2′Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors

Abstract

Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2¢fluoro (2¢F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2¢F pyrimidine. By contrast, incorporation of 2¢F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2¢F pyrimidine and 5¢ triphosphate (5¢ppp) increased cell death and interferon-b expression in human cancer cells compared with transfection with 2¢hydroxyl 5¢ppp RNAs, whereas RNAs containing 2¢O-methyl pyrimidine and 5¢ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2¢F and 2¢O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.

Aptamers as targeted therapeutics: current potential and challenges

“…Since the affinity/specificity and function of an aptamer is sensitive to its structure, post-SELEX modification may affect the inherent properties and folding structures of the original aptamers, thereby compromising the binding affinity. Therefore, it is necessary to precisely tailor modifications for the desired functions 76 . Unfortunately, universal rules are not available for all the aptamers, and laborious evaluation/optimization is often needed 77 .…”

“…Chemical modifications have proved to be a double-edged sword, since unnatural nucleotides may cause chemical toxic effects or become immunogenic. For example, severe hepatotoxicity was observed in LNA-modified nucleic acids 98 , and 2′-fluoropyridimine-modified RNAs differentially controlled the activation of pattern recognition receptors 76 . Thus, chemical modifications should be used cautiously, according to the desired therapeutic application of the aptamers.…”

Differential effects of toll-like receptor stimulation on mRNA-driven myogenic conversion of human and mouse fibroblasts

“…The cells were transfected with the IVT mRNAs using DharmaFECT ® Duo Transfection Reagent (Thermo Scientific) or Electro Square Porator ECM 830 (BTX, San Diego, CA) as described previously [17]. Briefly, DharmaFECT were mixed in OPTI-MEM I (Invitrogen) at a RNA:DharmaFECT:OPTI-MEMα ratio of 100 ng:0.4 μl:10 μl.…”

Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR

“…Modifications to RNA can also be introduced synthetically and have been used to prepare immunostimulatory RNAs (isRNA) for turning on the innate immune system to treat cancer and chronic infections. 40,41 …”

“…40 Notably, recent work from Lee, Sullenger, and co-workers illustrates that RNAs containing both 2’-fluoro pyrimidines and a 5’-triphosphate (so-called “2’F5’ppp RNA”) increase cell death in human cancer cells and that innate immune-sensing PRRs, including PKR, are strongly upregulated in this process. 41 …”