Shorter Is Better: The α-(<scp>l</scp>)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs

Matsuda, Shigeo; Bala, Saikat; Liao, Jen-Yu; Datta, Dhrubajyoti; Mikami, Atsushi; Woods, Lauren; Harp, Joel M.; Gilbert, Jason A.; Bisbe, Anna; Manoharan, Rajar M.; Kim, MaryBeth; Theile, Christopher S.; Guenther, Dale C.; Jiang, Yongfeng; Agarwal, Saket; Maganti, Rajanikanth; Schlegel, Mark K.; Zlatev, Ivan; Charisse, Klaus; Rajeev, Kallanthottathil G.; Castoreno, Adam; Maier, Martin; Janas, Maja M.; Egli, Martin; Chaput, John C.; Manoharan, Muthiah

doi:10.1021/jacs.3c04744

Cited by 13 publications

(4 citation statements)

References 58 publications

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“…The second nucleotide of the guide strand (AS2) constitutes a notable exception in this regard as modification there is limited to either 2′‐deoxy or 2′‐fluoro residues (Egli & Manoharan, 2019). A sharp turn in the seed region (AS2 to AS8) between nucleotides 6 and 7 seen in crystal structures of Ago2 complexes (Elkayam et al., 2012; Schirle & MacRae, 2012) led us to deploy modifications that destabilize seed pairing and preorganize the guide strand for this wrinkle, resulting in improvements in loading, mitigation of off‐target effects, and better clinical safety (Egli et al., 2023; Guenther et al., 2022; Matsuda et al., 2023; Schlegel et al., 2022). Lipophilic modification in the form of a 2′‐ O ‐hexadecyl conjugation at position 16 of the sense strand was shown to result in potent and durable silencing in the central nervous system (CNS) (Brown et al., 2022).…”

Section: Commentarymentioning

confidence: 99%

“…The absence of the second nucleotide together with PIWI residues of Ago2 that interact with the 5′‐terminal region of the guide strand expose limitations of MID complexes alone with regards to studying the conformation of that region of modified guides and interactions with Ago2. Thus, a computational model of miR‐20a with a 5′‐tTMP that adopts a C4′‐ exo pucker in place of UMP and bound to Ago2 demonstrates that TNA can stack on Y529 and mimic the tight turn between AS1 and AS3 in principle (Matsuda et al., 2023).…”

Section: Commentarymentioning

confidence: 99%

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Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His₆‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

Lei,

Harp,

Chaput

et al. 2024

Current Protocols

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The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5′‐terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman‐like beta1‐alpha1‐beta2‐alpha2‐beta3‐alpha3‐beta4‐alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one‐step approach to grow well‐diffracting crystals of hAgo2 MID‐nucleotide complexes by mixing purified His6‐SUMO‐MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2′‐3′ linked α‐L‐threofuranosyl thymidine‐3′‐triphosphate (tTTP) are presented. This article also describes fluorescence‐based assays to measure dissociation constants (Kd) of MID‐nucleotide interactions for nucleoside 5′‐monophosphates and nucleoside 3′,5′‐bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Crystallization of Ago2 MID‐nucleotide complexesBasic Protocol 2: Measurement of dissociation constant Kd between Ago2 MID and nucleotides

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Section: Commentarymentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His₆‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

Lei,

Harp,

Chaput

et al. 2024

Current Protocols

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“…So far, short RNAs are mainly produced chemically by assembling phosphoramidite building blocks on immobilized nucleosides. [5] While this method represents the workhorse for the production of therapeutic siRNAs and antisense oligonucleotides, [6] developing more efficient and sustainable alternatives granting access to longer RNA oligonucleotides remains an important challenge. [7] Indeed, success of solid-phase assembly of RNA oligonucleotides relies on the careful choice of the 2'-Oprotecting groups which directly affects the reactivity of phosphoramidite building blocks.…”

Section: Introductionmentioning

confidence: 99%

2’,3’‐Protected Nucleotides as Building Blocks for Enzymatic de novo RNA Synthesis

Pichon,

Levi‐Acobas,

Kitoun

et al. 2024

Chemistry A European J

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Besides being a key player in numerous, fundamental biological process, RNA also represents a versatile platform for the creation of therapeutic agents and efficient vaccines. The production of RNA oligonucleotides, especially those decorated with chemical modifications, cannot meet the exponential demand. Due to the inherent limits of solid‐phase synthesis and in vitro transcription, alternative, biocatalytic approaches are in dire need to facilitate the production of RNA oligonucleotides. Here, we present a first step towards the controlled enzymatic synthesis of RNA oligonucleotides. We have explored the possibility of a simple protection step of the vicinal cis‐diol moiety to temporarily block ribonucleotides. We demonstrate that pyrimidine nucleotides protected with acetals, particularly 2',3'‐O‐isopropylidene, are well‐tolerated by the template‐independent RNA polymerase PUP (polyU polymerase) and highly efficient coupling reactions can be achieved within minutes – an important feature for the development of enzymatic de novo synthesis protocols. Even though purines are not equally well‐tolerated, these findings clearly demonstrate the possibility of using cis‐diol‐protected ribonucleotides combined with template‐independent polymerases for the stepwise construction of RNA oligonucleotides.

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“…However, chemical modifications of the ribose-phosphate backbone can affect the RNA interference process, necessitating a balance between the number of modifications and the efficiency of RNAi. The tolerance of the RNAi system to modification of different positions in the antisense and sense strands is being actively studied [ 26 , 27 , 28 ]. Previously, our laboratory developed a highly efficient, selectively modified anti- MDR1 siRNA with 2′OMe modifications in nuclease-sensitive sites.…”

Section: Introductionmentioning

confidence: 99%

Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification

Chernikov,

Ponomareva,

Meschaninova

et al. 2024

Molecules

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Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3′ end of the sense strand of siRNA and cholesterol on the silencing activity of “light” and “heavy” modified siRNAs. All 3′-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5′-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3′-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

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Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs

Cited by 13 publications

References 58 publications

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His₆‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His₆‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

2’,3’‐Protected Nucleotides as Building Blocks for Enzymatic de novo RNA Synthesis

Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification

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Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs

Cited by 13 publications

References 58 publications

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His6‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His6‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

2’,3’‐Protected Nucleotides as Building Blocks for Enzymatic de novo RNA Synthesis

Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification

Contact Info

Product

Resources

About

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His₆‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth

Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His₆‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth