Binding of phosphorothioate oligonucleotides with RNase H1 can cause conformational changes in the protein and alter the interactions of RNase H1 with other proteins

Abstract

We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2′-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein–protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1

“…The reported interaction between NONO and 2′-F-PS-ASOs leads to a reduction in the nuclear abundance of NONO, likely due to targeted protein degradation ( 33 ). While the binding of the 2′-F-PS-ASOs to the NONO homodimer described in this study did not appear to compromise the integrity of the dimer, it is possible that the large-scale nuclear mRNP aggregates formed with ASO treatment elicit a defensive response to mitigate the formation of pathological aggregates, or that PS-ASO binding may affect the NONO interaction with other partner proteins ( 77 ). Ultimately, further investigation is required to explore what effect 2′-F-ASO binding to NONO containing dimers has within a cellular context.…”

RNA modifications can affect RNase H1-mediated PS-ASO activity

“…83 While 2 0 modifications have been shown previously not to affect RNase H1 binding affinity to the duplex, addition of the methyl group at the 2 0 position of the ribose would disrupt this series of interactions and require RNase H1 to traverse the hybrid to establish the necessary contacts for cleavage. 91 This alone does not suggest that overall cleavage should be reduced if RNase H1 is able to compensate by cleaving at alternative positions within the hybrid; however, in the case of the PS-ASO 558807 and its size-matched target RNA from the Cxcl12 gene, we do observe reduced cleavage over time. This could be because RNase H1 requires a minimum length of 7 to 10 RNA:DNA hybridized nucleotides to bind with its hybrid binding domain and cleave the RNA downstream.…”

Nanoparticles-Based Oligonucleotides Delivery in Cancer: Role of Zebrafish as Animal Model

“…In the case of RNase competent, the degradation of RNA is catalyzed by the RNase H enzyme RNASEH1 in presence of the RNA-DNA duplex formed by the binding of the DNA-based ONs with their specific mRNA transcripts. Thus, the target gene expression is silenced ( Figure 1 A) [ 8 , 9 ]. A central DNA-based “gap” surrounded by flanking regions, constituted by chemically modified RNA promoting target binding, constitutes the recently proposed RNase H-competent ASOs [ 10 ].…”