Allosterically Unstable NIR-Fbs for Intracellular Imaging
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Solution Overview
Problem
Current methods for visualizing and manipulating intracellular targets using nanobody-fusion proteins face challenges due to high background signal from unbound nanobodies, altered protein behavior from FP-fusions, and limited control over antigen-dependent expression, which complicates studies of heterogeneous cell populations.
Innovation Solution
Development of allosterically unstable NIR-Fbs, which are selectively stabilized and become brightly fluorescent when bound to antigens, allowing for antigen-dependent visualization and manipulation of intracellular targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nanobodies are used for visualizing intracellular targets, then specificity and ability to recognize intracellular antigens is improved, but high background signal from unbound nanobodies worsens the measurement precision
Solution Approach 1:
The patent introduces conditional stability through mutations in framework regions, changing the stability parameter of nanobodies based on antigen presence. This allows dynamic control of nanobody half-life, reducing background signal while maintaining detection capability
Solution Approach 2:
The patent creates a dynamic system where nanobody stability is not fixed but changes in response to antigen levels. The conditional stability mechanism allows the system to adapt between stable (when antigen present) and unstable (when antigen absent) states, optimizing signal-to-noise ratio
2Reliability
If FP-fusion constructs are used to ensure specificity, then ability to study protein dynamics in live cells is improved, but altered expression level and blocked functional domains worsen the reliability
Solution Approach 1:
The patent extracts the fluorescent protein tag from the traditional C-terminal or N-terminal fusion position and inserts it internally within the nanobody structure. This extraction from terminal positions reduces interference with functional domains while maintaining fluorescence capability
Solution Approach 2:
The patent embeds the fluorescent protein sequence within the nanobody structure, creating a nested configuration where the FP is contained inside the Nb framework. This nesting allows the FP to be part of the overall structure without dominating the functional properties
3Measurement precision
If stable cell lines with low Nb-FP fusion expression are developed, then background signal is reduced, but inability to study heterogeneous cell populations worsens the adaptability
Solution Approach 1:
The patent changes the stability parameter dynamically based on antigen presence, allowing the system to work effectively across heterogeneous populations with varying antigen levels. Each cell's nanobody stability is determined by its own antigen content rather than a uniform low expression level
Solution Approach 2:
The patent enables each cell to self-regulate its nanobody stability based on its own antigen levels. Cells with high antigen maintain stable nanobodies for detection, while cells with low or no antigen have unstable nanobodies that degrade, automatically optimizing signal-to-noise ratio in each cell type
4Quantity of substance
If mutations in framework regions are introduced to control intracellular Nb level, then expression level control is improved, but antigen-binding properties worsen
Solution Approach 1:
The patent applies mutations locally in specific framework regions (FR1, FR2, FR4) rather than uniformly across the entire nanobody structure. This localized approach allows control of stability while preserving antigen-binding properties in the CDR regions and critical framework areas
Solution Approach 2:
The patent introduces conditional parameter changes where the stability parameter is modified through specific mutations that only affect stability under certain conditions (antigen presence/absence). The mutations create allosteric effects that change stability without directly affecting binding affinity
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
Enables specific and efficient visualization and manipulation of intracellular targets with reduced background noise, facilitating studies in heterogeneous cell populations and deep-tissue imaging in living organisms.
Implementation Method 1
NIR-Fbs that are allosterically unstable and, consequently, degrade in mammalian cells. These NIR-Fbs become highly stabilized and, consequently, brightly fluorescent, when bound to the cognate intracellular antigen
Implementation Method 2
the unbound intrabodies fusions are translocated to the nucleus where the KRAB domain represses their transcription
Implementation Method 3
increased tissue penetration depths and better signal-to-noise ratio due to reduced light-scattering, tissue absorption and autofluorescence in NIR region
Implementation Method 4
increased tissue penetration depths and better signal-to-noise ratio due to reduced light-scattering, tissue absorption and autofluorescence in NIR region
Implementation Method 5
NIR FPs allow labeling of whole organisms, specific cell populations, organelles, or individual proteins
Data Source
AI summary
Provided herein are fusion proteins comprising a single domain antibody (sdAb) (including, but not limited to, a nanobody) that binds selectively to a specific antigen, wherein a second polypeptide is inserted into the single domain antibody, generating an internal fusion. Also provided are methods of making and methods of using the fusion proteins disclosed herein.