Call it Protein Logic Gate 2.0. The 1.0 version, which rewired native signaling pathways, was admirably direct, bypassing awkward rewiring at the DNA and RNA levels, but it wasn’t very scalable or extensible. It relied on a limited pool of building blocks, in this case, native proteins presenting interfaces open to fabricated protein-protein interactions. Artificial proteins can offer more flexibility. They can come in sets of modular units that interact with each other in clearly defined ways to read inputs, complete logical operations, and generate outputs.

The equivalent of Protein Logic Gate 2.0 has been launched by scientists based at the University of Washington (UW) School of Medicine. They’ve already run demonstrations of how it can be used to manipulate gene expression. These demonstrations, which involved cell-free extracts, yeast cells, and T cells, were presented in the journal Science, in an article titled, “De novo design of protein logic gates.”

The article describes how the same basic tools that allow computers to function are now being used to control life at the molecular level. Specifically, artificial proteins are being used as molecular logic gates, and these protein logic gates, like their electronic counterparts, can be used to program the behavior of complex systems. Protein logic gates, the article argues, have implications for future medicines and synthetic biology.

Whether electronic or biological, logic gates sense and respond to signals in predetermined ways. One of the simplest is the AND gate; it produces output only when one input AND another are present.

With the right gates operating inside living cells, inputs such as the presence of two different molecules—or one and not the other—can cause a cell to produce a specific output, such as activating or suppressing a gene.

“Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo–designed proteins,” the article’s author wrote. “Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates.”

This graphic table compares how electronic and protein AND logic gates respond when no input is present, when only A or B is present, and when both A and B are present. [UW Medicine Institute for Protein Design]

The article also described how protein logic gates were used to regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast, and in primary human T cells, where they control the expression of the TIM3 gene related to T-cell exhaustion.

The work with T cells suggests how protein logic gates could improve cell-based cancer immunotherapy. For example, protein logic gates could help chimeric antigen receptor (CAR) T-cell therapies succeed not only against blood cancers, but also solid tumors.

Thus far, targeting solid tumors with CAR-T cell therapies has been challenging because, scientists suspect, T cells suffer exhaustion. Genetically altered T cells can fight for only so long before they stop working. But what if CAR T cells were to incorporate protein logic gates that respond to exhaustion signals? According to the UW Medicine team, the activity of CAR T cells could be prolonged.

“Longer-lived T cells that are better programmed for each patient would mean more effective personalized medicine,” noted lead author Zibo Chen, a recent UW graduate student. He also emphasized that although the UW School of Medicine team’s protein logic gates have thus far accomplished only simple tasks, they are, nevertheless, are a key step toward programming complex biological circuits from scratch.

“Bioengineers have made logic gates out of DNA, RNA, and modified natural proteins before, but these are far from ideal,” added David Baker, the senior author of the current study and professor of biochemistry at the UW School of Medicine and director of the Institute for Protein Design. “Our logic gates built from de novo designed proteins are more modular and versatile.”

“In principle, it should be possible to design a wide range of logic gates de novo using a set of heterodimeric molecules,” the authors of the Science article concluded. “The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.”

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