Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs

Abstract

We report a generalized methodology for the one-pot production of chemically modified functional RNA nanoparticles during in vitro transcription with T7 RNA polymerase. The efficiency of incorporation of 2'-fluoro-dNTP in the transcripts by the wild type T7 RNA polymerase dramatically increases in the presence of manganese ions, resulting in a high-yield production of chemically modified RNA nanoparticles functionalized with siRNAs that are resistant to nucleases from human blood serum. Moreover, the unpurified transcription mixture can be used for functional ex vivo pilot experiments.

Figure 1

Cotranscriptional assembly of RNA NPs. Schematic representation of cotranscriptional assembly leading to the formation of RNA NP (nanoring) functionalized with six siRNAs and their further purification. Functional siRNAs can be released by Dicer nuclease.

Figure 2

Co-transcriptional assemblies of RNA nanoparticles (NP) with and without chemical modifications (2′-F-dUTP) and their further purifications. (a) Native-PAGE results representing cotranscriptional body-labeled assemblies leading to the formations of ring RNA NP 5′- or 3′-end functionalized with different numbers of siRNAs (0–6) and dynamic light scattering (DLS) result for assembly. (b) Membrane filtration and (c) native-PAGE purification of cotranscriptionally assembled functional RNA NP. (d) Presence of Mn²⁺ stimulates transcription with regular and chemically modified NTP substrates. The relative amounts of single-stranded RNAs produced in the presence of regular NTPs (blue trace) and ATP, CTP, GTP, and 2′-F-dUTP (red trace) are normalized to the standard yields of the nonmodified full-length transcript produced in the absence of MnCl₂ (conventional transcription buffer). The error bars show standard error from nine independent experiments. (e) Native-PAGE results illustrating the formation of cotranscriptionally assembled chemically modified (2′-F-dUTPs) functionalized ring RNA NP. Please note that conventional and 2′-Fl-dUMP-containing nanorings are produced with similar yields.

Figure 3

GFP knockdown assays for human breast cancer cells (MDA-MB-231/GFP) that stably express enhanced GFP (eGFP). Three days after the transfection of cells with cotranscriptionally assembled RNA NP functionalized with siRNA against eGFP, eGFP expression was observed by (a) fluorescence microscopy and (b) statistically (30 000 cells per sample) analyzed with flow cytometry experiments. Please note that the individual nonfunctionalized cotranscriptionally assembled RNA NP cause no decrease in eGFP production.