scholarly journals A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs

2018 ◽  
Vol 13 (3) ◽  
pp. 533-536 ◽  
Author(s):  
Travis Hardcastle ◽  
Irina Novosjolova ◽  
Venubabu Kotikam ◽  
Samwel K. Cheruiyot ◽  
Daniel Mutisya ◽  
...  
Keyword(s):  
Guide Strand ◽  
Amide Linkage ◽  
Passenger Strand

scholarly journals Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal

2015 ◽  
Vol 112 (38) ◽  
pp. E5271-E5280 ◽  
Author(s):  
Anna Y. Zinovyeva ◽  
Isana Veksler-Lublinsky ◽  
Ajay A. Vashisht ◽  
James A. Wohlschlegel ◽  
Victor R. Ambros
Keyword(s):  
Caenorhabditis Elegans ◽  
Structural Features ◽  
Guide Strand ◽  
Wild Type ◽  
Passenger Strand ◽  
Functional Transition ◽  
Mature Microrna ◽  
Microrna Processing ◽  
Mature Micrornas

MicroRNAs are regulators of gene expression whose functions are critical for normal development and physiology. We have previously characterized mutations in a Caenorhabditis elegans microRNA-specific Argonaute ALG-1 (Argonaute-like gene) that are antimorphic [alg-1(anti)]. alg-1(anti) mutants have dramatically stronger microRNA-related phenotypes than animals with a complete loss of ALG-1. ALG-1(anti) miRISC (microRNA induced silencing complex) fails to undergo a functional transition from microRNA processing to target repression. To better understand this transition, we characterized the small RNA and protein populations associated with ALG-1(anti) complexes in vivo. We extensively characterized proteins associated with wild-type and mutant ALG-1 and found that the mutant ALG-1(anti) protein fails to interact with numerous miRISC cofactors, including proteins known to be necessary for target repression. In addition, alg-1(anti) mutants dramatically overaccumulated microRNA* (passenger) strands, and immunoprecipitated ALG-1(anti) complexes contained nonstoichiometric yields of mature microRNA and microRNA* strands, with some microRNA* strands present in the ALG-1(anti) Argonaute far in excess of the corresponding mature microRNAs. We show complex and microRNA-specific defects in microRNA strand selection and microRNA* strand disposal. For certain microRNAs (for example mir-58), microRNA guide strand selection by ALG-1(anti) appeared normal, but microRNA* strand release was inefficient. For other microRNAs (such as mir-2), both the microRNA and microRNA* strands were selected as guide by ALG-1(anti), indicating a defect in normal specificity of the strand choice. Our results suggest that wild-type ALG-1 complexes recognize structural features of particular microRNAs in the context of conducting the strand selection and microRNA* ejection steps of miRISC maturation.

scholarly journals siRNAs containing 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2′-F-NMC NTPs

2021 ◽  
Author(s):  
Masaaki Akabane-Nakata ◽  
Namrata D Erande ◽  
Pawan Kumar ◽  
Rohan Degaonkar ◽  
Jason A Gilbert ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mitochondrial Rna ◽  
Seed Region ◽  
Guide Strand ◽  
Passenger Strand ◽  
Molecular Modeling Studies ◽  
Rnai Activity ◽  
Rna And Dna

Abstract We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2′-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2′-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5′ phosphate, suggesting that the 2′-F-NMC is a poor substrate for 5′ kinases. In mice, the 2′-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2′-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5′-phosphate mimic 5′-(E)-vinylphosphonate was attached to the 2′-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2′-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2′-F-NMC. Finally, the 5′-triphosphate of 2′-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

scholarly journals A study on the fundamental factors determining the efficacy of siRNAs with high C/G contents

2008 ◽  
Vol 13 (2) ◽  
Author(s):  
Jie-Ying Liao ◽  
James Yin ◽  
Fang Chen ◽  
Tie-Gang Liu ◽  
Jia-Chang Yue
Keyword(s):  
Gene Silencing ◽  
Structure And Function ◽  
Guide Strand ◽  
Target Mrna ◽  
Target Mrnas ◽  
Passenger Strand ◽  
Target Sequences ◽  
And Function ◽  
Necessary And Sufficient ◽  
Rnai Effect

AbstractAlthough there are many reports about the efficacy of siRNAs, it is not clear whether those siRNAs with high C/G contents can be used to silence their target mRNAs efficiently. In this study, we investigated the structure and function of a group of siRNAs with high C/G contents. The results showed that single siRNAs against the Calpain, Otoferlin and Her2 mRNAs could induce different silencing effects on their targets, suggesting that the accessibility to target sequences influences the efficacy of siRNA. Unexpectedly, a single siRNA could target its cognate sequence in the 3’UTR of EEF1D or the 5’UTR of hTRF2 or CDC6. Their interaction induced different modes of gene silencing. Furthermore, the introduction of mutations into the 3’ end of the passenger strand showed that the position and number of mutated nucleotides could exert some influence on the efficacy of siRNA. However, these mutations did not completely block the passenger strand from exerting its RNAi effect. Interestingly, our findings also indicated that the target mRNA might play essential roles in maintaining or discarding the guide strand in RISCs. Thus, the conclusion could be drawn that favorable siRNA sequences, accessible target structures and the fast cleavage mode are necessary and sufficient prerequisites for efficient RNAi.

RNA interference activity of single-stranded oligonucleotides linked between the passenger strand and the guide strand with an aryl phosphate linker

2021 ◽  
Vol 40 (6) ◽  
pp. 647-664
Author(s):  
Makoto Koizumi ◽  
Yasuhide Hirota ◽  
Makiko Nakayama ◽  
Masakazu Tamura ◽  
Wataru Obuchi
Keyword(s):  
Rna Interference ◽  
Guide Strand ◽  
Passenger Strand

scholarly journals Investigation of Strand-Selective Interaction of SNA-Modified siRNA with AGO2-MID

2020 ◽  
Vol 21 (15) ◽  
pp. 5218 ◽  
Author(s):  
Yukiko Kamiya ◽  
Yuuki Takeyama ◽  
Tomonari Mizuno ◽  
Fuminori Satoh ◽  
Hiroyuki Asanuma
Keyword(s):  
Nucleic Acid ◽  
Inhibitory Effect ◽  
Guide Strand ◽  
Argonaute 2 ◽  
Passenger Strand ◽  
Nuclease Resistance ◽  
Interfering Rna ◽  
Target Activity ◽  
Rnai Activity ◽  
Target Effects

Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5’-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5’ end of the passenger strand.

Regulation of miRNA strand selection: follow the leader?

2014 ◽  
Vol 42 (4) ◽  
pp. 1135-1140 ◽  
Author(s):  
Hedda A. Meijer ◽  
Ewan M. Smith ◽  
Martin Bushell
Keyword(s):  
Rna Binding ◽  
Current Knowledge ◽  
Guide Strand ◽  
Disease States ◽  
Protein Activator ◽  
Passenger Strand ◽  
Key Characteristics ◽  
Dependent Protein ◽  
Activation Response ◽  
The Many

miRNA strand selection is the process that determines which of the two strands in a miRNA duplex becomes the active strand that is incorporated into the RISC (RNA-induced silencing complex) (named the guide strand, leading strand or miR) and which one gets degraded (the passenger strand or miR*). Thermodynamic features of the duplex appear to play an important role in this decision; the strand with the weakest binding at its 5′-end is more likely to become the guide strand. Other key characteristics of human miRNA guide strands are a U-bias at the 5′-end and an excess of purines, whereas the passenger strands have a C-bias at the 5′-end and an excess of pyrimidines. Several proteins are known to play a role in strand selection [Ago (Argonaute), DICER, TRBP (trans-activation response RNA-binding protein), PACT (protein activator of dsRNA-dependent protein kinase) and Xrn-1/2]; however, the mechanisms by which these proteins act are largely unknown. For several miRNAs the miR/miR* ratio varies dependent on cell type, developmental stage and in different disease states, suggesting that strand selection is a tightly controlled process. The present review discusses our current knowledge regarding the factors and processes involved in strand selection and the many questions that still remain.

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