Origins of the enhanced affinity of RNA–protein interactions triggered by RNA phosphorodithioate backbone modification

Yang, Xianbin; Abeydeera, Nalin; Liu, Feng‐Wu; Egli, Martin

doi:10.1039/c7cc05722a

Cited by 14 publications

(18 citation statements)

References 33 publications

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“…These calculations also help explain the increased thermodynamic stability of the dithiophosphate-substituted SRL relative to the wild-type molecule, which is dominated by the enthalpic component in the experimental measurements. Taken together, these two independent studies further demonstrate the significant impact that the incorporation of dithiophosphate at strategic RNA backbone positions may have on stability and/or intermolecular interactions. − By extension, the underlying reasons for the increased stability of the PS2-modified SRL also apply to the R p-PSO SRL.…”

Section: Discussionmentioning

confidence: 79%

“…We recently observed that regiospecific incorporation of a dithiophosphate moiety (PS2) in place of phosphate (PO2) into the backbones of RNA molecules (e.g., viral RNA and siRNA) resulted in dramatically improved binding affinities of the complexes with their respective target proteins. − Anti-thrombin and anti-VEGF RNA aptamers with a single PS2 exhibited a K d of ∼1 pM or 1000-fold tighter than that of the corresponding native RNAs with thrombin and VEGF-165, respectively . Interestingly, monothiophosphate (PSO) incorporation at the same sites resulted in a much more modest gain (thrombin) or an actual loss (VEGF) of binding affinity.…”

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confidence: 99%

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Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

Pallan¹,

Lybrand²,

Schlegel

et al. 2020

Biochemistry

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GNRA (N = A, C, G, or U; R = A or G) tetraloops are common RNA secondary structural motifs and feature a phosphate stacked atop a nucleobase. The rRNA sarcin/ricin loop (SRL) is capped by GApGA, and the phosphate p stacks on G. We recently found that regiospecific incorporation of a single dithiophosphate (PS2) but not a monothiophosphate (PSO) instead of phosphate in the backbone of RNA aptamers dramatically increases the binding affinity for their targets. In the RNA:thrombin complex, the key contribution to the 1000-fold tighter binding stems from an edge-on contact between PS2 and a phenylalanine ring. Here we investigated the consequences of replacing the SRL phosphate engaged in a face-on interaction with guanine with either PS2 or PSO for stability. We found that PS2···G and Rp-PSO···G contacts stabilize modified SRLs compared to the parent loop to unexpected levels: up to 6.3 °C in melting temperature T m and −4.7 kcal/mol in ΔΔG°. Crystal structures demonstrate that the vertical distance to guanine for the closest sulfur is just 0.05 Å longer on average compared to that of oxygen despite the larger van der Waals radius of the former (1.80 Å for S vs 1.52 Å for O). The higher stability is enthalpy-based, and the negative charge as assessed by a neutral methylphosphonate modification plays only a minor role. Quantum mechanical/molecular mechanical calculations are supportive of favorable dispersion attraction interactions by sulfur making the dominant contribution. A stacking interaction between phosphate and guanine (SRL) or uracil (U-turn) is also found in newly classified RNA tetraloop families besides GNRA.

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Section: Discussionmentioning

confidence: 79%

mentioning

confidence: 99%

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

Pallan¹,

Lybrand²,

Schlegel

et al. 2020

Biochemistry

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“…***REVISED*** 2 ammonium O,O'-diethyl dithiophosphate (DETP(S¯)=S) and 5'-Omethoxyphosphorodithioyl-2'-deoxyguanosine (G-P(S¯)=S) were selected as models (Figure 1). [14,15] Employing these compounds, ESR spectroscopy, pulse radiolysis, and theoretical calculation (Figure 1 for the radicals investigated in this work) led to several important findings: (a) Reaction of the one-electron oxidant (e.g., Cl2 •¯) with DETP(S¯)=S and G-P(S¯)=S did not result in the formation of a two-center three-electron σ 2 -σ* 1 -bonded adduct radical, P(2S∸Cl), as found for phosphorothioates. [9] Instead, oxidation of DETP(S¯)=S led to formation of a dithiyl radical, P-2S•, in which the unpaired spin is equally shared by both sulfurs.…”

Section: Full Papermentioning

confidence: 99%

One Way Traffic: Base‐to‐Backbone Hole Transfer in Nucleoside Phosphorodithioate

Kaczmarek

Ward

Debnath

et al. 2020

Chemistry A European J

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“…They found that the anionic sulfur of the PS linkage makes contacts with hydrophobic and cationic amino acids. Similarly Egli showed that the related phosphorodithioate (PS2) linkage enhances binding of the MS2 coat protein and its cognate RNA hairpin by a combination of electrostatic and hydrophobic interactions . While the reports above provide insights into how a single PS can interact with proteins, structural insights into interactions of ASOs with multiple PS linkages in conjunction with additional sugar modifications are lacking.…”

Section: Introductionmentioning

confidence: 99%

Origins of the Increased Affinity of Phosphorothioate-Modified Therapeutic Nucleic Acids for Proteins

et al. 2020

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The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2′-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5′-terminal 2′-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein–protein interactions causative of cellular toxicity.

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Origins of the enhanced affinity of RNA–protein interactions triggered by RNA phosphorodithioate backbone modification

Cited by 14 publications

References 33 publications

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost

One Way Traffic: Base‐to‐Backbone Hole Transfer in Nucleoside Phosphorodithioate

Origins of the Increased Affinity of Phosphorothioate-Modified Therapeutic Nucleic Acids for Proteins

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