One-Pot Production of RNA Nanoparticles via Automated Processing and Self-Assembly

Abstract

From the original sequencing of the human genome, it was found that about 98.5% of the genome did not code for proteins. Subsequent studies have now revealed that a much larger portion of the genome is related to short or long noncoding RNAs that regulate cellular activities. In addition to the milestones of chemical and protein drugs, it has been proposed that RNA drugs or drugs targeting RNA will become the third milestone in drug development (Shu, Y.; et al. Adv. Drug Deliv. Rev. 2014, 66, 74.). Currently, the yield and cost for RNA nanoparticle or RNA drug production requires improvement in order to advance the RNA field in both research and clinical translation by reducing the multiple tedious manufacturing steps. For example, with 98.5% incorporation efficiency of chemical synthesis of a 100 nucleotide RNA strand, RNA oligos will result with 78% contamination of aborted byproducts. Thus, RNA nanotechnology is one of the remedies, because large RNA can be assembled from small RNA fragments via bottom-up selfassembly. Here we report the one-pot production of RNA nanoparticles via automated processing and self-assembly. The continuous production of RNA by rolling circle transcription (RCT) using a circular dsDNA template is coupled with self-cleaving ribozymes encoded in the concatemeric RNA transcripts. Production was monitored in real-time. Automatic production of RNA fragments enabled their assembly either in situ or via one-pot co-transcription to obtain RNA nanoparticles of desired motifs and functionalities from bottom-up assembly of multiple RNA fragments. In combination with the RNA nanoparticle construction process, a purification method using a largescale electrophoresis column was also developed.

Circular Nucleic Acids: Discovery, Functions and Applications

“…Recently, Guo et al. reported the synthesis of RNA nanoparticles by self‐cleaving ribozyme‐coupled rolling circle transcription using circular double‐stranded DNA as a template (Figure B) . Purified RNA nanoparticles with desired motifs and functionalities can be obtained by automatic production and the bottom‐up assembly of multiple RNA fragments.…”

Virus-associated ribozymes and nano carriers against COVID-19

“…Transport of non-toxic LMW-PEI-mediated ribozymes resulted in a significant reduction in tumour growth [120]. Besides, nanoparticular systems for the delivery of siRNA, ribozymes and/or aptamers containings were developed [121]. The packaging RNA (pRNA) monomer sequence containing a central 3-way junction (3WJ) domain contains ribozymes, siRNA and folate (FA).…”

Opportunities and challenges for the clinical translation of structuredDNAassemblies as gene therapeutic delivery and vaccine vectors

“…In the field of nanotechnology‐formulated drugs, the production of a formulation equivalent to that of at least 1 kg of API is a typical prerequisite for a successful clinical program. The costs summarized in the table compare DNA origami with some of the most common nanotechnology platforms and are based on references (Ebiohippo, n.d.; Halford, 2005; Jasinski, Binzel, & Guo, 2019;Nanocomposix, n.d.; Praetorius et al, 2017; TTScientific, n.d.; US‐Nano, n.d.). The costs were converted to U.S. dollars (USD) based on the rates available on April 12, 2019.…”