Post-Poly A Signal RNA Reduces Epigenetic Silencing
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Solution Overview
Problem
Gene silencing is a long-standing problem in synthetic biology and genetic engineering, particularly when synthetic gene circuits are integrated into mammalian genomes, and it is exacerbated for large and complex genetic circuits.
Innovation Solution
An engineered nucleic acid is designed to include a nucleotide sequence encoding a post-poly A signal (post-PAS) RNA, which is positioned 3' to a terminator region, incorporating elements such as a poly A signal, a poly A tail, or a synthetic poly A mimic, along with RNA cleavage sites and regulatory elements like IRES and caps, to enhance stability and expression of functional RNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If synthetic gene circuits are integrated into mammalian genomes, then functional RNA expression is achieved, but epigenetic silencing occurs reducing circuit reliability
Solution Approach 1:
The patent introduces a post-poly A signal RNA as an intermediary element positioned downstream of the poly A signal. This residual RNA transcript acts as a protective mediator that prevents epigenetic silencing of the synthetic gene circuit by interfering with the recruitment of silencing machinery to the integrated circuit, thereby maintaining reliable functional RNA expression.
Solution Approach 2:
The patent employs preliminary action by pre-establishing epigenetic marks on endogenous genes before synthetic circuit integration. By integrating the synthetic circuit into regions with predetermined epigenetic states and using post-poly A signal RNA to maintain these states, the system prevents subsequent silencing and ensures sustained circuit function.
2Adaptability or versatility
If large and complex genetic circuits are integrated, then functional capability is enhanced, but epigenetic silencing is exacerbated
Solution Approach 1:
The patent segments the synthetic genetic circuit into modular units, each potentially protected by its own post-poly A signal RNA element. This segmentation allows complex circuits to be built from smaller functional modules while maintaining epigenetic stability, as each module can be independently regulated and protected from silencing.
Solution Approach 2:
For complex circuits, the patent uses multiple post-poly A signal RNA intermediaries distributed throughout the circuit architecture. These residual RNAs act as protective agents that collectively prevent silencing of large genetic constructs, enabling enhanced functional capability without proportionally increased silencing vulnerability.
3Stability of the object's composition
If residual RNA transcript is produced downstream of poly A signal, then transcription termination is complete, but RNA stability is reduced due to unprotected ends
Solution Approach 1:
The patent converts the previously harmful residual RNA transcript into a beneficial element. By deliberately designing and positioning post-poly A signal sequences, the system transforms the unstable residual RNA into a protective intermediary that prevents epigenetic silencing, thereby converting a stability problem into a functional advantage for circuit maintenance.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered nucleic acid effectively reduces epigenetic silencing of synthetic gene circuits, allowing for sustained expression of functional RNA, including protein-coding, miRNA, and gRNA, while maintaining the regulation of endogenous genetic programs.
Implementation Method 1
the terminator further includes a poly A tail or a synthetic poly A mimic 3′ to the poly A signal... a nucleotide sequence encoding a RNA cleavage site between the poly A signal and the poly A tail or the synthetic poly A mimic, wherein the RNA cleavage site is capable of being cleaved by Cleavage and polyadenylation specificity factor (CPSF)
Implementation Method 2
the residual RNA is expected to be short-lived due to unprotected 5′ and 3′ ends which are recognized by exonucleases
Implementation Method 3
a nucleotide sequence encoding a 5′ cap... The 5′ cap is a clover-leaf
Implementation Method 4
a nucleotide sequence encoding an internal ribosome entry site (IRES)
Data Source
AI summary
The present disclosure is related to an engineered nucleic acid encoding a post-poly A signal RNA 3′ to a terminator for expression of protein, and/or non-coding RNA. Also provided herein are methods for reducing epigenetic silencing, genetic modification, transcriptional regulation of the engineered nucleic acid described herein.