pH-Sensitive Protein Substrate for Stable Translation
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Solution Overview
Problem
Current protein array technologies face challenges in efficiently translating and stabilizing proteins on substrates while maintaining the ability to bind specific agents, particularly under varying pH conditions and with the need for precise spatial addressing.
Innovation Solution
The method involves providing a substrate with nucleic acids encoding hybrid amino acid sequences and affinity tags, along with binding agents, and using translation effectors to express proteins on the substrate, while employing pH-sensitive reagents and surface modifications to facilitate binding and removal of nucleic acids, and using hetero-functional groups for covalent and non-covalent interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If proteins are translated and stabilized on substrates using conventional methods, then protein expression is achieved, but binding specificity and stability under varying pH conditions deteriorate
Solution Approach 1:
The patent introduces pH-sensitive reagents as intermediaries between the protein and substrate. These reagents mediate the binding interaction by forming reversible complexes that are stable at physiological pH but dissociate under denaturing pH conditions, thereby enabling proteins to maintain binding specificity across varying pH environments without direct covalent attachment to the substrate
Solution Approach 2:
The patent utilizes changes in pH as a parameter to control protein binding and release. By adjusting pH conditions, the system transitions between bound and unbound states, allowing proteins to be stabilized at physiological pH and released under denaturing conditions, thus achieving both stability and pH adaptability
2Productivity
If nucleic acids are permanently attached to substrates for protein translation, then translation efficiency is improved, but substrate reuse and cleaning capability deteriorate
Solution Approach 1:
The patent transforms the static attachment of nucleic acids to substrates into a dynamic, reversible interaction. Nucleic acids are attached through pH-sensitive reagents that allow the nucleic acid-substrate complex to transition between bound and unbound states, enabling the substrate to be reused after protein translation by simply changing pH conditions to release the nucleic acids
Solution Approach 2:
The patent enables the recovery and reuse of substrates by discarding (releasing) the nucleic acids after they have served their translation function. By using pH-sensitive attachment mechanisms, the nucleic acids can be released from the substrate through pH changes, allowing the substrate to be regenerated and reused for subsequent translation cycles
3Stability of the object's composition
If binding agents are used to stabilize proteins on substrates, then protein immobilization is improved, but spatial addressing precision and binding agent removal deteriorate
Solution Approach 1:
The patent introduces removable binding agents as intermediaries that temporarily mediate between the protein and substrate during translation. These binding agents provide stable immobilization during the translation process but can be selectively removed afterward through specific conditions (such as pH changes or competitive displacement), thereby preserving spatial addressing precision while achieving stable protein immobilization during the critical translation phase
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 the efficient translation and stabilization of proteins on substrates, allowing for precise binding of specific agents and maintaining protein stability across varying pH conditions, enhancing the functionality and versatility of protein arrays.
Implementation Method 1
a binding agent that recognizes the affinity tag
Implementation Method 2
contacting the substrate with a translation effector to thereby translate the hybrid amino acid sequence
Implementation Method 3
removing the nucleic acid includes digesting the nucleic acid with an enzyme, e.g., an RNase or DNase
Implementation Method 4
The nucleic acid can be attached to the substrate, e.g., non-covalently attached by a reagent whose affinity for nucleic acid varies with buffer conditions. For example, the reagent binds nucleic acid at a pH of less than 7.5, but releases nucleic acid at a pH greater than 8
Implementation Method 5
providing a substrate that includes a reactive surface having a homo-functional group capable of reacting with proteins (e.g., a primary amino group, an aldehyde group, an epoxy group, or a carboxyl group)
Implementation Method 6
a non-protein agent that binds to the Fc region of an antibody (e.g. a boronate group, or a metal chelating group)
Data Source
AI summary
The disclosure features methods that include: providing a substrate that includes (i) a nucleic acid (e.g., DNA or RNA) encoding a hybrid amino acid sequence including a test amino acid sequence and an affinity tag, and (ii) a binding agent that recognizes the affinity tag; contacting the substrate with a translation effector to thereby translate the hybrid amino acid sequence; maintaining the substrate under conditions permissive for the hybrid amino acid sequence to bind the binding agent; and removing the nucleic acid from the substrate. In one embodiment, the substrate includes a plurality of positionally-distinguishable addresses, for example, each include a different nucleic acid. The addresses can be located a regularly or irregularly spaced locations.