Prokaryotic Argonaute Proteins as a Tool for Biotechnology

Abstract

Programmable nucleases are the most important tool for manipulating the genes and genomes of both prokaryotes and eukaryotes. Since the end of the 20th century, many approaches were developed for specific modification of the genome. The review briefly considers the advantages and disadvantages of the main genetic editors known to date. The main attention is paid to programmable nucleases from the family of prokaryotic Argonaute proteins. Argonaute proteins can recognize and cleave DNA sequences using small complementary guide molecules and play an important role in protecting prokaryotic cells from invading DNA. Argonaute proteins have already found applications in biotechnology for targeted cleavage and detection of nucleic acids and can potentially be used for genome editing.

Structural basis for sequence-specific recognition of guide and target strands by the Archaeoglobus fulgidus Argonaute protein

“…However, unlike eAgos, which canonically use RNA guides for recognition of RNA targets in a process called RNA interference (RNAi) 19 – 21 , different long pAgos may use either RNA or DNA guides and/or targets 13 , 14 , 18 , and in vitro may associate with phosphorylated (e.g. AaAgo, PfAgo, and RsAgo from Aquifex aeolicus, Pyrococcus furiosus, and Rhodobacter sphaeroides , respectively) 13 , non-phosphorylated guide strands (e.g., MpAgo and TpAgo from Marinitoga piezophila and Thermotoga profunda , respectively) or lack preference for 5′-phosphorylation (CbAgo, LrAgo, KjAgo from Clostridium butyricum , Limnothrix rosea , Kordia jejudonensis , respectively) 13 , 22 – 24 . Interestingly, the recently described KmAgo from Kurthia massiliensis can utilize both DNA and RNA guides to cleave DNA and RNA targets in vitro, albeit with different efficiencies 25 , 26 .…”

Programmable Proteins: Target Specificity, Programmability and Future Directions

“…Several types of programmable nucleases of natural and synthetic origin have been reported [17][18][19][20]. Two groups of such enzymes are used for genome engineering: protein- Such programmable proteins are widely used in biology, synthetic biology, and medicine.…”

“…Several types of programmable nucleases of natural and synthetic origin have been reported [17][18][19][20]. Two groups of such enzymes are used for genome engineering: proteinguided nucleases that recognize the specific sequence using protein module-DNA interactions (e.g., TALEN: Section 2.1) and nucleic-acid-guided nucleases that recognize the specific sequence via an attached short complementary DNA or RNA (e.g., Cas9: Sections 2.2-2.6).…”

“…Fok1, discovered in Flavobacterium okeanokoites, is a type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a DNA cleavage domain (~200 amino acids) at the C-terminus. However, earlier approaches to create programmable nucleases such as meganucleases did not gain popularity because of their technical limitations (e.g., modification of homing enzymes and FEN1 (Flap structure-specific endonuclease-1)) [19,20,22]. Subsequently, engineered zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were invented as two types of protein-guided programmable nucleases.…”

Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification

“…Argonaute (Ago) proteins are widely present in almost all organisms, indicating their ancient origin and the importance of their functions, such as host defense against invading nucleic acids. − Among them, the LongA group of prokaryotic Argonautes (pAgos) such as Thermus thermophilus argonaute (TtAgo) are nucleic acid-mediated programmable endonucleases, which can precisely identify and cleave target sequences between the 10th and 11th nucleotides under the guidance of single strand DNA or RNA. − pAgos-mediated methods have been developed to detect nucleic acids in a programmable manner as well as with high efficiency and single-nucleotide discrimination specificity. − pAgos usually possess four crucial domains, among them, the MID domain ,, and PAZ (PIWI-Argonaute-Zwille) domain − is responsible for binding the 5′ and 3′ terminus of the nucleic acid strand, respectively, which endow pAgos strong binding affinity with single strand nucleic acids. , Recently, scientists further find that thermophilic pAgos (e.g., TtAgo), exhibit a stronger binding ability with nucleic acids than that of mesophilic pAgos (e.g., Clostridium butyricum Ago (CbAgo)) . Despite the progress, the binding affinity of pAgos with nucleic acids remains unexplored for promoting the specificity of the amplification system.…”