scholarly journals Chemical Modification of the siRNA Seed Region Suppresses Off-Target Effects by Steric Hindrance to Base-Pairing with Targets

ACS Omega ◽  
2017 ◽  
Vol 2 (5) ◽  
pp. 2055-2064 ◽  
Author(s):  
Hanna Iribe ◽  
Kengo Miyamoto ◽  
Tomoko Takahashi ◽  
Yoshiaki Kobayashi ◽  
Jastina Leo ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Steric Hindrance ◽  
Seed Region ◽  
Base Pairing ◽  
Target Effects

scholarly journals Correction to “Chemical Modification of the siRNA Seed Region Suppresses Off-Target Effects by Steric Hindrance to Base-Pairing with Targets”

ACS Omega ◽  
2017 ◽  
Vol 2 (6) ◽  
pp. 2482-2482
Author(s):  
Hanna Iribe ◽  
Kengo Miyamoto ◽  
Tomoko Takahashi ◽  
Yoshiaki Kobayashi ◽  
Justina Leo ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Steric Hindrance ◽  
Seed Region ◽  
Base Pairing ◽  
Target Effects

scholarly journals SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P. K. Bravo ◽  
Stijn H. P. Prinsen ◽  
...  
Keyword(s):  
Immune Response ◽  
Diagnostic Tool ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Base Pairing ◽  
Specific Detection ◽  
Type Iii ◽  
Single Host ◽  
Target Rna

AbstractCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5′ end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.

scholarly journals Thermodynamic and structural characterization of 2′-nitrogen-modified RNA duplexes

2004 ◽  
Vol 32 (11) ◽  
pp. 3446-3455 ◽  
Author(s):  
John W. Pham ◽  
Ishwar Radhakrishnan ◽  
Erik J. Sontheimer
Keyword(s):  
Molecular Modeling ◽  
Nmr Spectroscopy ◽  
Nucleic Acids ◽  
Chemical Modification ◽  
Structural Characterization ◽  
Cross Linking ◽  
Base Pairing ◽  
Alkyl Groups ◽  
Rna Duplexes

Abstract 2′-aminonucleosides are commonly used as sites of post-synthetic chemical modification within nucleic acids. As part of a larger cross-linking strategy, we appended alkyl groups onto the N2′ position of 2′-amino-modified RNAs via 2′-ureido and 2′-amido linkages. We have characterized the thermodynamics of 2′-amino, 2′-alkylamido and 2′-alkylureido-modified RNA duplexes and show that 2′-ureido-modified RNAs are significantly more stable than analogous 2′-amido-modified RNAs. Using NMR spectroscopy and NMR-based molecular modeling of 2′-modified RNA duplexes, we examined the effects that 2′-nitrogen modifications have on RNA helices. Our data suggest that the 2′-ureido group forms a specific intra-nucleoside interaction that cannot occur within 2′-amido-modified helices. These results indicate that 2′-ureido modifications are superior to analogous 2′-amido ones for applications that require stable base pairing.

scholarly journals The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects

2015 ◽  
Vol 11 (12) ◽  
pp. e1004656 ◽  
Author(s):  
Piotr J. Kamola ◽  
Yuko Nakano ◽  
Tomoko Takahashi ◽  
Paul A. Wilson ◽  
Kumiko Ui-Tei
Keyword(s):  
Seed Region ◽  
Target Sequences ◽  
Target Effects

scholarly journals Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis

2014 ◽  
Vol 15 (1) ◽  
pp. 192 ◽  
Author(s):  
Bahar Yilmazel ◽  
Yanhui Hu ◽  
Frederic Sigoillot ◽  
Jennifer A Smith ◽  
Caroline E Shamu ◽  
...  
Keyword(s):  
Large Scale ◽  
Rnai Screen ◽  
Seed Region ◽  
Region Analysis ◽  
Target Effects

scholarly journals Critical contribution of 3' non-seed base pairing to the in vivo function of the evolutionarily conserved let-7a microRNA

2021 ◽  
Author(s):  
Ye Duan ◽  
Isana Veksler-Lublinsky ◽  
Victor Ambros
Keyword(s):  
Systematic Investigation ◽  
Seed Region ◽  
Seed Sequence ◽  
Base Pairing ◽  
Non Coding Rna ◽  
Evolutionarily Conserved ◽  
Microrna Function

MicroRNAs are endogenous regulatory non-coding RNA that exist in all multi-cellular organisms. Base-pairing of the seed region (g2-g8) is essential for microRNA targeting, however the in vivo functions of 3' non-seed region (g9-g22) are less well understood. Here we report the first systematic investigation of the in vivo roles of 3' non-seed nucleotides in microRNA let-7a, whose entire g9-g22 region is conserved among bilaterians. We found that the 3' non-seed sequence functionally distinguishes let-7a from its family paralogs. The complete pairing of g11-g16 is essential for let-7a to fully repress multiple key targets in vivo, including evolutionarily conserved lin-41, daf-12 and hbl-1. Nucleotides at g17-g22 are less critical but may compensate for mismatches in the g11-g16 region. Interestingly, we find that 3' non-seed pairing of let-7a can be functionally required even with sites that permit perfect seed pairing. These results provide evidence that the specific configurations of both seed and 3' non-seed base-pairing can critically influence microRNA function in vivo.

scholarly journals Small-hairpin RNAs cause target-independent microRNA dysregulation in neurons and elicit global transcriptomic changes

2020 ◽  
Author(s):  
Rafi Kohen ◽  
Katherine T. Baldwin ◽  
Patricia Marie Garay ◽  
Takao Tsukahara ◽  
Alex Chen ◽  
...  
Keyword(s):  
Protein Composition ◽  
Careful Consideration ◽  
Global Changes ◽  
Seed Region ◽  
Efficient Gene ◽  
Non Coding Rnas ◽  
Genetic Deletion ◽  
Transcriptomic Changes ◽  
Microrna Dysregulation ◽  
Target Effects

Small hairpin RNAs (shRNAs) allow highly efficient gene knockdown. Here we employed different shRNAs to knock down the reticulon RTN4A/NogoA in primary neurons. Depletion of NogoA correlates with altered synaptic protein composition and spontaneous neurotransmission. However, similar phenotypes are not observed upon genetic deletion of Nogo or its receptors. Step-wise introduction of mismatches in the seed region of shNogoA provides further evidence that synaptic phenotypes are NogoA-independent. RNA sequencing revealed global changes in the neuronal transcriptome of cultures transduced with the original shNogoA or closely related variants. Transcriptomic changes are shRNA seed sequence dependent, but not target-specific. Parallel sequencing of small non-coding RNAs revealed dysregulation of microRNAs. Computational analysis shows that the altered miRNA composition correlates with changes in mRNA expression and preferentially affects protein-protein networks that function at synapses. Thus, off-target effects associated with shRNAs are an inherent property, and in particular, altered miRNA composition needs careful consideration.

scholarly journals Viral miRNA adaptor differentially recruits miRNAs to target mRNAs through alternative base-pairing

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Carlos Gorbea ◽  
Tim Mosbruger ◽  
David A Nix ◽  
Demián Cazalla
Keyword(s):  
Seed Region ◽  
Base Pairing ◽  
Recognition Mechanism ◽  
Target Mrnas ◽  
Non Coding Rna ◽  
Infected Cells ◽  
Nucleotide Resolution ◽  
Rna Interaction ◽  
General Method

HSUR2 is a viral non-coding RNA (ncRNA) that functions as a microRNA (miRNA) adaptor. HSUR2 inhibits apoptosis in infected cells by recruiting host miRNAs miR-142–3p and miR-16 to mRNAs encoding apoptotic factors. HSUR2’s target recognition mechanism is not understood. It is also unknown why HSUR2 utilizes miR-16 to downregulate only a subset of transcripts. We developed a general method for individual-nucleotide resolution RNA-RNA interaction identification by crosslinking and capture (iRICC) to identify sequences mediating interactions between HSUR2 and target mRNAs in vivo. Mutational analyses confirmed identified HSUR2-mRNA interactions and validated iRICC as a method that confidently determines sequences mediating RNA-RNA interactions in vivo. We show that HSUR2 does not display a ‘seed’ region to base-pair with most target mRNAs, but instead uses different regions to interact with different transcripts. We further demonstrate that this versatile mode of interaction via variable base-pairing provides HSUR2 with a mechanism for differential miRNA recruitment.

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