Optimal design of synthetic circular RNAs

Abstract

Circular RNAs are an unusual class of single-stranded RNAs whose ends are covalently linked via back-splicing. Due to their versatility, the need to express circular RNAs in vivo and in vitro has increased. Efforts have been made to efficiently and precisely synthesize circular RNAs. However, a review on the optimization of the processes of circular RNA design, synthesis, and delivery is lacking. Our review highlights the multifaceted aspects considered when producing optimal circular RNAs and summarizes the available options for each step of exogenous circular RNA design and synthesis, including circularization strategies. Additionally, this review describes several potential applications of circular RNAs.

Fig. 1. Types of endogenous and synthetic circRNA backbones.

a Endogenous circularization methods that have been used or mimicked. b A highly efficient circularization method that leaves a scar sequence in the product. c RNAs have been designed to enable the effective circularization of scarless circRNAs. d The sequence of interest has been permuted; thus, it resembles exonic sequences in the T4 td gene. The yellow and orange boxes indicate natural introns and ribozymes, respectively. Sequences that bring the splice sites together are shown as cyan boxes. Genes of interest are represented as green boxes. Promoters and SV40 terminators are shown as gray circles and red hexagons, respectively. DNA constructs are depicted with black lines, and RNA transcripts are depicted with gray lines. RNA transcription is indicated with blue arrows. FL flag, NLS nuclear localization signal, PUF PUF binding motif, DIM dimerization domain, FPG split GFP, H homology arm, E1 and E2 exonic sequences, CDS coding sequence, L ligation sequence, GOI gene of interest.

Fig. 2. Rational design of circRNA sequences.

Each factor to be considered is presented for each region of the vector of interest. Spacers were added to lessen the structural hindrance between the IRES and the gene of interest or the splice junction. Adding motifs for RBPs that enhance translation is beneficial. Several IRESs with stronger activity than the canonically used IRESs and additional codon optimization can result in faster and more abundant protein production.

Fig. 3. Biogenesis and regulation of circRNAs during their lifespan.

a Epigenetic and transcriptional regulation of circRNAs. b Mechanisms of circRNA biogenesis. c CircRNA translation and subcellular localization. d Mechanisms of circRNA decay. e Cellular export of circRNAs.

Fig. 4. Application of circRNAs.

CircRNAs can be used as mRNA vaccines (a) and guide RNAs for DNA and RNA editing (b, c). Additionally, circRNAs function as miRNA sponges or siRNA mimics (d, e) and can be used to isolate proteins (f).