Subscriber access provided by UNIVERSITY OF CONNECTICUT
Article
Origami Arrays as Substrates for the Determination of Reaction Kinetics using High Speed Atomic Force Microscopy Masudur Rahman, Brian Scott Day, David Neff, and Michael L. Norton Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01556 • Publication Date (Web): 05 Jul 2017 Downloaded from http://pubs.acs.org on July 7, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
Origami Arrays as Substrates for the Determination of Reaction Kinetics using High Speed Atomic Force Microscopy Masudur Rahman, B. Scott Day, David Neff and Michael L. Norton Department of Chemistry, Marshall University, Huntington, West Virginia, USA
ABSTRACT: DNA Nanostructures (DN) are powerful platforms for the programmable assembly of nanomaterials. As applications for DN both as a structural material and as a support for functional biomolecular sensing systems develop, methods enabling the determination of reaction kinetics in real time become increasingly important. In this report, we present the study of the kinetics of streptavidin binding onto biotinylated DN constructs enabled by these planar structures. High-speed AFM was employed at a 2.5 frame/sec rate to evaluate the kinetics and indicates that the binding fully saturates in less than 60 seconds. Fitting the data with an adsorption limited kinetic model, a forward rate constant of 5.03 x 105 s-1 was found.
The binding between the tetrameric protein streptavidin (SA) and the vitamin biotin is one of the tightest noncovalent interactions found in biological systems. Because of the extremely low dissociation constant of 10-15M, resistance to high temperatures, extremes of pH, organic denaturants, and proteolytic enzymes,1 the SA-biotin conjugate has become a popular linker for assembling species ranging from single nanoparticles2 to long carbon nanotubes3 onto DNA Nanostructures (DNs). Many chemical and biological processes are too complex to be fully understood using conventional bulk or ensemble techniques.4, 5 In vivo biochemical reactions occur with single enzymes, nucleic acids, and/or protein, in an isolated fashion. Thus, singlemolecule analysis is necessary to comprehend any chemical or bio-chemical reaction.6 Atomic force microscopy (AFM) enables the direct observation of biomolecules at nanoscale resolution under physiological conditions.7 To facilitate the observation of single biomolecules, a versatile observation scaffold is needed for the precise analysis of interactions and reactions. The scaffolded DNs called DNA origami have provided an optimal platform to serve as templates for organizing nanoparticles including various biologically
Figure 1: Schematic illustration representing a) self-assembly of m13 single-stranded phase DNA, biotin labeled strands and staple strands to form biontinylated DNA origami; b) and c) present 3D images of DNA origami and SA labeled origami, respectively, on mica.
relevant molecules8, 9, metal nanoparticles10, quantum dots11 and carbon nanotubes3 with extreme precision and control. These structures hold great promise for leading to advances in many fields, including nanoelectronics, nanorobotics and nanoscale signal transducers. DNA origami have also been extensively used for single-molecule studies ranging from chemical,12 photochemical,13 and biochemical reactions14 to the determination of photo-physical properties,15 recognition of single-nucleotide polymorphisms,16 conformational changes in DNA17 and DNA nanodevice18 through imaging the reaction position on origami via AFM. Recently Endo et al. observed the mechanical behavior of enzymes14, 19 using High-speed AFM (HS-AFM).This high-speed imaging technology can record 40 images per second, enabling one to image the dynamic motions of biomolecules in real time at molecular resolution.7 In a previous study, the binding of individual SA molecules to functionalized origami was characterized using time-lapse AFM.20 In the studies reported here,
ACS Paragon Plus Environment
Langmuir
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 6
Figure 2: Time-laps HS-AFM images of SA labeling on biotinylated origami; (SI Movie2) Frame 1 shows origami immobilized on mica before SA exposure in solution; Frame 4 shows after 1.6 seconds and Frame 148 after 60 seconds of exposure to a buffered solution of streptavidin. Scale bar 100nm
the binding of individual SA protein to biotin on modified DNA molecules precisely placed on closely spaced arrays of origami platforms was observed using HS-AFM, providing a very rapid means of measuring the binding rate using a sensing region only 400 X 400 nm2 in size. To measure the kinetics on a bulk scale and for comparison purposes, surface plasmon resonance (SPR) was also employed. Two sites on the DN constructs were coded for addressing SA by adding biotin labeled strands complementary to the M13 single stranded phage DNA scaffold. At each of the two sites, a pair of biotins were positioned with ~2 nm spacing, comparable to the spacing between biotin binding sites in SA.21 The schematic illustration in Fig. 1a shows the two sites in which the biotin pairs are positioned along the midline region on the origami construct at a separation distance of 68nm.21, 22 The topographical images in Figures 1b (before SA exposure) and 1c (after SA exposure) demonstrate SA labeling. Having successfully addressed the SA to the binding sites at fixed distances, the same technique was implemented with 1D DN constructs. The AFM image in Fig. S1a demonstrates long 1D constructs which were successfully formed in solution then immobilized on mica. Figure S1b shows 1D origami successfully labeled with SAs with ~68 nm spacing and ~97 nm pitch. AFM line profile measurements reflect a height of ~2.3 nm for SA and the height measured for DNA origami is ~1.2 nm. The spacing is consistent with the designed biotin locations on the origami and the observed SA height is consistent with our prior observations.2 The HS-AFM imaging system was operated at 0.4 sec/frame.23 SA binding was completed in less than 60 seconds as indicated by Frame 148 in Fig. 2. The entire
HS-AFM analysis is shown in SI Movie1. The HSAFM video (SI Movie2, the frame numbers are labeled in the upper left corner) clearly indicates that assembly reached near saturation (~90% occupancy) in less than 14 seconds, and by 52 seconds, the origami are fully occupied (31 binding sites is observed) by SA. In the final frame of the HS-AFM scan series two sites appear to have ambiguous occupations, and were therefore not included in the analysis. One of the targeted sites was covered by an adjacent DO. Another site is impossible to evaluate, perhaps because the origami is not firmly immobilized on the mica and waved in solution, obscured by a scan artifact throughout the series. The best-fit line shown in Fig. 3 is based on the equation to predict the behavior of irreversible adsorption of protein molecules onto a self-assembled monolayer.23 Data are expected to be consistent with this equation only when the following criteria are met: Binding is irreversible: we find that this data set and other similar sets show almost no (
