Chemical Modification of siRNA Bases To Probe and Enhance RNA Interference

Peacock, Hayden; Kannan, Arunkumar; Beal, Peter A.; Burrows, Cynthia J.

doi:10.1021/jo2012225

Cited by 89 publications

(74 citation statements)

References 65 publications

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“…Four major classes of chemical modifications (backbone, sugar, base, and terminal) have been developed to resist nuclease-catalyzed degradation of oligonucleotides and to improve siRNA stability in both fetal bovine and human serum (10)(11)(12)(13)(14)(15)26). To test the hypothesis that enzymatic hydrolysis of SNA-1 takes places at a site near the AuNP-facing terminus of the siRNA, thus leading to oligonucleotide fragments too short to remain hybridized and associated to SNA-1, we designed and analyzed SNA-1-(2′-H) 5 and SNA-1-(2′-OMe) 5 , where the RNA nucleotides of siRNA AR at positions 1-5 for both the senseand AS oligonucleotide (i.e., the AuNP-facing terminus) are substituted with 2′-H or 2′-OMe nucleotides and are therefore not susceptible to RNase-catalyzed hydrolysis (Fig.…”

Section: Degradation Of Sna-1 In Solutions Of Fbs Via Loss Of the Asmentioning

confidence: 99%

“…Chemically modified nucleotides [e.g., 2′-O-methyl (2′-OMe), 2′-deoxy-2′-fluoro (2′-F)] are commonly used to increase the stability of siRNA in serum-containing media (10)(11)(12)(13)(14)(15), and research efforts to develop therapeutic siRNA often use "design rules" and patterns for the placement of chemically modified nucleotides to enhance serum lifetimes. For example, multiple reports have shown that modifying all pyrimidines in the siRNA sequence with 2′-F modifications provides high serum stability without compromising activity (16)(17)(18)(19).…”

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confidence: 99%

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Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Barnaby

Lee

Mirkin

2014

Proc. Natl. Acad. Sci. U.S.A.

100

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Small interfering RNA (siRNA) is a powerful and highly effective method to regulate gene expression in vitro and in vivo. However, the susceptibility to serum nuclease-catalyzed degradation is a major challenge and it remains unclear whether the strategies developed to improve the stability of siRNA free in serum solution are ideal for siRNA conjugated to nanoparticle surfaces. Herein, we use spherical nucleic acid nanoparticle conjugates, consisting of gold nanoparticles (AuNPs) with siRNA chemisorbed to the surface, as a platform to study how a model siRNA targeting androgen receptor degrades in serum (SNA-siRNA AR ). In solutions of 10% (vol/vol) FBS, we find rapid endonuclease hydrolysis at specific sites near the AuNP-facing terminus of siRNA AR , which were different from those of siRNA AR free in solution. These data indicate that the chemical environment of siRNA on a nanoparticle surface can alter the recognition of siRNA by serum nucleases and change the inherent stability of the nucleic acid. Finally, we demonstrate that incorporation of 2′-O-methyl RNA nucleotides at sites of nuclease hydrolysis on SNA-siRNA AR results in a 10-fold increase in siRNA lifetime. These data suggest that strategies for enhancing the serum stability of siRNA immobilized to nanoparticles must be developed from a dedicated analysis of the siRNAnanoparticle conjugate, rather than a reliance on strategies developed for siRNA free in solution. We believe these findings are important for fundamentally understanding interactions between biological media and oligonucleotides conjugated to nanoparticles for the development of gene regulatory and therapeutic agents in a variety of disease models.biological recognition | nanotechnology | OliGreen | polyacrylamide gel electrophoresis

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Section: Degradation Of Sna-1 In Solutions Of Fbs Via Loss Of the Asmentioning

confidence: 99%

mentioning

confidence: 99%

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Barnaby

Lee

Mirkin

2014

Proc. Natl. Acad. Sci. U.S.A.

100

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“…A thorough review of the chemical modifications is beyond the scope of this report but can be found elsewhere (Snove and Rossi, 2006;Peacock et al, 2011;Engels, 2013;Nolte et al, 2013). There are two common strategies, to reduce off-targeting -(I) Chemical modifications on the passenger strand preventing its loading, hence eliminating off-targeting caused by the passenger strand Snead et al, 2013) and (II) Chemical modifications in the seed region, which interfere with miRNA-like target recognition but do not prevent specific RNAi targeting.…”

Section: Mismatch Introductionmentioning

confidence: 99%

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

Pačes

Nič²,

Novotný³

et al. 2017

EFS3

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This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on~1,400 cumulative references chosen from~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter.

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“…Several modifications were introduced. The thio (−SH), hydroxyl (−OH), or iodo (−I) can modify bases in specific sites or utilize the pseudouracil base in siRNA, which would augment potency of naked siRNA [31]. There are three most popular chemical modification sites on siRNA structure containing the phosphodiester backbone, ribose 2′-hydroxyl group (R-2′-OH), and ribose ring.…”

Section: Stabilizing the Sirna Deliverymentioning

confidence: 99%

RNAi Therapeutic Potentials and Prospects in CNS Disease

Cho¹,

Kim²

2016

RNA Interference

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Over the past 20 years, RNA interference (RNAi) technology has provided a new regulatory paradigm in biology. This technique can efficiently suppress target genes of interest in mammalian cells. Small non-coding RNAs play important roles in gene regulation, including both in post-transcriptional and in translational regulation. For in vivo experiments, continuous development has resulted in successful new ways of designing, identifying, and delivering small interfering RNAs (siRNAs). Proof-of-principle studies in vivo have clearly demonstrated that both viral and non-viral delivery methods can provide selective and potent target gene suppression without any clear toxic effects. There are also the persistent problems with off-target effects (OTEs), competition with cellular RNAi components, and effective delivery in vivo. Although recent researches and trials from a large number of animal model studies have confirmed that most OTEs are not dangerous, other important issues need to be addressed before RNAi-based drugs are ready for clinical use. Currently, RNAi may be harnessed as a new therapeutic modality for brain diseases. Finally, there are already several RNAi-based human clinical trials in progress. It is hoped that this technology will have also effective applications in human central nervous system (CNS)-related disease.

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Chemical Modification of siRNA Bases To Probe and Enhance RNA Interference

Cited by 89 publications

References 65 publications

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

RNAi Therapeutic Potentials and Prospects in CNS Disease

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