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DNA Nanodevices As Mechanical Probes of Protein Structure and Function

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applied sciences Perspective DNA Nanodevices as Mechanical Probes of Protein Structure and Function Nicholas Stephanopoulos 1,2,* and Petr Šulc 1,2 1 School of Molecular Sciences, Arizona State University, Tempe, AZ 85281, USA; [email protected] 2 Biodesign Center for Molecular Design and Biomimetics, Arizona State University, Tempe, AZ 85281, USA * Correspondence: [email protected] Abstract: DNA nanotechnology has reported a wide range of structurally tunable scaffolds with precise control over their size, shape and mechanical properties. One promising application of these nanodevices is as probes for protein function or determination of protein structure. In this perspective we cover several recent examples in this field, including determining the effect of ligand spacing and multivalency on cell activation, applying forces at the nanoscale, and helping to solve protein structure by cryo-EM. We also highlight some future directions in the chemistry necessary for integrating proteins with DNA nanoscaffolds, as well as opportunities for computational modeling of hybrid protein-DNA nanomaterials. Keywords: DNA nanotechnology; protein-DNA biomaterials; mechanical nanodevices 1. Introduction The ability to probe protein structure and function at the single-molecule level has Citation: Stephanopoulos, N.; Šulc, P. been a key motivation of biology, chemistry and physics for decades. Innovations like DNA Nanodevices as Mechanical super-resolution microscopy, optical tweezers, atomic force microscopy (AFM) probing, Probes of Protein Structure and and Förster resonance energy transfer (FRET) studies have opened new windows into Function. Appl. Sci. 2021, 11, 2802. the biological world by enabling analysis of single protein molecules with unprecedented https://doi.org/10.3390/app11062802 precision. Structural biology, ranging from X-ray crystallography and nuclear magnetic resonance (NMR) to the recent revolution in cryo-electron microscopy (cryo-EM), has Academic Editor: Alexander also played an indispensable role in determining the three-dimensional conformation of E. Marras proteins, and thereby elucidating their function at the molecular level. In the past 15 years or so, DNA mechanical nanodevices have emerged as powerful new alternative tools Received: 13 February 2021 for probing proteins with single-molecule resolution. The DNA origami technique [1,2], Accepted: 13 March 2021 Published: 21 March 2021 by which a long DNA scaffold strand is folded into a well-defined, addressable, and mechanically robust nanoobject, has in particular been a boon for designing structures on Publisher’s Note: MDPI stays neutral a similar length scale as proteins, which can immobilize them or apply forces to them in with regard to jurisdictional claims in interesting ways to elucidate their function. In this perspective, we outline recent advances published maps and institutional affil- in the use of rigid DNA origami objects and lattices to probe the structure of proteins, or to iations. interrogate their function at the single-molecule level. We focus on three key properties that make DNA nanoscaffolds particularly well-suited to this task (Figure1): (1) their rigidity, which in turn allows for protein attachment with a controlled orientation for structural biology studies; (2) their ability to space multiple proteins with nanoscale control, or to confine them within a defined volume; and (3) their ability to impart forces at the nanoscale, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and thereby probe the biophysics of attached proteins. We then close with a brief discussion This article is an open access article of two areas for future investigation for improving DNA nanomechanical devices: (1) rigid, distributed under the terms and multipoint attachment of proteins on a DNA scaffold and (2) computational modeling of conditions of the Creative Commons hybrid protein-DNA nanomaterials. Our goal is to highlight some key recent advances in Attribution (CC BY) license (https:// these areas and to focus on how the mechanical nature of DNA scaffolds can facilitate new creativecommons.org/licenses/by/ insights into protein science, not to provide an exhaustive overview of the rich intersection 4.0/). of protein science and DNA nanotechnology. For a more complete treatment of hybrid Appl. Sci. 2021, 11, 2802. https://doi.org/10.3390/app11062802 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 18 Appl. Sci. 2021, 11, 2802 2 of 18 folds can facilitate new insights into protein science, not to provide an exhaustive over- view of the rich intersection of protein science and DNA nanotechnology. For a more com- protein-DNAplete treatment nanomaterials of hybrid pr beyondotein-DNA the scope nanomaterials of this perspective, beyond we the refer scope the interestedof this perspec- readertive, we to severalrefer the key interested reviews [ 3reader–6]. to several key reviews [3–6]. FigureFigure 1. 1.Scope Scope of of this this Perspective. Perspective. ( A(A)) Controlling Controlling protein protein orientation orientation by by attachment attachment to to rigid rigid DNADNA nanostructures nanostructures that that enables enables visualization visualizatio byn another by another technique technique (e.g., cryo-EM). (e.g., cryo-EM). (B) Controlling (B) Control- proteinling protein spacing spacing (at the (at nanometer the nanometer scale) and scale) the and number the number or type ofor proteintype of attached.protein attached. (C) Using (C a) Us- DNAing a nanostructure DNA nanostructure to either to apply either a force apply to, a or force measure to, or the measure force exerted the force by, anexerted attached by, protein.an attached protein. 2. Structural Characterization of Proteins on DNA Nanoscaffolds 2. StructuralThe original Characterization goal of DNA nanotechnology, of Proteins on as DNA conceived Nanoscaffolds by Ned Seeman in 1982 [7], was to generate a three-dimensional crystal from the self-assembly of individual DNA The original goal of DNA nanotechnology, as conceived by Ned Seeman in 1982 [7], strands linked by immobile Holliday junctions. Such a crystal could, in turn, be used to was to generate a three-dimensional crystal from the self-assembly of individual DNA scaffold proteins in a repeating lattice in space and solve their structure using X-ray crystal- lographystrands withoutlinked by having immobile to crystalize Holliday the junctions. proteins (Figure Such2 aA) crystal [ 8]. Key could, to this in endeavor turn, be isused to thescaffold rigid attachment proteins in of a therepeating proteins lattice to a mechanically in space and robust, solve their 3D DNA structure latticework, using X-ray since crys- eventallography slight variations without inhaving their orientationto crystalize would the pr resultoteins in (Figure poor diffraction 2A) [8]. Key and to preclude this endeavor high-resolutionis the rigid attachment characterization. of the proteins The first to rationally a mechanically designed, robust, self-assembled 3D DNA DNAlatticework, crystal since waseven reported slight byvariations Seeman, Maoin their and orientation coworkers in would 2009, based result on in a poor tensegrity diffraction triangle and motif— preclude ahigh-resolution nanoscale analogue characterization. of a mechanically The rigid first macroscopic rationally designed, object—comprised self-assembled of three DNADNA crys- strandstal was (Figure reported2B) [by9]. Seeman, This crystal Mao was and characterized coworkers toin 4–14 2009, Å based resolution on a (depending tensegrity ontriangle 3 themotif—a design), nanoscale with the largestanalogue cavities of a mechanical surpassing 1100ly rigid nm macroscopicin volume. Inobject— 2016 and comprised 2017, of Yan,three Seeman DNA andstrands coworkers (Figure reported 2B) [9]. a secondThis crys DNAtal crystalwas characterized design, once again to 4–14 employing Å resolution three strands but relying on four stacked duplexes linked by Holliday junctions as a key (depending on the design), with the largest cavities surpassing 1100 nm3 in volume. In motif (Figure2C,D) [ 10,11]. These motifs diffracted to ~3 Å, and a subsequent report that 2016 and 2017, Yan, Seeman and coworkers reported a second DNA crystal design, once optimized the Holliday junction sequence yielded crystals that diffracted to 2.6 Å, which again employing three strands but relying on four stacked duplexes linked by Holliday allowed for visualization of individual purine-pyrimidine base stacks (Figure2E,F )[12]. Critically,junctionsthis as a latter key motif design (Figure allowed 2C,D) for crystal[10,11]. cavities These motifs with ~50% diffracted larger to edges ~3 Å, (albeit and a sub- atsequent a reduced report resolution) that optimized and volumes the Holliday of 1250 nm ju3nction, paving sequence the way foryielded incorporation crystals that of dif- largerfracted proteins. to 2.6 Å, which allowed for visualization of individual purine-pyrimidine base stacks (Figure 2E,F) [12]. Critically, this latter design allowed for crystal cavities with ~50% larger edges (albeit at a reduced resolution) and volumes of 1250 nm3, paving the way for incorporation of larger proteins. Appl. Sci. 2021, 11, 2802 3 of 18 Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 18 FigureFigure 2. Self-assembled 2. Self-assembled DNA DNA crystal crystal lattices.lattices. (A ()A Original) Original concept concept of DNA of DNA nanotechnology nanotechnology as conceived as concei by Seeman:ved by using Seeman: usinga
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