The Problem
Proteins are well known as essential orchestrators of life, but what is less well-understood is their post-translational modifications (PTMs). These modifications can include the attachment of chemical groups, carbohydrates, or lipids to the proteins, which affect their folding, stability, and functions. Certain protein PTMs have been linked to diabetes, cancer, and neurodegenerative disease, but studying them is a challenge. Many PTMs are unstable and relatively rare in the body, making it difficult to detect and isolate them.
Our Solution
DNA nanoswitch calipers (DNCs) offer a novel method to deeply probe the structures of proteins rapidly and cheaply, revolutionizing PTM research. This transformative approach to proteomics marries DNA nanotechnology with single-molecule manipulation techniques, enabling high-throughput analysis of protein PTMs, which could unlock profound advances in our understanding of health and disease, as well as biomedicine.
Product Journey
DNCs are the product of years of collaboration between Wyss Core Faculty members William Shih and Wesley Wong, both members of the Institute’s Molecular Robotics platform. Molecular Robotics harnesses the unique physical qualities of DNA to create programmable nanoscale tools that can perform a variety of functions, and interact with biological molecules.
DNCs were born from the researchers’ goal of creating something that would let them interact with single molecules to probe their structures and functions, ultimately leading to new insights that could advance medicine and manufacturing. DNCs are based on the fundamental technology of the DNA nanoswitch: a single strand of DNA with molecular “handles” attached to it at multiple points along its length. When two of these handles bind to each other, they create a loop in the DNA strand, and the overall length of the strand is shortened. When force is applied to pull the handles apart, the strand extends back to its original length.
Members of Shih and Wong’s groups realized that rather than binding to each other, the handles of a DNA nanoswitch could also “pinch” a biomolecule between themselves like a caliper. By measuring how the addition of the molecule between the handles changes the overall length of the DNA nanoswitch in its looped vs. unlooped states, the size of the molecule could be measured with nanoscale precision.
To create DNCs, the team crafted a DNA nanoswitch with one “strong” handle to which a target molecule is bound. Then, they attached several “weak” handles to the target molecule that can bind to the other end of the DNA nanoswitch. By moving both ends of the DNA nanoswitch closer together, a bond is formed between one of the target molecule’s weak handles and the nanoswitch, creating a looped state. Applying a small amount of force to the DNC by pulling the ends apart causes the weak handle to release its bond, returning the DNC to its longer, unlooped state. Because the target molecule contains multiple weak handles, repeated cycles of binding and releasing creates a series of distance measurements between the strong handle and the weak handles that are unique to each molecule measured. This “fingerprint” can be used to identify a known molecule within a sample, or to learn structural information about an unknown molecule with angstrom-level precision (ten times smaller than the width of a DNA double helix).
Because a PTM can be as tiny as the addition of a few atoms to a protein’s structure, distinguishing between a modified and unmodified protein – or between different types of modifications – is a significant challenge that DNCs are poised to overcome. Based on its potential to unlock immense amounts of knowledge about proteins, this project was named a Wyss Validation Project in 2023 and 2024. Its foundational DNA nanoswitch technology was also selected to receive funding through the Northpond Labs-supported Laboratory for Bioengineering Research in 2023.
The team is currently is focusing on generating molecular fingerprints of glycosylation and branched ubiquination on intact proteins, as these are areas that are currently extremely difficult to analyze via mass spectrometry. They are especially interested in meeting collaborators or supporters who would like to focus on these modifications in the context of neurodegenerative disease.