Odorant Receptor Identification via Single-Cell Sequencing
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Solution Overview
Problem
Current methods for identifying odorant and malodor receptors, such as indole and skatole receptors, are inefficient due to the retention of olfactory receptors in the endoplasmic reticulum of non-olfactory cells, preventing proper interaction with odorants and limiting the comprehensive decoding of combinatorial interactions between odorants and receptors.
Innovation Solution
A method involving the dissociation of olfactory epithelium into single cells, loading with indicator dyes, contacting with odorants, measuring neuronal activity, amplifying mRNA, and sequencing using Next-Generation Sequencing to identify activated receptors directly from native olfactory neurons, thereby bypassing the limitations of non-olfactory cell assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional deorphanization methods using non-olfactory cells are used, then high-throughput screening is enabled, but olfactory receptors are retained in the endoplasmic reticulum and cannot interact with odorants
Solution Approach 1:
The patent uses a heterologous expression system where olfactory receptors are expressed in non-olfactory cells (HEK293 cells) as an intermediary approach. While the cells are non-olfactory, the system enables high-throughput screening by expressing human olfactory receptors in a controlled cellular environment, allowing identification of ligands despite the ER retention issue
Solution Approach 2:
The patent applies parameter changes by modifying cellular conditions and using different cell lines (HEK293, CHO, COS-7) to express olfactory receptors. By changing the expression system parameters and using pharmacological tools like forskolin and IBMX to enhance cAMP signaling, the method enables functional assessment of receptors that would otherwise be retained in the ER
2Loss of information
If comprehensive screening of all olfactory receptors is performed, then complete decoding of odorant-receptor interactions is achieved, but the complexity and time required increases significantly
Solution Approach 1:
The patent performs preliminary action by expressing multiple olfactory receptors in parallel in heterologous cell lines before conducting the actual odorant screening. This pre-preparation of receptor-expressing cell lines allows for systematic and efficient screening of odorants against multiple receptors simultaneously, reducing the overall time required for comprehensive decoding
Solution Approach 2:
The patent segments the olfactory receptor repertoire into individual receptor types, each expressed in separate cell lines. This segmentation allows for systematic screening where each receptor can be individually assessed for odorant binding, enabling complete decoding while maintaining organizational efficiency and reducing the complexity of simultaneous multi-receptor analysis
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 allows for the rapid and reliable identification of odorant and malodor receptors, enhancing the throughput and accuracy in decoding the olfactory code and identifying receptors activated by specific odorants, including indole and skatole, which can lead to the development of effective malodor counteractants.
Implementation Method 1
loading the olfactory cells with an indicator dye that allows for the measurement of odorant or aroma receptor binding activity
Data Source
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AI summary
Provided here are new methods to identify specific families of mammalian odorant receptors for odorants or aroma, particularly indole and skatole malodors and their use in assays that may be used to discover compounds that modulate (blocking, enhancing, masking or mimicking compounds) their activity. Orphan mouse odorant receptors are identified from olfactory sensory neurons that respond to target compounds. The resulting receptors as well as their human counterparts can be screened in assays against test compounds to confirm their identity as odorant or aroma receptors, particularly malodor receptors and subsequently discover for example modulators that inhibit the perception of the malodor in humans.