Enhanced affinity of racemic phosphorothioate DNA with transcription factor SATB1 arising from diastereomer-specific hydrogen bonds and hydrophobic contacts

Abstract

Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.

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

“…63 Favorable PSO-mediated protein−DNA and −RNA interactions compared to the parent all-PO2 species are commonly attributed to diastereoisomer-specific hydrophobic contacts. 64,65 However, in this study, the key contribution to the increased stability afforded by the Rp-PSO− and PS2−base face-on interaction stems primarily from enhanced dispersion attraction, as suggested by the calculations and dramatically supported by the thermodynamic measurements for the Rp-PSO stereoisomer that forms a face-centered complex with guanine. The observed similar stabilities of the PO2− and MePO−G interactions further support the conclusion that hydrophobics cannot be the main driver for the stability increases seen with thiophosphates.…”

Beyond ribose and phosphate: Selected nucleic acid modifications for structure–function investigations and therapeutic applications

“…Particularly, as far as improving metabolic stability, pairing properties (RNA affinity), protein binding and transport/cellular uptake are concerned, chemical modifications are a prerequisite for the discovery and development of oligonucleotide therapeutics [11][12][13][14][15]. Thus, the natural PS and 2'-OMe backbone modifications provide improved resistance to degradation by exo-and endonucleases and they both affect protein binding [16,17]. Eight of the now approved 13 oligonucleotide drugs feature the PS modification in the backbone and all four approved siRNA therapeutics: ONPATTRO ® (patisiran, 2018), GIVLAARI ® (givosiran, 2019), OXLUMO ® (lumasiran, 2020) and LEQVIO ® (inclisiran, 2020) have 2'-OMe modifications [18][19][20][21] (https:// www.oligotherapeutics.org/20th-anniversary-of-rna-interference-in-mammalian-cells/).…”

Molecular recognition between bacterial phosphorothioate DNA and sulfur-binding domain (SBD): competition between the water cage and chalcogen-hydrophobic packet

“…Several high contact numbers are observed in outstanding inhibitions of the artificial phosphorothioate nucleotide complexes, e.g. , S2 sulfur in (poxvirus immune nuclease), 61 S1 and S2 in (transcription factor SATB1), 20 S2 in (DNA lyase), 62 (DNA polymerase I), 63 and (translation initiation factors), S1 in 64 (CRISPR-Cas Phi effector), and (hydrolase SAMHD1) 66 (see Table S6, ESI†). Many PS-nucleotide drug/inhibitor target proteins possess fewer than five contacts, such as in , 18 , 19 , 21 , 25 , , 65 , 67 , 68 , 69 70 and , 71 revealing that the sulfur atom accidentally (man-forcedly) replaces the “natural” phosphate non-bridging oxygen atom due to artificial reasons in drug development—it is opposite to virtually replace the natural sulfur atom of bacterial phosphorothioate DNA in the theoretical studies.…”

“…17,18 The phosphorothioate (PS) and phosphorodithioate (PS2) investigations suggest that hydrophobic contacts between the PT-modified backbone and protein side chains enhance their affinities to the functional proteins (see Table S1, ESI †). 19,20 For instance, Seth et al reported a crystal structure for the human transcriptional cofactor PC4-DNA binding domain with a phosphorothioate 2 0 -OMe gap-mer antisense oligonucleotide, 21 in which the PS site has electrostatic and hydrophobic contacts with R70, R100, and K101. Similarly, Egli et al found that PS2 linkage increases the binding affinity of the MS2 coat protein binding with cognate RNA hairpin also by electrostatic and hydrophobic interactions.…”