Viral xrRNA Protective Elements for Nucleic Acid Stability
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Solution Overview
Problem
Nucleic acid molecules in synthetic biology are prone to degradation by nucleases, which limits their functionality and requires either chemical modification or increased expression levels, both of which have drawbacks such as metabolic toxicity and finite supply issues.
Innovation Solution
The use of nucleic acid protective elements (PELs) derived from viral exoribonuclease-resistant RNA motifs, which form mechanical blocks to inhibit nuclease degradation, enhancing the stability and performance of nucleic acids in synthetic biology applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If chemically modified nucleic acids are used to protect against degradation, then nucleic acid stability is improved, but the finite supply and need for repeated delivery events worsen the practical applicability
Solution Approach 1:
The cell's own transcription machinery is harnessed to continuously produce the protected nucleic acid sequences. The PEL-containing DNA template is transcribed by host RNA polymerase, providing self-sustaining production without external chemical modification delivery, resolving the finite supply issue while maintaining stability
Solution Approach 2:
The invention changes the structural parameter of the nucleic acid by incorporating PEL sequences that form mechanical blocks, transforming the degradation resistance mechanism from chemical modification to structural protection, enabling both stability and continuous supply
2Quantity of substance
If expression levels of degraded RNAs are increased to ensure sufficient quantities survive, then functional availability is improved, but metabolic toxicity worsens
Solution Approach 1:
The PEL is placed at the 5' end of the RNA sequence before degradation can occur, creating a protective mechanical block in advance. This preliminary structural protection prevents degradation without requiring increased expression levels, thereby avoiding metabolic toxicity while ensuring sufficient surviving RNA quantity
3Ease of manufacture
If unmodified RNA is expressed within the cell to avoid chemical modifications, then ease of synthesis is improved, but degradation resistance worsens
Solution Approach 1:
The invention merges the simplicity of unmodified RNA synthesis with the protection against degradation by combining PEL sequences with the coding sequence. The resulting construct is transcribed as a single unmodified RNA molecule that possesses both ease of synthesis and degradation resistance through the PEL-induced mechanical block
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
PELs significantly reduce nucleic acid degradation, increasing their longevity and functional availability, thereby improving the performance and dynamic range of nucleic acid-based systems in medicine, agriculture, and energy applications.
Implementation Method 1
PELs are derived from all or part of a viral xrRNA structural motif and/or sequence... PELs comprise a structured region that reduces non-enzymatic degradation of a protected nucleic acid 5' and/or 3' of the PEL... PELs significantly enhance the performance of nucleic acid synthetic biology, protecting nucleic acid regulatory and/or structural elements from degradation
Data Source
AI summary
Nucleic acids (DNA and RNA) provide a versatile platform for engineering synthetic biology in a variety of technology areas including medicine, science, agriculture, and energy. In many settings, degradation of nucleic acid molecules poses a significant engineering challenge as the molecules do not function if they have been degraded. In some embodiments, nucleic acid protective elements (PELs) are used to protect chemically synthesized or expressed nucleic acid molecules from degradation. PELs may be derived from all or part of a viral xrRNA sequence and/or structural motif, PELs may include rationally designed sequences and/or structural motifs, PELs may be engineered using directed evolution, and in some embodiments, PELs comprise a mixture of biologically derived, rationally designed sequence and/or structural motifs, and/or sequences and/or structural motifs that are engineered by directed evolution. In some embodiments, PELs significantly enhance the performance of nucleic acid synthetic biology, protecting nucleic acid regulatory and/or structural elements from degradation to increase regulatory dynamic range, fractional dynamic range, fold-change, and/or other performance metrics. In some embodiments, PELs that reduce nucleic acid degradation provide a platform technology for enhancing the performance of synthetic biology, with applications including therapeutics, diagnostics, biological research tools, vaccines, crop protection, molecular manufacturing, sustainable energy production, and other areas involving nucleic acids.