Click Biology: How to Click and Play to Explore Biology

This post was written by Professor Mark Howarth, investigator at the University of Cambridge. His research focuses on protein innovations for therapeutic and vaccine design.

We all want tools that expand what we can achieve, but are simple to pick up and work well the first time. But biology is complicated — so many different parts and species, with components quickly losing activity. I set out to collect the features that would define a good tool for sticking together biological components. Nearly all of us played with LEGO as a child (some of us still do), so we are used to the idea of general interfaces and the freedom to test out lots of combinations, to end up finding something surprisingly useful. To apply these principles in a biological setting, we first need to define what features would allow for the best performance in the most contexts (Figure 1). Together the features with optimal biological performance are described as Click Biology (Howarth, 2025).

Figure 1: Modular assembly tools, alongside criteria for Click Biology. Created with BioRender.com.

Principles of Click Biology

Physical scientists have been talking about Click Chemistry since 2001, making use of robust reactions that work in just about any situation (Devaraj & Finn, 2021). In one of the most common Click Chemistry reactions, one component bearing an azide reacts covalently with another component bearing an alkyne; the two components lock together rapidly and selectively in all sorts of contexts. But the reactive groups of Click Chemistry are almost completely absent from living systems. What are the possibilities for modular assembly using the reactive groups naturally present in a cell?

By analogy to Click Chemistry, Click Biology can be defined as a set of reactions derived from the regular building-blocks of living cells, rapidly forming covalent bonds to specific partners under cell-friendly conditions. Using the natural amino acids or nucleic acid bases already present throughout living organisms, Click Biology reactions can add new functionality to existing genes, while being accessible simply by dialing up the right nucleic acid sequence (Addgene can help with this!). Besides requiring stable covalent reactions, other required parameters of the coupling system include:

• Wide fusion-tolerance — Click Biology tags should be similarly useful across the diverse kinds of proteins that one finds in cells.
• High yield of reaction — generating product without a lot of left-over starting components.
• High rate constant— time is money and things move fast in biology. Slow reactions are only good enough if the reacting parts are present at high concentrations, but in biology one often works with parts in the micromolar or nanomolar concentration range.
• Reactions to happen under lots of conditions — in the cytosol and nucleus, nearly all cysteines are reduced; in the ER and Golgi apparatus and outside the cell, most cysteines are oxidized in disulfide bonds. Reactions also can happen at both body temperature and on ice, when we have cracked open the cells. The tools should react in all of these situations.
• Specificity — coupling must happen in the right place in living systems when there are lots of other biomolecules floating around that must not be touched.

Click Biology Tags

The broad functionality of Click Biology is made possible through the utilization and development of genetically engineered components, like protein tags (Figure 2). Through the work of many groups, there are systems like split inteins or SpyTag, which are useful for connecting proteins in the test tube or in cells. There are also SNAP-tag and HaloTag modules, which can connect proteins to any kind of small molecule probes with different functionalities, such as fluorescence or triggering new cellular behavior like degradation or electrophile signaling. Many of these tools can be found in Addgene's repository (Table 1).

Figure 2: Adding function to My Favorite Protein (MFP) through rapid covalent coupling. Created with BioRender.com.

 

Table 1: A handful of Click Biology tools available at Addgene.

Addgene Link	Click Biology Tag	Description
Humberg et al. (2025)	Split inteins	Cystein-less, fast split intein; N-terminal CLm and C-terminal Aes
gp41-1	Split inteins	A small, fast split intein
SpyTag003/SpyCatcher003	SpyTag/SpyCatcher	Rapid covalent bond formation between a genetically encoded peptide (SpyTag) and protein (SpyCatcher)
SnoopTag/SnoopCatcher	SpyTag/SpyCatcher	Rapid covalent bond formation between a genetically encoded peptide (SnoopTag) and protein (SnoopCatcher); no cross-reaction with SpyTag/SpyCatcher, allows for iterative expansion
DogTag/DogCatcher	SpyTag/SpyCatcher	Rapid covalent bond formation between a genetically encoded peptide (DogTag) and protein (DogCatcher); ideal for protein loop regions
CRISPaint Gene Tagging Kit	SpyTag/SpyCatcher	CRISPR-assisted insertion tagging Kit; integration of large DNA fragments at user-defined locations
NEB Cell-Imaging Collection	SNAP-tag, CLIP-tag	Plasmids for visualization of proteins in live or fixed cells using SNAP-tags or CLIP-tags
Tetbow	SNAP-tag, CLIP-tag, HaloTag	Based on the Brainbow system, adds Tetracycline (Tet) transactivator to increase fluorescent protein expression
C234-E25: attB2r-SNAP-attB3	SNAP-tag	Gateway entry clone to add C-terminal SNAP-tag
Promega Plasmid Collection	HaloTag	Collection of research tools from Promega, including many HaloTag plasmids
Exchangeable HaloTag Ligands	HaloTag	Reversible binding of fluorescent ligands to HaloTag
PCV-Cas9, Cas9-PCV	HUH-tag	Cas9 plasmids with N- or C-terminal PCV2 HUH-tag; increase homology-directed repair efficiency by linking the repair template
VirD2	HUH-tag	For creating DNA-protein conjugates
rHUH	HUH-tag (RNA)	Covalent protein tag for RNA labeling

Click Into Biological Building

Click Biology describes the search for robust, reliable reactions, using only nature’s building blocks. Applications are quickly expanding with these kinds of Click Biology reactions. For basic research, the stable and selective reactions of these tags can help image, purify, or solve the structure of proteins. Split inteins are particularly useful for a kind of cellular computation, making possible multiple logic-gated steps. Beyond the lab, these systems can also assemble vaccines, target gene therapy vectors, or lock together smart biomaterials. There are many convenient tools out there, not just relating to proteins but also covalent reactions to DNA (with HUH-tag or VirD2) or RNA (with rHUH). Many of these tools are already deposited at Addgene in multiple formats: for use in different organisms, targeted to particular compartments, or fused to other effectors from nanobodies to fluorescent proteins. So, I hope you will take full advantage and have lots of fun building and clicking.

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References and Resources

References

Devaraj, N. K., & Finn, M. G. (2021). Introduction: Click Chemistry. Chemical Reviews, 121(12), 6697–6698. https://doi.org/10.1021/acs.chemrev.1c00469 

Howarth, M. R. (2025). Click biology highlights the opportunities from reliable biological reactions. Nature Chemical Biology. https://doi.org/10.1038/s41589-025-01944-x  

Additional Resources on the Addgene Blog

• Live and Let Dye: Self-Labeling Protein Tags
• Plasmids 101: Degron Tags

Additional Resources on addgene.org

• New England Biolabs Cell-Imaging Plasmid Collection
• Promega Plasmid Collection