Stabilizing Poly(dA:dT) Sequences with Random Linkers
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Solution Overview
Problem
The 3' polyadenyl cassette in DNA sequences encoding RNA for therapeutic use is prone to shortening during bacterial propagation, leading to instability and reduced translational efficiency, necessitating extensive screening of bacterial clones to ensure the correct length.
Innovation Solution
Incorporating a 10-nucleotide random sequence as a linker within the 3' poly(dA:dT) region, specifically between positions 30 and 50, stabilizes the poly(dA:dT) sequence, maintaining RNA stability and translational efficiency without affecting immunological responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a 3' polyadenyl cassette with consecutive dA:dT base pairs is used to encode the 3' poly(A) sequence, then RNA stability and translational efficiency are improved, but the DNA sequence is subject to shortening during bacterial propagation in E.coli
Solution Approach 1:
The poly(dA:dT) cassette is divided into multiple segments separated by interrupter sequences. Instead of a continuous stretch of dA:dT base pairs, the sequence is segmented into several shorter dA:dT regions interspersed with non-dA:dT sequences, preventing slippage while maintaining the encoded poly(A) functionality.
Solution Approach 2:
Interrupter sequences act as intermediaries between adjacent dA:dT segments. These interrupter sequences consisting of non-dA:dT base pairs serve as spacers that break the continuity of the poly(dA:dT) stretch, thereby preventing bacterial DNA polymerase slippage while allowing the overall sequence to still encode a functional poly(A) tail in the RNA transcript.
2Productivity
If a continuous poly(dA:dT) cassette is used to ensure proper poly(A) encoding, then RNA translational efficiency is maintained, but extensive screening of bacterial clones is required to find clones with correct sequence length
Solution Approach 1:
The poly(dA:dT) cassette is pre-modified with interrupter sequences before cloning into the expression vector. This preliminary design modification ensures sequence stability during bacterial propagation, eliminating the need for extensive clone screening later in the workflow.
Solution Approach 2:
The sequence composition parameter of the poly(dA:dT) cassette is changed by introducing non-dA:dT interrupter sequences at specific positions. This parameter modification maintains the encoded poly(A) length while dramatically improving sequence stability during E.coli propagation, thereby reducing clone variability.
3Duration of action of stationary object
If the poly(dA:dT) cassette length is increased to encode longer poly(A) sequences for improved RNA stability, then RNA half-life is extended, but the sequence becomes more prone to shortening mutations during bacterial propagation
Solution Approach 1:
Long poly(dA:dT) cassettes are segmented into shorter regions separated by interrupter sequences. This segmentation prevents slippage mutations that would otherwise occur in long continuous stretches, thereby maintaining sequence fidelity while still encoding sufficiently long poly(A) tails for RNA stability.
Solution Approach 2:
Interrupter sequences serve as protective intermediaries that prevent the formation of slippage-prone secondary structures in long poly(dA:dT) regions. By inserting these non-dA:dT spacers, the overall sequence length can be maintained for RNA stability while preventing shortening mutations during propagation.
Data Source
AI summary
The present invention relates to nucleic acid molecules containing poly(dA:dT) regions which are stabilized in E.coli, methods of propagating such nucleic acid molecules in E.coli, methods of obtaining RNA, peptides or proteins using such nucleic acid molecules and to RNA which is obtained from such nucleic acid molecules and its use. In particular, the poly(dA:dT) regions contain at least one disruption by a sequence not encoding a sequence solely composed of A residues.