1. Scope

This practice covers the procedures for the preparation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) for assembly into discrete nucleic acid nanoparticles (NANPs), including the most representative NANPs such as DNA cube, RNA cube, and RNA ring. The programmed assemblies of these specific shapes, sizes, and compositions of nucleic acids are determining criteria for their recognition by the immune system for downstream applications in nanomedicine. RNA oligonucleotides are synthesized by the in vitro transcription of polymerase chain reaction (PCR)-amplified DNA templates. All oligonucleotides are purified by denaturing PAGE. Afterwards, oligonucleotides are precipitated in ethanol and dried. The concentration of each sequence is calculated based on the absorbance at 260 nm. Oligonucleotides are then combined in equimolar ratios in one-pot assemblies in Type 1 water which is double deionized (ddiH2O), heated to melt, and set to undergo additional heating steps based on the NANP. To verify assembly, NANPs are visualized via EMSA on a native-PAGE stained with ethidium bromide. Based on its migration through the gel, the presence of a major single band confirms the oligonucleotides have assembled into uniform NANPs. Validated NANPs can then be used for downstream experimental applications

Keywords

nucleic acids, nanoparticles, NANPs, synthesis, physicochemical characterization

Rationale

NANPs are being increasingly utilized in biosensors, biocomputing, nanoscaffolding, and for the controlled delivery of targeted therapeutics. Their design and scaled-up production are becoming more commonplace among an international and interdisciplinary cohort of research groups which work in the field of nucleic acid nanotechnology. While a variety of therapeutic nucleic acids are moving towards clinical use, there are still barriers to the clinical translation of NANPs in this arena. Standardized protocols are one route which has been identified has a means to facilitate their further development. More systemic studies are needed, especially to overcome inefficient communication between the academic, industrial, and clinical sectors.The unknown immunostimulatory profiles of therapeutic nucleic acids have been a major detriment to their clinical progression. For NANPs, recent studies to classify NANPs immune stimulation have demonstrated that the induction of type I and type III interferons vary in response to their physicochemical properties. Therefore, the opportunity for NANP formulations to be personalized for optimal immunomodulation is a promising avenue of this technology. The NANPs shown here have been thoroughly characterized in primary human peripheral blood mononuclear cells (PBMCs) isolated from multiple donors which is an established method of analysis7. Their relative immunorecognition has been utilized to experimentally compare other variables, including platforms for delivery.Additional systematic studies coming from individual labs will aid in accelerating the adaptation of NANP technology. With additional systematic studies, a unified foundational knowledge and established Good Laboratory Practices (GLP) will allow for the possibility of regulatory submissions. Therefore, standardized preparation and assembly protocols of established and well-characterized NANPs such as the DNA cube, RNA cube, and RNA ring are needed. The practice is intended for industry, academic research who plan on translating their NANPs to clinical use and regulatory agencies such as FDA.

The title and scope are in draft form and are under development within this ASTM Committee.