Circularized RNA Production Using DNA Templates for Yield and Stability
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Solution Overview
Problem
Existing methods for producing circularized RNA are limited by quantity and size, hindering their therapeutic application.
Innovation Solution
A method involving 5′ and 3′ complement-reverse complement (CRC) sequences, random nucleotides, and polyA sequences facilitates the enzymatic circularization of RNA, enabling higher yields and stability of circularized RNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If existing methods for producing circularized RNA are used, then RNA circularization can be achieved, but the quantity and size of circularized RNA are limited
Solution Approach 1:
The patent introduces a DNA template as an intermediary to enable in vitro transcription that produces circularized RNA. The DNA template contains specific structural features (inverted repeats, terminal redundancy, or hairpin structures) that direct the RNA polymerase to synthesize circularized RNA directly, bypassing the need for post-transcriptional circularization steps and enabling high-yield production of large quantities of circularized RNA
Solution Approach 2:
The patent modifies the transcription parameters by using specially designed DNA templates with specific structural features (inverted repeats, terminal redundancy, hairpin structures) that change the outcome of in vitro transcription from linear to circularized RNA. This parameter change in the template structure enables the production of circularized RNA in high quantities without size limitations
2Length of stationary object
If existing methods for producing circularized RNA are used, then RNA circularization can be achieved, but the size of RNA that can be circularized is limited
Solution Approach 1:
The DNA template serves as an intermediary that can accommodate any size of RNA sequence to be circularized. By designing the DNA template with appropriate length and structural features, the patent enables the production of circularized RNA of any desired size through in vitro transcription, eliminating the size constraints of previous methods
Solution Approach 2:
The patent creates a universal method using DNA templates with specific structural features that can circularize RNA molecules of any size. The same basic approach (using a DNA template with inverted repeats, terminal redundancy, or hairpin structures) works for both small and large RNA molecules, making the method universally applicable without size limitations
3Stability of the object's composition
If linear RNA is used for therapeutic applications, then transient gene therapy can be achieved, but stability is insufficient
Solution Approach 1:
The patent applies the principle of curvature by circularizing the RNA molecule, forming a closed loop structure without free ends. This circular configuration protects the RNA from exonuclease degradation and increases stability in vivo, while the circularization is achieved through relatively simple DNA template design and in vitro transcription, avoiding excessive structural complexity
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 method produces circularized RNA with improved stability and translation efficiency, suitable for therapeutic applications, including protein expression and vaccination.
Implementation Method 1
The 5' CRC sequence is at least partially complementary to the 3' CRC sequence, e.g., complementary to the 3' CRC sequence
Data Source
AI summary
The present invention provides circularized RNA and methods of making and using same.