Nucleic Acid-Protein Conjugation via Segmented Peptide Linkers
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Solution Overview
Problem
Current methods for coupling proteins to DNA face challenges such as protein instability and aggregation due to suboptimal reaction conditions, and lack of specificity and stoichiometry, especially when working with thermo-sensitive proteins.
Innovation Solution
A two-step method involving the conjugation of nucleic acids to synthetic peptides using bio-orthogonal click chemistry, followed by transpeptidase-mediated coupling to proteins, allowing for protein-compatible conditions and preserving protein function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional chemistries (di-sulfide linkages, thiol-primary-amine linkages) are used to couple proteins to DNA, then conjugation can be achieved, but protein instability and aggregation occur due to suboptimal reaction conditions
Solution Approach 1:
The conjugation process is divided into two distinct steps: first, nucleic acids are conjugated to synthetic peptides using bio-orthogonal click chemistry under controlled conditions; second, the peptide-nucleic acid conjugate is coupled to the protein of interest using transpeptidase enzymes under protein-compatible conditions. This segmentation allows each step to be optimized independently, preserving protein stability while achieving reliable conjugation.
Solution Approach 2:
Synthetic peptides serve as intermediaries between the nucleic acids and proteins. The peptides are first conjugated to nucleic acids, then used as substrates for transpeptidase enzymes that transfer the peptide-nucleic acid conjugate to the protein. This intermediary approach allows the protein to interact with the conjugate under mild, physiological conditions rather than direct coupling under harsh conditions.
2Manufacturing precision
If bio-orthogonal click chemistry is used to overcome specificity issues, then conjugation specificity improves, but protein aggregation and precipitation occur under required non-physiological conditions
Solution Approach 1:
The process separates the high-specificity chemistry step (click chemistry between nucleic acids and peptides) from the protein conjugation step (transpeptidase-mediated). The click chemistry can proceed with high specificity under controlled conditions, while the subsequent protein coupling occurs under physiological conditions, preventing aggregation and precipitation.
Solution Approach 2:
Synthetic peptides act as intermediaries that can tolerate the non-physiological conditions required for bio-orthogonal click chemistry. Once the peptide is conjugated to the nucleic acid, it serves as a stable carrier that can be transferred to the protein under mild conditions, thus protecting the protein from exposure to harsh reaction environments.
3Productivity
If direct conjugation methods are used, then conjugation can be achieved in one step, but stoichiometry control and purification are difficult
Solution Approach 1:
The two-step process allows for better stoichiometry control: first, nucleic acids are conjugated to peptides in a controlled ratio; second, the peptide-nucleic acid conjugate is transferred to the protein in a separate step with controlled stoichiometry. This segmentation enables independent optimization of each conjugation step's stoichiometry and facilitates purification by allowing selective removal of unreacted components.
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
This approach enables robust and specific conjugation of nucleic acids to proteins with minimal loss of protein activity, maintaining at least 75% of the protein's specific activity and facilitating purification, suitable for both thermostable and non-thermostable proteins.
Implementation Method 1
conjugating the nucleic acid-peptide conjugate to a protein of interest using a transpeptidase (e.g., a sortase enzyme) reaction
Implementation Method 2
groups have worked on developing bio-orthogonal techniques such as copper-free click-chemistry
Data Source
AI summary
Provided are methods and compositions relating to conjugation of nucleic acids and proteins of interest under conditions that maintain protein activity. The nucleic acid-protein conjugates may be used in nucleic acid nanostructures such as those generated using DNA origami methods.