Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

Abstract

The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes.

DOI: http://dx.doi.org/10.7554/eLife.15312.001

eLife digest

Biologists often wish to study the role of a particular cell type within an organism, but such studies are often not possible due to the lack of reagents that allow one to gain control of the cell type of interest. One method that can be used to detect and manipulate the cells that express specific proteins uses molecules called antibodies. An antibody can strongly bind to a specific part of a protein, and a diversity of antibodies that bind to different proteins can be isolated by animal immunization, or by using molecular or cell-based methods.

Antibodies from camelid species (which include camels and llamas) are increasingly being used to detect and manipulate proteins in living cells. The variable region of these antibodies – also known as the nanobody – recognises the proteins that the antibody binds to, and often just this fragment of the antibody is used in protein detection experiments. However, nanobodies are stable even in cells that do not contain their target proteins, which makes it difficult to use nanobodies to study just a specific cell type within an organism.

Tang, Drokhlyansky et al. have now developed a way of engineering the sequence of a nanobody so that it is broken down in living cells unless it is bound to its protein target inside the cell. Any protein that is tethered to the engineered nanobody is also broken down. For example, some tethered proteins with useful biological activities are fluorescent proteins and enzymes that can modify DNA. When one of these engineered nanobodies binds to a protein target of interest, the activity of the nanobody-tethered protein can be turned on in just those cells that produce the targeted protein. Thus, this strategy of engineering allows “conditionally stable” tools to be generated.

A core set of sequence alterations can be used to modify different nanobodies that target different proteins. Tang, Drokhlyansky et al. have demonstrated the uses of several of the resulting conditionally stable nanobodies. In one application, the nanobodies were used to target specific cell types in the mouse brain in a way that allowed the activity of these cells to be controlled by light. Another application of the technique enables live human cells that have been infected with HIV to be detected and isolated.

The conditionally stable nanobody tools can be used to detect and manipulate cells that express any protein for which a camelid antibody exists. Tang, Drokhlyansky et al. therefore hope that biologists who work in a wide range of fields will find the tools useful for studying many different types of organisms and biological processes.

DOI: http://dx.doi.org/10.7554/eLife.15312.002