HomeNewsNanotechnologyFingerprinting proteins with pressure

Fingerprinting proteins with pressure

Oct 21, 2021

(Nanowerk News) As scientists have probed the mysteries of life all the way down to smaller and smaller scales, they’ve invented instruments to assist them perceive what they observe. Determining the identification of DNA and RNA molecules has now grow to be commonplace because of the business growth of next-generation sequencing applied sciences, however the identical just isn’t but true of proteins, that are critically vital gamers in practically all organic processes. Proteins are way more advanced than DNA and RNA, and are sometimes chemically modified, making the purpose of simply figuring out single proteins inside a pattern (single-molecule proteomics) difficult to attain. Now, scientists working on the Molecular Robotics Initiative inside the Wyss Institute at Harvard University, the Blavatnik Institute at Harvard Medical School (HMS), and Boston Children’s Hospital (BCH) have used DNA, the elemental stuff of life itself, to create what will be the world’s tiniest ruler for measuring proteins. Dubbed “DNA Nanoswitch Calipers” (DNC), this expertise permits researchers to carry out distance measurements on single peptides (the constructing blocks of proteins) with excessive precision by making use of small quantities of pressure. By quickly making many distance measurements on the identical molecule, DNC creates a novel “fingerprint” that can be utilized to determine it in subsequent experiments. The achievement is reported in Nature Nanotechnology (“Single-Molecule Mechanical Fingerprinting with DNA Nanoswitch Calipers”).

“When you’re trying to understand something in biology, there are two main methods of inquiry: you can observe your subject in its natural state, or you can perturb it and see how it reacts. Observations can provide lots of great biological information, but sometimes the best way to learn about something is to physically interact with it,” stated co-corresponding writer Wesley Wong, Ph.D., an Associate Faculty Member on the Wyss Institute and Associate Professor at HMS who can also be an Investigator at BCH. “Determining the pattern of amino acids within a peptide molecule by applying force is a new paradigm in the ongoing scientific quest for techniques that will enable us to sequence proteins as easily as we currently sequence DNA.”

Use the pressure

DNC relies on the underlying expertise of the DNA nanoswitch: a single strand of DNA with molecular “handles” connected to it at a number of factors alongside its size. When two of those handles bind to one another, they create a loop within the DNA strand, and the general size of the strand is shortened. When pressure is utilized to drag the handles aside, the strand extends again to its unique size. The distinction between the size of the strand in its looped and unlooped states displays the dimensions of the loop, and thus the gap between the handles. The analysis crew realized that they might take DNA nanoswitches one step additional: in the event that they as an alternative engineered the handles to bind to a biomolecule, the handles may successfully “pinch” the molecule between themselves like the 2 ideas of a caliper, moderately than binding to one another. By measuring how the addition of the goal molecule between the handles modified the general size of the DNA nanoswitch in its looped vs. unlooped states, the crew hypothesized that they might successfully measure the dimensions of the molecule. “In some ways, DNA nanoswitches harness one of the most classical, mechanical methods for measuring objects: just apply force to something and see how it changes in response,” stated co-first writer Darren Yang, a Postdoctoral Researcher on the Wyss Institute and BCH. “It’s an approach that we haven’t really seen used in the field of single-molecule proteomics, because applying force to such small objects is incredibly challenging. But we were up to the challenge.” To flip their concept of a brand new, force-based measuring method into actuality, Yang and his colleagues first connected two several types of handles to a goal molecule: one “strong” deal with to firmly anchor the molecule to at least one finish of the DNC, and a number of other “weak” handles that would connect to the opposite finish of the DNC. They then tethered each ends of the DNC to 2 “optically trapped” beads suspended in laser beams. By transferring the beads nearer collectively, they induced one of many goal molecule’s weak handles to bind to the DNC, making a looped state. When they then elevated the pressure by transferring the beads additional aside, the weak deal with ultimately launched its bond, returning the DNC to its longer, unlooped state. To create their DNA Nanoswitch Calipers, the crew first connected sturdy (purple) and weak (crimson) “handles” to a goal strand of DNA (a). Then, they connected the sturdy deal with and one weak deal with to every finish of the DNA Nanoswitch Calipers held between two optically trapped beads. Applying pressure to the beads (b) triggered the weak deal with to launch, altering the general size of the construction. Repeated cycles of binding and releasing (c) create a novel “fingerprint” for the goal molecule based mostly on the placement of the handles. They later repeated this course of utilizing peptides because the goal molecule. (Image: Wyss Institute at Harvard University) (click on on picture to enlarge) The crew first examined this system on easy, single-stranded DNA (ssDNA) molecules, and confirmed that the change in distance measurements between the DNC’s looped and unlooped state immediately correlated with the size of the goal molecule. These size modifications might be measured with angstrom-level precision (that’s ten instances smaller than the width of a DNA double helix), enabling the identification of modifications in size as small as that of a single nucleotide. Because the goal molecule comprises a number of weak handles that may bind to the DNC, repeated cycles of binding and breaking these handles creates a sequence of distance measurements between the sturdy deal with and the weak handles which are distinctive to every molecule measured. This “fingerprint” can be utilized to determine a identified molecule inside a pattern, or to deduce structural details about an unknown molecule.

Probing proteins

Having confirmed that DNC may reliably measure the dimensions of DNA molecules, the researchers shifted focus to their actual purpose: proteins. They designed an artificial peptide (a brief chain of amino acids) with a identified size and sequence and repeated the experiment, attaching it to at least one finish of the DNC by way of the sturdy deal with and repeatedly attaching and breaking the bonds between its weak handles and the DNC by making use of totally different quantities of pressure. They discovered that all the distances their device measured between the sturdy and weak handles matched the distances anticipated based mostly on the size of the DNC and the lengths of the amino acids within the peptide. They additionally bought related outcomes once they used the DNC to measure a naturally occurring linearized peptide referred to as NOXA BH3. This course of additionally generated distinctive measurement fingerprints for every peptide. The crew created a pc mannequin to foretell what number of human proteins might be uniquely recognized utilizing this methodology, and located that over 75% of the proteins in a generally used protein database might be recognized by way of fingerprints with a likelihood of a minimum of 90%. “We were actually somewhat surprised by how well this technique worked,” stated co-first writer Prakash Shrestha, Ph.D., a Postdoctoral Fellow on the Wyss Institute and BCH. “Optical tweezers have been around for decades and cycling DNA between a looped and unlooped state has been around for about 10 years, and we weren’t sure whether we could get sufficiently high-resolution measurements by combining those ideas. But it turned out that these fingerprints are very effective for identifying proteins.” Identifying single protein molecules is a powerful feat itself, however with the ability to try this for a number of proteins concurrently is the true holy grail for single-molecule proteomics. The crew additional demonstrated that by changing the optical beads with a magnetic tweezer system, they have been capable of carry out measurements on a number of totally different peptides in parallel, in addition to decide the relative concentrations of various molecules. “Single-molecule proteomics is still largely a pipe dream due to challenges in scaling and resolution. Our present work shows that force-based sequence fingerprinting has the potential to realize this dream,” stated co-corresponding writer William Shih, Ph.D., a Core Faculty Member on the Wyss Institute and Professor at HMS and the Dana-Farber Cancer Institute. “Our ultimate ambition is to efficiently read not just protein sequences, but also protein structures in a high-throughput manner.” The scientists’ subsequent step towards that purpose is validating their calipers for low-force structural measurements on folded proteins and their complexes, investigating their potential use for structural biology and proteomics. They are additionally engaged on rising the expertise’s throughput to additional pace up the evaluation of combined samples. “This research integrates molecular biophysics with cutting-edge DNA nanotechonlogy pioneered here at the Wyss Institute to allow us to interact with and analyze biological molecules in a truly novel way. When William and Wesley first posed this idea as a core challenge for the newly formed Molecular Robotics Initiative, it truly seemed like science fiction, but that is precisely the type of project we want to take on at the Wyss. I’m very proud of the team for making this technology a reality – it has the potential to totally change how we do science and develop therapeutics,” stated Wyss Founding Director Don Ingber, M.D., Ph.D., who can also be the Judah Folkman Professor of Vascular Biology at Harvard Medical School and BCH, and Professor of Bioengineering on the Harvard John A. Paulson School of Engineering and Applied Sciences.

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