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The authors of the paper stated that the complexity of the new protein design is close to the real protein assembly.

(this study) , It is a key step in programming complex biological circuits.

Also, according to the first author Chen Zibo, the team has already begun to study whether this method can be used to form a multilevel circuit.

Control biological functions with logic gates

The basic input of the protein circuit is controlled by CIPHR (co-inducible protein heterodimers) .

The so-called CIPHR is an aggregate of two different proteins, and each protein corresponds to an input at a specific location. Different combinations are equivalent to 1 at different locations of the circuit.

CIPHR logic gates are portable, which also means that we can use logic gates to control different biological functions.

In the experiment, the researchers designed four pairs of heterodimer modules and constructed two logic gates with different control functions. One is used to isolate the catalytic activity of luciferase, and the other is used for gene expression in primitive human T cells.

In the Control of luciferase system , they set a protein on the corresponding nanosite, and test whether the logic gate is open through in vitro translation and monitoring luminescence.

The logic gate that controls the expression of human immune T cell genes is handed over to the isolated TALE-KRAB suppression system.

If you use a heterodimer, as long as you enter (1), inhibition (0) will occur, which is equivalent to a NOT gate.

If a protein monomer is used, as long as any one of the proteins enters (1), the original TALE-KRAB binding will be destroyed and the inhibition (1) will be cancelled, thereby realizing the function of the OR gate.

Negative and OR gates are used to control the expression of TIM3 protein in human T cells and are detected by flow cytometry.

The above is only a case of two inputs. It is similar if you increase the input to three. But more dimers are used, and more proteins are used inside the logic gates.

For the 3-way AND gate, 4 pairs of proteins are shared, and there are 1 and 2 ‘in the logic gate. Only when 1’-4 ‘, 4-3′, and 3′-2 are input simultaneously to form 1: 1’-4 ‘: 4-3′: 3’-2: 1, the logic gate can be activated.

The principle of the 3-input OR gate is similar.

Although protein-designed logic gates have long existed, the heterodimer module is a completely new technology that takes advantage of protein design.

By redesigning the protein modelBlocks can produce more logic gate components with almost the same overall topology.

Specific encoding of protein hydrogen bond networks allows for broad binding affinity between monomers with similar structures, which in turn allows the construction of more complex gates based on competitive binding.

Because the design element of the logic gate is an ultra-stable protein, no additional cellular mechanical structure is required. The protein logic gate can work whether it is inside or outside the cell.

The first author of the article, Chen Zibo, said: “We have demonstrated its function with cell-free extracts, yeast cells, and purified components from T cells.”

How to design?

So how is such a “carbon-based circuit” specifically designed?

In principle, various logic gates can be designed from scratch using a set of heterodimer molecules.

For example, given a heterodimer pair A and A ‘, B and B’, C and C ‘. By genetically fusing A ‘and B, and B’ and C, an AND gate that regulates the relationship between A and C ‘can be constructed.

The conditions involved are:

First, there should be many orthogonal heterodimer pairs so that the complexity of the gate is not limited by the number of individual elements.

Second, the components should be modular and similar in structure.

Third, a single component should have the ability to combine with multiple different, affinity-adjustable partners so that the input can perform negation by disrupting pre-existing low-affinity interaction operating.

Fourth, the interaction should be cooperative (cooperative) The gate activation is insensitive to the imbalance of stoichiometry in the input.

Specifically in this study, in order to satisfy condition 1, the researchers used a heterodimer set with hydrogen bond network-mediated specificity and orthogonal designs to construct logic gates.

The heterodimer interfaces share the same 4 spiral bundle topology to satisfy condition 2.

The shared interaction interface limits the amount of crosstalk between heterodimer pairs, so that the binding affinity hierarchy can satisfy condition 3.

Finally, the condition is satisfied by constructing a monomer fusion (A ‘and B, B’ and C mentioned in the above example) 4.

Hereinafter, a system such as A + A’-B + B ‘is called an induced dimerization, A and B’ are called monomers, and A’-B is called a dimer.

The schematic diagram of the induced dimerization system (6 residue linker) is as follows.

In the experiment, the dimers showed synergistic binding.

So, using nMS, the researchers explored a two-input AND gate consisting of two dimers 1′-3 and 3-2 ‘, and monomers 1 and 2 as inputs. (AND) .

In the experiments, researchers only observed very small amounts of heterotrimers and heterodimers, further demonstrating the high synergy of the method.

Researchers also constructed a three-input AND gate of 1′-4 ‘, 4-3’, and 3-2 ‘. The result is similar to the quantity input AND gate. The abundance of the complete pentameric complex is only Slight decline at concentrations greater than the input stoichiometric concentration.

Based on this, the researchers constructed a variety of 2-input and 3-input CIPHR logic gates.

The specific method is that the monomers of each DHD are linked to the target effector protein through genetic fusion, so that the input (linked heterodimer subunits) < / span> controls colocalization or dissociation of effector proteins.

Logic gates are constructed by homologous binding between designed protein pairs and competitive binding involving multispecific interactions.

Extend the life of immune cells

Now we can use the same operation as a computer to controlThis has important implications for the future of medicine and synthetic biology.

The University of Washington research team has begun the next step of preparing the technology for cancer treatment.

CAR-T cell therapy targeted therapies for treating cancer now have many difficulties, which scientists believe are related to T cell failure.

The so-called CAR-T cells are chimeric antigen receptor T cells, and the chimeric antigen receptor is a bioengineered protein. The line of scientists extracted T cells from the human body, and genetically modified them to specifically target cancer cell surface antigens, which were then imported back into patients.

But transgenic T cells can only survive for a short period of time, and the University of Washington team hopes that the use of protein logic gates that attenuate signal responses can prolong CAR-T cell activity.

Chen Zibo said that using programming methods to make long-lived T cells for each patient means that more effective personalized medicine will be born.

About the author

Chen Zibo is 29 years old. He is a postdoc in the Elowitz laboratory at the California Institute of Technology. His current research direction is to build logic circuits from scratch with proteins.

He graduated from the National University of Singapore with a Bachelor of Life SciencesMajor, received his PhD from the University of Washington in 2019. Last year, he was also selected by MIT Technology Review 35 people under 35.

Corresponding author David Baker, professor of biochemistry and director of the Institute of Protein Design at the University of Washington School of Medicine.

It is worth mentioning that David Baker is also one of the authors of the protein design game Foldit. The protein designed by Foldit has been cited many times by scientific researchers and is now also used to design against the new crown virus.

Paper address: https://science.sciencemag.org/content/368/6486/78

This article is from WeChat public account: qubit (ID: QbitAI) author: policy side, the fish sheep