Biology subjects

Kropocheva, E.

Publications and source records attributed to Kropocheva, E..

3 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↗

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↗

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↗