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Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants

Artificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miR...

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Autores principales: Kuo, Yen‐Wen, Falk, Bryce W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9129084/
https://www.ncbi.nlm.nih.gov/pubmed/35113475
http://dx.doi.org/10.1111/pbi.13786
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author Kuo, Yen‐Wen
Falk, Bryce W.
author_facet Kuo, Yen‐Wen
Falk, Bryce W.
author_sort Kuo, Yen‐Wen
collection PubMed
description Artificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear‐replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic‐replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non‐viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.
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spelling pubmed-91290842022-05-26 Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants Kuo, Yen‐Wen Falk, Bryce W. Plant Biotechnol J Research Articles Artificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear‐replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic‐replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non‐viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects. John Wiley and Sons Inc. 2022-02-19 2022-06 /pmc/articles/PMC9129084/ /pubmed/35113475 http://dx.doi.org/10.1111/pbi.13786 Text en © 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Research Articles
Kuo, Yen‐Wen
Falk, Bryce W.
Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
title Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
title_full Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
title_fullStr Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
title_full_unstemmed Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
title_short Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
title_sort artificial microrna guide strand selection from duplexes with no mismatches shows a purine‐rich preference for virus‐ and non‐virus‐based expression vectors in plants
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9129084/
https://www.ncbi.nlm.nih.gov/pubmed/35113475
http://dx.doi.org/10.1111/pbi.13786
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