Table 1. . Overview of NANP production methods.

Synthesis	Characteristics	Multistrand NANP assembly†	Adaptability for NANP production
Chemical	- Phosphoramidite chemistry with solid-phase supports	No	The synthesis of pure short RNA or DNA oligonucleotides ready to use for NANP assembly is especially useful. This is an easy and versatile way to attach various chemical modifications (fluorophores, linkers, etc.) or produce DNA oligonucleotides for further enzymatic synthesis or cloning for biological applications
Enzymatic	- PCR - PCT - IVT	NA NA Yes	- PCR is predominantly used for amplification of DNA templates/parts for IVT or cloning, respectively. - PCT has not been yet evaluated for NANP production, but is potentially promising. - IVT is the current golden standard for production of individual RNA strands for NANP assemblies
Biological	- Phagemid amplification of ssDNA - Extracellular RNA production - Intracellular RNA production - Vesicular/viral RNA production	Yes Yes Yes Yes	- Amplification of various ssDNAs in phagemid particles can be done, as one strand is very cost effective with the currently largest yield. No similar variant is available for RNA. - Intracellular overexpression of DNA/RNA strands from recombinant vectors may provide a means of production of RNA in the cell, allowing for the transport of RNA to media or its export within a vesicle or virus. Co-transcription of multiple strands can lead to in vivo assembly of NANPs

^†Possibility of multistrand NANP assembly during one pot production.

IVT: In vitro transcription; NANP: Nucleic acid nanoparticle; PCR: Polymerase chain reaction; PCT: Polymerase chain transcription; ssDNA: Single-stranded DNA.