Peptide-Oligonucleotide Conjugation via Carbodiimide Activation
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Solution Overview
Problem
Current methods for preparing peptide-oligonucleotide conjugates (POCs) are inefficient and inconvenient, limiting their therapeutic applications due to instability and low yields, particularly when using unstable phosphodiester linkages or ester linkages for conjugating peptides to oligonucleotides.
Innovation Solution
A method involving reacting nucleic acids with a 5′-monophosphate using an activating agent, followed by mixing with an alcohol to form an intermediate, and then dissolving in a buffer containing EDTA and a nucleophile for conjugation, which stabilizes the POCs with high purity and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If post solid-phase synthesis or stepwise solid-phase synthesis is used to prepare POCs, then peptide-oligonucleotide conjugates can be formed, but the preparation is inefficient and inconvenient with limited reactions available
Solution Approach 1:
The invention changes the chemical parameters of the conjugation reaction by using carbodiimide chemistry (EDC/NHS activation) instead of traditional phosphodiester or ester linkage methods. This allows conjugation at room temperature in aqueous buffers with high yields and stability, making the process efficient and convenient for typical research laboratories
Solution Approach 2:
The invention introduces carbodiimide (EDC) and NHS as intermediary reagents that facilitate the conjugation between oligonucleotides and peptides. These intermediaries enable stable amide bond formation without requiring specialized solid-phase synthesis equipment, thereby improving both ease of manufacture and productivity
2Ease of manufacture
If unstable phosphodiester linkages or ester linkages are used to conjugate peptides to oligonucleotides, then POCs can be formed, but the linkages are unstable and yields are low
Solution Approach 1:
The invention changes the chemical nature of the linkage from unstable phosphodiester or ester bonds to stable amide bonds through carbodiimide-mediated coupling. This parameter change in bond type provides superior stability while maintaining ease of formation under mild conditions
Solution Approach 2:
The invention creates a composite conjugation linkage system using EDC/NHS chemistry that combines the reactivity of carboxyl groups with the stability of amide bonds. This composite approach yields stable POCs with high purity and yields, overcoming the instability issues of traditional linkages
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 method facilitates the preparation of stable POCs with improved biological stability and cellular uptake, enhancing their therapeutic potential by overcoming the limitations of existing conjugation techniques.
Implementation Method 1
reacting the nucleic acid having a 5'-monophosphate with an activating agent in a first buffer to form a solution
Implementation Method 2
dissolving the intermediate in a second buffer containing an ethylenediaminetetraacetic acid (EDTA)
Data Source
AI summary
A method for conjugating a nucleic acid with a molecule is provided. The method includes steps of (a) reacting the nucleic acid having a 5′-monophosphate with an activating agent in a first buffer to form a solution; (b) mixing an alcohol with the solution formed in the step (a) to obtain an intermediate; and (c) dissolving the intermediate in a second buffer containing an ethylenediaminetetraacetic acid (EDTA) and adding a nucleophile thereinto to react the intermediate with the nucleophile.