Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA

Abstract

The recent identification and development of RNA-guided enzymes for programmable cleavage of target nucleic acids offers exciting possibilities for both therapeutic and biotechnological applications. However, critical challenges such as expensive guide RNAs and inability to predict the efficiency of target recognition, especially for highly-structured RNAs, remain to be addressed. Here, we introduce a programmable RNA restriction enzyme, based on a budding yeast Argonaute (AGO), programmed with cost-effective 23-nucleotide (nt) single-stranded DNAs as guides. DNA guides offer the advantage that diverse sequences can be easily designed and purchased, enabling high-throughput screening to identify optimal recognition sites in the target RNA. Using this DNA-induced slicing complex (DISC) programmed with 11 different guide DNAs designed to span the sequence, sites of cleavage were identified in the 352-nt human immunodeficiency virus type 1 5′-untranslated region. This assay, coupled with primer extension and capillary electrophoresis, allows detection and relative quantification of all DISC-cleavage sites simultaneously in a single reaction. Comparison between DISC cleavage and RNase H cleavage reveals that DISC not only cleaves solvent-exposed sites, but also sites that become more accessible upon DISC binding. This study demonstrates the advantages of the DISC system for programmable cleavage of highly-structured, functional RNAs.

A programmable omnipotent Argonaute nuclease from mesophilic bacteria Kurthia massiliensis

“…Previous studies of eAgos and several mesophilic pAgos showed that mismatches between the guide and target strands may have large effects on the cleavage efficiency and precision ( 25 , 32 ). To study the effect of mismatches on target cleavage, we designed a set of DNA and RNA guides, each containing a single mismatched nucleotide at a certain position ( Supplementary Table S1 ), and tested them in the cleavage reaction with KmAgo (Figure 5 and Supplementary Figure S7 ).…”

“…Dayeh et al. reported that HIV-1 ΔDIS 5′UTR is a highly structured RNA (see Supplementary Table S2 ) ( 32 ). The HIV-1 ΔDIS 5′UTR was in vitro transcribed using T7 RNA polymerase and synthetic DNA templates carrying a T7 promoter sequence.…”

“…Transcripts used for cleavage assays was DNase I-treated and gel-purified. Guide DNAs (gDNAs) for experiments targeting the HIV-1 ΔDIS 5′UTR sequence ( Supplementary Table S3 ) were designed as described in ( 32 ) with minor modifications. Briefly, Dayeh et al.…”

An RNase P‐Based Assay for Accurate Determination of the 5′‐Deoxy‐5′‐azidoguanosine‐Modified Fraction of in Vitro‐Transcribed RNAs

“…Our work should motivate studies to test other G analogues (especially non‐azide/alkyne initiators such as 5′‐hydrazinyl‐5′‐deoxyguanosine), and determine whether a common 10‐nt leader/initiating sequence rich in A, C, or U would afford high yields of RNAs initiated with different G analogues. The use of programmable RNases (e.g., Argonautes loaded with defined DNA guides) for site‐specific cleavage of large RNAs and the generation of smaller cleavage products bearing the 5′‐G analogue initiator also merits consideration.…”

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

“…Some truncated versions of approximately half the size (short Agos) exist in nature22 . We speculate that it will be possible to truncate them further as Ago-PAINT only relies on the property of pre-forming the helix structure of the imager strand and a variant from Kluyveromyces polysporus that contains only the C-lobe was reported to retain almost all the binding properties of the untruncated version 35.…”