Toolkit/RGEPO1

RGEPO1

Construct Pattern·Research·Since 2025

Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.

Summary

RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively.

Usefulness & Problems

Why this is useful

RGEPO1 is a red genetically encoded potassium indicator targeted to the extracellular membrane. It produces a positive K+-specific fluorescence response and was used for extracellular potassium imaging.; extracellular membrane-targeted potassium imaging; real-time visualization of extracellular K+ dynamics

Source:

RGEPO1 is a red genetically encoded potassium indicator targeted to the extracellular membrane. It produces a positive K+-specific fluorescence response and was used for extracellular potassium imaging.

Source:

extracellular membrane-targeted potassium imaging

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real-time visualization of extracellular K+ dynamics

Problem solved

It provides a tool for monitoring extracellular potassium dynamics in real time, including in intact neural preparations and awake mice.; providing a red genetically encoded indicator for extracellular potassium monitoring

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It provides a tool for monitoring extracellular potassium dynamics in real time, including in intact neural preparations and awake mice.

Source:

providing a red genetically encoded indicator for extracellular potassium monitoring

Problem links

providing a red genetically encoded indicator for extracellular potassium monitoring

Literature

It provides a tool for monitoring extracellular potassium dynamics in real time, including in intact neural preparations and awake mice.

Source:

It provides a tool for monitoring extracellular potassium dynamics in real time, including in intact neural preparations and awake mice.

Published Workflows

Sensitive red fluorescent indicators for real-time visualization of potassium ion dynamics in vivo.

2025

Objective: Develop red genetically encoded potassium indicators suitable for real-time visualization of intracellular and extracellular K+ dynamics in intact biological systems and in vivo.

Why it works: The workflow combines microbial directed evolution with subsequent mammalian-cell optimization, then applies the resulting indicators in relevant neural and in vivo contexts. The abstract also states that molecular dynamics simulations were used to interpret potassium-binding mechanisms.

potassium binding-linked fluorescence responsedistinct potassium-binding pockets and structural featuresdirected evolution in Escherichia colioptimization in mammalian cellsmolecular dynamics simulation

Stages

  1. 1.
    Directed evolution in Escherichia coli(selection)

    The abstract states that the indicators were developed through directed evolution in Escherichia coli as an initial engineering stage.

    Selection: Development of novel red genetically encoded potassium indicators through directed evolution.

  2. 2.
    Optimization in mammalian cells(secondary_characterization)

    The abstract explicitly states that optimization in mammalian cells followed directed evolution in E. coli.

    Selection: Subsequent optimization of indicator performance in mammalian cells.

  3. 3.
    Cell-based characterization in HEK293FT cells(functional_characterization)

    The abstract reports affinities and localization-specific responses in HEK293FT cells before broader biological deployment.

    Selection: Measure K+-specific fluorescence response and affinity of RGEPO1 and RGEPO2 in HEK293FT cells.

  4. 4.
    Application in neural preparations and awake mouse(confirmatory_validation)

    The abstract describes deployment of the indicators in progressively more intact neural systems to demonstrate real-time potassium imaging capability.

    Selection: Demonstrate real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and awake mouse brain.

  5. 5.
    Molecular dynamics analysis of potassium-binding mechanisms(secondary_characterization)

    The abstract states that molecular dynamics simulations provided insights into potassium-binding mechanisms and distinct binding pockets.

    Selection: Use molecular dynamics simulations to analyze potassium-binding mechanisms and structural features of RGEPO1 and RGEPO2.

Steps

  1. 1.
    Perform directed evolution in Escherichia coliengineered indicators

    Generate improved red genetically encoded potassium indicator variants.

    The abstract presents directed evolution in E. coli as the first engineering step before mammalian-cell optimization.

  2. 2.
    Optimize indicator variants in mammalian cellsengineered indicators

    Improve performance of the indicators in mammalian cellular context.

    This step follows E. coli directed evolution because the abstract explicitly states subsequent optimization in mammalian cells.

  3. 3.
    Measure localization-specific K+-responsive fluorescence and affinity in HEK293FT cellsindicators under characterization

    Quantify K+-specific fluorescence response and affinity in a mammalian cell line.

    The abstract reports HEK293FT-cell characterization after mammalian-cell optimization and before deployment in neural and in vivo systems.

  4. 4.
    Deploy RGEPOs in cultured neurons, astrocytes, acute brain slices, and awake mice for real-time K+ imagingimaging indicators

    Validate real-time monitoring of subsecond potassium dynamics in relevant biological systems.

    This application step follows cell-line characterization to test the indicators in more physiologically relevant and intact systems.

  5. 5.
    Use molecular dynamics simulations to analyze potassium-binding mechanismssimulated indicators

    Interpret structural features and potassium-binding pockets of the indicators.

    The abstract presents molecular dynamics simulations as a later analysis that provided mechanistic insight into the developed indicators.

Taxonomy & Function

Primary hierarchy

Mechanism Branch

Architecture: A reusable architecture pattern for arranging parts into an engineered system.

Target processes

No target processes tagged yet.

Implementation Constraints

cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor

It requires cellular expression and extracellular membrane targeting, along with fluorescence imaging. The reported affinity was measured in HEK293FT cells.; requires extracellular membrane targeting; requires expression in cells for fluorescence readout

Independent follow-up evidence is still limited. Validation breadth across biological contexts is still narrow. Independent reuse still looks limited, so the evidence base may be fragile. No canonical validation observations are stored yet, so context-specific performance remains under-specified.

Validation

Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication

Supporting Sources

Ranked Claims

Claim 1application scopesupports2025Source 1needs review

RGEPOs were used for real-time monitoring of subsecond potassium dynamics in cultured neurons, astrocytes, acute brain slices, and awake mice in intracellular and extracellular environments.

We employed RGEPOs for real-time monitoring of subsecond K+ dynamics in cultured neurons, astrocytes, acute brain slices, and the awake mouse in both intracellular and extracellular environments.
Claim 2engineering workflowsupports2025Source 1needs review

RGEPO1 and RGEPO2 were developed through directed evolution in Escherichia coli followed by optimization in mammalian cells.

through a combination of directed evolution in Escherichia coli and subsequent optimization in mammalian cells
Claim 3first visualizationsupports2025Source 1needs review

Using RGEPOs, the authors visualized intracellular and extracellular potassium transients during seizures in the brains of awake mice.

Using RGEPOs, we were able, for the first time, to visualize intracellular and extracellular potassium transients during seizures in the brains of awake mice.
Claim 4localization and affinitysupports2025Source 1needs review

RGEPO1 is targeted to the extracellular membrane and shows a positive K+-specific fluorescence response with 2.4 mM affinity in HEK293FT cells.

RGEPO1, targeted to the extracellular membrane... exhibited positive K+-specific fluorescence response with affinities of 2.4 ... mM in HEK293FT cells
affinity 2.4 mM
Claim 5localization and affinitysupports2025Source 1needs review

RGEPO2 is localized in the cytoplasm and shows a positive K+-specific fluorescence response with 43.3 mM affinity in HEK293FT cells.

RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of ... 43.3 mM in HEK293FT cells
affinity 43.3 mM
Claim 6mechanistic insightsupports2025Source 1needs review

Molecular dynamics simulations suggested that RGEPO1 and RGEPO2 have distinct potassium-binding pockets and structural features.

molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features
Claim 7tool developmentsupports2025Source 1needs review

The study developed two novel red genetically encoded potassium indicators, RGEPO1 and RGEPO2.

In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2

Approval Evidence

1 source4 linked approval claimsfirst-pass slug rgepo1
RGEPO1, targeted to the extracellular membrane, and RGEPO2, localized in the cytoplasm, exhibited positive K+-specific fluorescence response with affinities of 2.4 and 43.3 mM in HEK293FT cells, respectively.

Source:

engineering workflowsupports

RGEPO1 and RGEPO2 were developed through directed evolution in Escherichia coli followed by optimization in mammalian cells.

through a combination of directed evolution in Escherichia coli and subsequent optimization in mammalian cells

Source:

localization and affinitysupports

RGEPO1 is targeted to the extracellular membrane and shows a positive K+-specific fluorescence response with 2.4 mM affinity in HEK293FT cells.

RGEPO1, targeted to the extracellular membrane... exhibited positive K+-specific fluorescence response with affinities of 2.4 ... mM in HEK293FT cells

Source:

mechanistic insightsupports

Molecular dynamics simulations suggested that RGEPO1 and RGEPO2 have distinct potassium-binding pockets and structural features.

molecular dynamics simulations provided new insights into the potassium-binding mechanisms of RGEPO1 and RGEPO2, revealing distinct K+-binding pockets and structural features

Source:

tool developmentsupports

The study developed two novel red genetically encoded potassium indicators, RGEPO1 and RGEPO2.

In this study, we developed two novel red genetically encoded potassium indicators (RGEPOs), RGEPO1 and RGEPO2

Source:

Comparisons

Source-backed strengths

positive K+-specific fluorescence response; extracellular membrane targeting; affinity reported in HEK293FT cells

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positive K+-specific fluorescence response

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extracellular membrane targeting

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affinity reported in HEK293FT cells

Compared with carbohydrate-centered glycoconjugates

RGEPO1 and carbohydrate-centered glycoconjugates address a similar problem space.

Shared frame: same top-level item type

Compared with cell membrane-coated nanoparticles

RGEPO1 and cell membrane-coated nanoparticles address a similar problem space.

Shared frame: same top-level item type

Compared with self-complementary AAV genomes

RGEPO1 and self-complementary AAV genomes address a similar problem space.

Shared frame: same top-level item type

Ranked Citations

  1. 1.
    StructuralSource 1MED2025Claim 1Claim 2Claim 3

    Extracted from this source document.