Phosphorothioate Nucleic Acids: Artificial Modification Envisaged by Nature

Pawłowska, Róża; Guga, Piotr

doi:10.1007/978-981-16-1313-5_51-1

Cited by 2 publications

(1 citation statement)

References 81 publications

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“…The T7 RNA polymerase is also used to introduce modifications into the internucleoside linkages, such as phosphorothioate (PS), borano, or phosphoroselenoate , modification. Although phosphorothioate is commonly introduced into synthetic RNA and DNA to enhance their biological activity, interestingly, this modification has also been detected in naturally occurring nucleic acids, − which makes it particularly interesting.…”

Section: Introductionmentioning

confidence: 99%

Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP

Pawlowska,

Graczyk,

Radzikowska-Cieciura

et al. 2024

ACS Omega

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Modified nucleotides are commonly used in molecular biology as substrates or inhibitors for several enzymes but also as tools for the synthesis of modified DNA and RNA fragments. Introduction of modification into RNA, such as phosphorothioate (PS), has been demonstrated to provide higher stability, more effective transport, and enhanced activity of potential therapeutic molecules. Hence, in order to achieve widespread use of RNA molecules in medicine, it is crucial to continuously refine the techniques that enable the effective introduction of modifications into RNA strands. Numerous analogues of nucleotides have been tested for their substrate activity with the T7 RNA polymerase and therefore in the context of their utility for use in in vitro transcription. In the present studies, the substrate preferences of the T7 RNA polymerase toward β,γ-hypophospho-modified ATP derivatives for the synthesis of unmodified RNA and phosphorothioate RNA (PS) are presented. The performed studies revealed the stereoselectivity of this enzyme for α-thio-β,γ-hypo-ATP derivatives, similar to that for α-thio-ATP. Additionally, it is demonstrated herein that hypodiphosphoric acid may inhibit in vitro transcription catalyzed by T7 RNA polymerase.

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Section: Introductionmentioning

confidence: 99%

Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP

Pawlowska,

Graczyk,

Radzikowska-Cieciura

et al. 2024

ACS Omega

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Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo

Pawlowska,

Radzikowska-Cieciura,

Jafari

et al. 2024

Sci Rep

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Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue—alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure–activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist—AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial–mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

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Phosphorothioate Nucleic Acids: Artificial Modification Envisaged by Nature

Cited by 2 publications

References 81 publications

Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP

Substrate Specificity of T7 RNA Polymerase toward Hypophosphoric Analogues of ATP

Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo

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