A Genetic Circuit Design for Targeted Viral RNA Degradation

Figures

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  Figure 1. RNA-binding protein recognizing RNA sequence for processing. Different binding motifs on the viral RNA sequence are represented with different colors. (A) The RNA-binding protein recognizes the cognate motifs (blue) and facilitates further steps in the RNA processing either for degradation, translation, localization, or stability. (B) RNA without the recognizable blue motifs prevents binding of the RBP to the RNA, thereby truncating RNA processing.

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  Figure 2. Principles of a simple genetic circuit design. (A) An AND logic gate with two promoters (PInA and PInB) for respective inputs (InA and InB). Expressed products (InA and InB) of both genes form a complex (InAInB Complex) that acts as a transcription activator to bind to the promoter (POut) of the output. The final product (OutQ) is predetermined in most cases to perform specific functions. The promoter regions are subject to fine-tuning to regulate the expression of the inputs or the output or both. (B) When both inputs A and B are expressed, the circuit is switched on and the expected output would be significantly expressed. The expression of either or none of the inputs would lead to low or no output. (C) When the circuit is switched on, the expected output increases significantly, and vice versa when switched off (D).

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  Figure 3. Proposed genetic circuit for viral RNA degradation. (A) When the virus infects a new cell, it triggers the expression of the upregulation of the RBP zinc finger antiviral protein (ZAP). The synthetic promoter in the circuit also allows the viral particles to trigger the expression of non-coding RNA (ncRNA) containing a sequence motif specific to protein. The protein–RNA complex acts as a secondary transcription activator to trigger the expression of the RNA-degrading enzyme, which is designed to specifically target the viral RNA in the host cell for degradation. (B) A recombinase such as serine integrase can act as a biological device to perform site-specific DNA inversion at specific att (attachment) sites to switch ON/OFF the expression of the non-coding RNA.

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  Figure 4. Zinc finger antiviral protein (ZAP) bound to an RNA molecule. Coordinates of the crystal structure of ZAP (PDB ID: 3U9G) were downloaded from www.rcsb.org, and HDOCK (http://hdock.phys.hust.edu.cn/, (accessed on 23 November 2023) was used to dock the RBP on the RNA with the 5′-CGUCGU-3′ binding motifs for ZAP [84]. The best model obtained was visualized using PyMOL version 2.5 [85].