Incorporation of Biaryl Units into the 5′ and 3′ Ends of Sense and Antisense Strands of siRNA Duplexes Improves Strand Selectivity and Nuclease Resistance

Yoshikawa, Kayo; Ogata, Aya; Matsuda, Chiho; Kohara, Michinori; Iba, Hideo; Kitade, Yukio; Ueno, Yoshihito

doi:10.1021/bc100301w

Cited by 19 publications

(11 citation statements)

References 32 publications

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“…1. The last column indicates the ability of siRNAs to suppress gene expression, as determined by a dual-luciferase reporter assay with Renilla and firefly luciferase genes as reporter genes [22], [28]–[32]. All measured data are at 1 nM of siRNA concentration.…”

Section: Resultsmentioning

confidence: 99%

“…Several siRNA 3' overhang modifications were developed in our lab [22], [26]–[32]. The structure of these compounds (as shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…In this context, several modifications of the nucleotides at the 3' end have been thoroughly investigated [10], [22]–[25]. However, it is not well understood whether compounds with stronger or weaker binding with PAZ domain could enhance or hinder the whole RNAi process.…”

Section: Introductionmentioning

confidence: 99%

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Computational Analysis of siRNA Recognition by the Ago2 PAZ Domain and Identification of the Determinants of RNA-Induced Gene Silencing

Kandeel

Kitade

2013

PLoS ONE

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RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.

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Section: Resultsmentioning

confidence: 99%

“…Several siRNA 3' overhang modifications were developed in our lab [22], [26]–[32]. The structure of these compounds (as shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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Computational Analysis of siRNA Recognition by the Ago2 PAZ Domain and Identification of the Determinants of RNA-Induced Gene Silencing

Kandeel

Kitade

2013

PLoS ONE

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“…16,17 Other types of modifications at guide position 1 are also known to decrease RNAi potency. 42–44 The source of decreased potency from modification of positions 3, 6, 18 and 10 are unknown at this time, but the latter may be a result of modification of the guide/target duplex major groove directly across from the cleavage site of Ago2. 17 In addition, the inhibitory effects seen from modification of guide position 3 or position 6 mirror Terrazas and Kool’s observation that C5-propynyl uridine modification at multiple positions in the 5′ half of the guide strand was detrimental to potency.…”

Section: Resultsmentioning

confidence: 99%

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

et al. 2012

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Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide/alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activty of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

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“…Opportunistic synthetic optimization of the overhang region by means of a diol library had led to the identification of a biphenyl-containing overhang . It proved more effective than the alternative aliphatic overhangs. − Thus, we decided to create a subset of biphenyl-containing compounds from the Novartis Compound Library . We used docking to prioritize compounds for NMR assay.…”

Section: Introductionmentioning

confidence: 99%

How to Computationally Stack the Deck for Hit-to-Lead Generation: In Silico Molecular Interaction Energy Profiling for de Novo siRNA Guide Strand Surrogate Selection

Greenidge

Blommers

Priestle

et al. 2019

J. Chem. Inf. Model.

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The Argonaute-2 protein is part of the RNA-induced silencing complex (RISC) and anchors the guide strand of the small interfering RNA (siRNA). The 3′-end of the RNA contains two unpaired nucleotides (3′-overhang) that interact with the PAZ (PIWI/Argonaute/Zwille) domain of the protein. Theoretical and experimental evidence points toward a direct connection between the PAZ/3′-overhang binding affinity and siRNA’s potency and specificity. Among the challenges to overcome when deploying siRNA molecules as therapeutics are their ready degradation under physiological conditions and off-target effects. It has been demonstrated that nuclease resistance can be improved via replacement of the dinucleotide overhang by small molecules which retain the interactions of the RNA guide strand with the PAZ domain. Most commonly, nucleotide analogues are used to substitute the siRNA overhang. However, in this study we adopt a de novo approach to its modification. The X-ray structure of human Argonaute-2 PAZ domain served to perform virtual screening and molecular interaction energy profiling (i.e., decomposition of the force field calculated protein–ligand interaction energies) of tailored-to-purpose fragment libraries. The binding of fragments to the PAZ domain was validated experimentally by NMR spectroscopy. The in silico guided protocol led to the efficient discovery of a number of PAZ domain ligands with affinities comparable to that of a reference dinucleotide (UpU, K d = 33 μM). Originally starting from a generic fragment library, hits progress from 930 μM down to 14 μM within three iterations for the fragments selected via in silico molecular interaction energy profiling from a bespoke library. These dinucleotide siRNA guide strand surrogates represent potential new siRNA-based therapeutics (when attached to siRNA to form bioconjugates) featuring improved efficacy, specificity, stability, and cellular uptake. This project yielded a portfolio of seven patent applications, four of which have been granted to date.

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Incorporation of Biaryl Units into the 5′ and 3′ Ends of Sense and Antisense Strands of siRNA Duplexes Improves Strand Selectivity and Nuclease Resistance

Cited by 19 publications

References 32 publications

Computational Analysis of siRNA Recognition by the Ago2 PAZ Domain and Identification of the Determinants of RNA-Induced Gene Silencing

Computational Analysis of siRNA Recognition by the Ago2 PAZ Domain and Identification of the Determinants of RNA-Induced Gene Silencing

7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove

How to Computationally Stack the Deck for Hit-to-Lead Generation: In Silico Molecular Interaction Energy Profiling for de Novo siRNA Guide Strand Surrogate Selection

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