidal cell response with a unique range of nuclease activities, creating additional opportunities for biotechnologies.

molecular biology↗

bioRxiv2022

Bacterial Argonaute proteins aid cell division in the presence of topoisomerase inhibitors in Escherichia coli

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix roseae (LrAgo) act as DNA-guided DNA nucleases in Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin. Both pAgos are preferentially loaded with small DNA guides derived from the sites of replication termination. The amount of pAgo-associated small DNAs (smDNAs) from the termination sites is increased in the presence ciprofloxacin, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin also enhances asymmetry in the distribution of smDNAs around Chi-sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases help to complete replication of chromosomal DNA by targeting the sites of termination, and may switch their functional activities when expressed in different host species.

molecular biology↗

bioRxiv2022

Bacterial Argonaute nucleases reveal different modes of DNA targeting in vitro and in vivo

Prokaryotic Argonaute proteins (pAgos) are homologs of eukaryotic Argonautes (eAgos) that were similarly proposed to play a role in cell defense against invaders. However, pAgos are much more diverse than eAgos and very little is known about their functional activity and target specificity in vivo. Here, we describe five pAgo proteins from mesophilic bacteria that act as DNA-guided DNA endonucleases and analyze their ability to target chromosomal and invader DNA. In vitro, the analyzed proteins use small guide DNAs for precise cleavage of single-stranded DNA at a wide range of temperatures. Upon their expression in Escherichia coli, all five pAgos are loaded with small DNAs preferentially produced from plasmid DNA and from chromosomal regions of replication termination. One of the tested pAgos, EmaAgo from Exiguobacterium marinum can induce DNA interference between multicopy sequences resulting in targeted processing of homologous plasmid and chromosomal loci. EmaAgo also protects bacteria from bacteriophage infection and is preferentially loaded with phage guide DNAs suggesting that the ability of pAgos to target multicopy elements may be crucial for their protective function. The wide spectrum of pAgo activities suggests that they may have diverse functions in vivo and paves the way for their use in biotechnology.

molecular biology↗

bioRxiv2022

Specific targeting of plasmids with Argonaute enables genome editing

Prokaryotic Argonautes (pAgos) are programmable nucleases involved in cell defense against invading DNA. Recent studies showed that pAgos can bind small single-stranded guide DNAs (gDNAs) to recognize and cleave complementary DNA in vitro. In vivo pAgos preferentially target plasmids, phages and multicopy genetic elements. Here, we reveal that CbAgo nuclease from Clostridium butyricum can be used for genomic DNA cleavage and engineering in bacteria. CbAgo-dependent targeting of genomic loci with plasmid-derived gDNAs promotes recombination between plasmid and chromosomal DNA. Efficient genome cleavage and recombineering depends on the catalytic activity of CbAgo, its interactions with gDNAs, and the extent of homology between plasmid and chromosomal sequences. Specific targeting of plasmids with Argonautes can be used to integrate plasmid-encoded sequences into the chromosome thus enabling genome editing. One-Sentence SummaryProkaryotic Argonaute nuclease induces DNA interference between plasmid and chromosomal DNA to promote genome recombineering.

molecular biology↗

bioRxiv2021

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique KmAgo nuclease from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo preferentially binds 16-20 nt long 5'-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

molecular biology↗