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. 2018 Jan 31;11:41–56. doi: 10.1016/j.omtn.2018.01.006

Figure 1.

Figure 1

Design and Functional Investigation of trans-Splicing-Based Suicide RNA

(A) Design of tsRNA for 5′ and 3′ exon replacement (ER) and structure of an AFP mini-gene. The AFP mini-gene encompasses AFP exons 3–6 (E3–E6), including introns 3 and 5 (I3 and I5) but lacking intron 4. The tsRNAs comprise three functional domains. First, computationally selected 50 nt binding domains (BDs) harboring central 2 nt target mismatches, which are fused to the other domains via spacers (blue); the BDs of the 5′ or 3′ ER tsRNAs bind to AFP I3 or I5. Second, splicing domains composed of an intronic splice enhancer (ISE) and a consensus splice donor (SD) site (CAG/GT) for 5′ ER or an ISE, a consensus YNYURAC branchpoint (BP) sequence, an extensive polypyrimidine tract (PPT), and a consensus splice acceptor (SA) site (AG/G) for 3′ ER (red). Third, HSVtk coding domain occuring with or without a start codon for 5′ or 3′ ER, including a novel exonic splice enhancer (ESE), as well as a β-globin mini-intron (orange). The tsRNA for 5′ ER additionally harbors a tertiary structure-stabilized hammerhead ribozyme (HHRz) downstream of the BDs; the tsRNA for 3′ ER is featured with a P2A proteolytic cleavage site upstream of the HSVtk (magenta). For detection of trans-splicing, positioning of primers (arrows) and probes (open troughs) for TaqMan probe-based real-time RT-PCR detection is indicated in magenta (5′ ER) and green (3′ ER). (B–D) Real-time RT-PCR quantification of trans- and cis-spliced RNA isolated from transfected HepG2 cells. Indicated are raw Ct values, mean ± SEM (n = 3). (B) Detection of trans-splicing toward the overexpressed and endogenous AFP message in cells transfected with the parental 5′ ER construct 5-mBDopen and/or the AFP mini-gene p-AFP (E3–E6). (C) Detection of trans-splicing toward the overexpressed and endogenous AFP message in cells transfected with the parental 3′ ER construct 3-mBDopen and/or the AFP mini-gene p-AFP (E3–E6). (D) Detection of trans-splicing toward the endogenous AFP message in cells transfected with the 5′ or 3′ ER construct. (E) RT-PCR (35 + 35 cycles) detection of endogenous AFP mRNA after cis-splicing (C) or trans-splicing (T) via 5′ ER (upper panel) or 3′ ER (lower panel) on 1% agarose gels. See also Figure S1 and Table S1.