Dynamic Modulation of DNA Hybridization Using Allosteric DNA

Subscriber access provided by University of Michigan-Flint

Article

Dynamic modulation of DNA hybridization using allosteric DNA tetrahedral nanostructures Ping Song, Min Li, Juwen Shen, Hao Pei, Jie Chao, Shao Su, Ali Aldalbahi, Lihua Wang, Jiye Shi, Shiping Song, Lianhui Wang, Chunhai Fan, and Xiaolei Zuo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01373 • Publication Date (Web): 20 Jul 2016 Downloaded from http://pubs.acs.org on July 21, 2016

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.

Analytical Chemistry 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 21

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

Analytical Chemistry

Dynamic modulation of DNA hybridization using allosteric DNA tetrahedral nanostructures Ping Songa, Min Lia, Juwen Shenb, Hao Peib, Jie Chaoc, Shao Suc, Ali Aldalbahid, Lihua Wanga, Jiye Shie, Shiping Songa, Lianhui Wangc, Chunhai Fana, Xiaolei Zuoa,* a. Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China b. School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China c. Key Laboratory for Organic Electronics & Information Displays (KLOEID), Institute of Advanced Materials (IAM) and School of Materials Science and Engineering, Nanjing University of Posts & Telecommunications, Nanjing, 210046, China d. Chemistry Department, King Saud University, Riyadh 11451, Saudi Arabia e. Kellogg College, University of Oxford, Oxford, OX2 6PN, UK * Email: [email protected], Fax: 86-21-39194173

ACS Paragon Plus Environment

1


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 21

ABSTRACT The fixed dynamic range of traditional biosensors limits their utility in several real applications. For example, viral load monitoring requires the dynamic range spans several orders of magnitude; whereas, monitoring of drugs requires extremely narrow dynamic range. To overcome this limitation, here, we devised tunable biosensing interface using allosteric DNA tetrahedral bioprobes to tune the dynamic range of DNA biosensors. Our strategy takes the advantage of the readily and flexible structure design and predictable geometric reconfiguration of DNA nanotechnology. We reconfigured the DNA tetrahedral bioprobes by inserting the effector sequence into the DNA tetrahedron, through which, the binding affinity of DNA tetrahedral bioprobes can be tuned. As a result, the detection limit of DNA biosensors can be programmably regulated. The dynamic range of DNA biosensors can be tuned (narrowed or extended) for up to 100-fold. Using the regulation of binding affinity, we realized the capture and release of biomolecules by tuning the binding behavior of DNA tetrahedral bioprobes.

KEYWORDS Allosteric , DNA nanostructure , Tetrahedral, DNA biosensor, Dynamic range

Introduction The dynamic range of biosensors is critically important in several real applications such as disease diagnostics and therapeutic monitoring of drugs 1-14. A typical application of viral (e.g., HIV) load monitoring, which depends on quantitative measurement of nucleic acids, require the dynamic range spans over 5-order of magnitude. Whereas, in the monitoring of therapeutic drugs (e.g., cyclosporine), the dynamic range is narrow to less than one order of magnitude

7,11,15

.

Beyond biosensoring applications, the performances of bio-fuel cells and molecular logic gates are highly related to the dynamic range of the bio-molecule recognition5. Therefore, the ability to tune the dynamic range of biosensors would improve the biosensing performances in real


2

Page 3 of 21

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


applications. However, a traditional biosensor that based on the immobilization of bioprobes is characterized with a fixed dynamic range 1-13,15, which originated from the single-site binding property of bioprobe-target binding. Hence, the fixed dynamic range limits the utility of biosensors in many real applications. In natural system, the allosteric effect was employed to optimize many cellular processes by tuning the binding behavior of biological receptors (e.g., tuning the dynamic range of receptors in real time), in which the binding of one ligand can influence the binding affinity of the others 16-23

. An elegant example of heterotropic allostery is that the binding of bicarbonate ions to

crocodile hemoglobin drastically reduced the oxygen binding affinity of crocodile hemoglobin (by shifting the midpoint of binding curves to higher oxygen concentration, figure 1a and 1b)19. Inspired by the natural allosteric effect, we here devised allosteric DNA tetrahedral nanostructures to dynamically modulate the DNA hybridization, which, in turn, modulate the dynamic range of DNA biosensors (figure 1c). DNA nanotechnology is considered as one of the most promising nanotechnologies with superior characteristics such as highly programmable design, highly predictable geometric structure and highly precise dimensions 24-33. In addition, the state-of-the-art DNA synthetic technology allows flexible modifications at 3 terminus (or 5 terminus) and any arbitrary position of DNA. Based on these advantages, various DNA structures in 1-dimension (1-D), 2-D and 3-D were successfully designed and assembled

24-34

. With these unique properties of DNA nanotechnology, DNA

nanostructures can be excellent candidates for designing the mimetic allosteric bioprobes. We could precisely design the active sites and remote sites on DNA nanostructures and predict the conformational change of the DNA nanostructures, through which the requirements of designing mimetic allosteric bioprobes are met. To explore the potential of DNA nanotechnology in


3


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 4 of 21

mimicking the allosteric control, here, we have demonstrated a strategy to rationally engineer the DNA tetrahedral bioprobes that can dynamically modulate the binding affinity of DNA tetrahedral bioprobes to their targets (figure 1c). In our previous studies, we have demonstrated the superior properties of biosensing interface with DNA tetrahedral structures for improving the mass transport and biosensing performances 31,33,34

. Here, we further demonstrated that the surface confined DNA tetrahedral bioprobes can

be reconfigured, through which the binding affinity of DNA tetrahedral bioprobes can be allosterically modulated (figure 1). As a mimetic system of natural allosteric system, we demonstrated the capture and release of target molecules by tuning the binding behavior of DNA tetrahedral bioprobes. Experimental section Materials DNA oligonucleotides shown in table S1 and S2 were synthesized and purified by Sangon Biotechnology Inc. (Shanghai, China). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and hexaammineruthenium (III) chloride (RuHex) were purchased from Sigma. The TMB substrate (where TMB = 3,3’,5,5’- tetrame- thylbenzidine) was purchased from Neogen in the format of a ready-to-use reagent (K-blue custom activity substrate, H2O2 included). Horseradish peroxidase-conjugated avidin (avidin-HRP) was from Roche Diagnostics (Mannheim, Germany). The enzyme diluent was universal casein diluent/block which was purchased from fitzgerald industries international Inc. All other chemicals were of analytical grade, and all chemicals were used without further purification. All solutions were prepared with Milli-Q water from a Millipore system. Preparation of DNA tetrahedral bioprobe


4

Page 5 of 21

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


Concentrations of single-stranded DNA (ss-DNA) stock solutions were calculated based on their absorbance at 260 nm using UV–Vis spectroscopy (Hitachi U-3010). Extinction coefficients were estimated by the SciTools from Integrated DNA Technologies (IDT). Equal molar quantities (1 µM of final concentration) of four strands for the formation of the tetrahedrons were mixed in buffer (20 mM Tris, 50 mM MgCl2, pH 8.0) at 95 °C and then cooled to 4 °C by using PTC-200 (MJ. Research Inc., SA). The final concentration of the DNA tetrahedral nanostructure is 1 µM. Polyacrylamide gel electrophoresis (PAGE) The DNA solution mixed with 6× loading buffer (TEK buffer, pH 8.0, 50% glycerol, 0.25% bromphenol blue) was analyzed in 8% native polyacrylamide gel. The electrophoresis was conducted in 1 × TBE ( Mg2+ concentration: 12.5 mM; pH 8.0) at a constant voltage of 80 V for 2 h. The gels were scanned by a UV transilluminator (SynGene) after staining with Gel Red. And the productivities of the five structures were calculated by the GeneTools from SynGene. Electrochemical measurements All electrochemical measurements were carried out with a CHI 630b electrochemical workstation (CH Instruments Inc.). A three-electrode configuration was employed in all experiments and involved a gold working electrode, a platinum wire counter electrode and an Ag/AgCl reference electrode. Gold electrodes (2 mm in diameter, CH Instruments Inc.) were first electrochemically cleaned in 0.5 M NaOH. Then, the gold electrodes were polished on microcloth with 0.3 µm and 0.05 µm Gamma alumina for 3 min. The polished electrodes were then sonicated in ethanol and Mill-Q water for 2 min, respectively. Finally, the electrodes were electrochemically cleaned. In the fifth step, the charge associated with this process (the area


5


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 6 of 21

under the reduction peak) was converted to the electrode area using the parameter of 422 µC/cm2. Gold electrodes were incubated with 3 µL of tetrahedral bioprobe (1 µM) overnight at room temperature before electrochemical measurements. And then washed with 0.1 M PBS, the target detect process was referenced by published work34. Cyclic voltammetry (CV) was carried out at a scan rate of 100 mV/s and the potential was from 0 to 0.7 V. In addition, Amperometric detection was fixed at 100 mV, and the electroreduction current was measured for 100 s after the HRP redox reaction reached steady state. Assembly density of tetrahedral bioprobe on gold electrode Prior to measurements, the 10 mM phosphate buffer (pH 7.4) was deoxygenated via purging with nitrogen gas for 20 min, and the cell was blanketed with nitrogen for the duration of the experiments. Cyclic voltammetry (CV) and chronocoulometry (CC) were performed without Ru3+ and with Ru3+ (final concentration of 300µM), and then calculate the density of the TDN assembled on the surface of the gold electrode. The following parameters were employed: CV, sweep rate, 100 mV/s; CC, pulse period, 500 ms, pulse width, 500 mV. Dynamic process In the dynamic reconfiguring process of tetra A26 to tetra A50, we first immobilized tetra A26 on the gold surface. In the hybridized process with target, we added effector sequence to reconfigure tetra A26 to tetra A50. The target capture process and transform process were at the same time. The dynamic reconfiguring process of tetra A26 to tetra E was the same as tetra A26 to tetra A50.


6

Page 7 of 21

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


Results and discussion To design DNA tetrahedral bioprobes with different binding affinity to their targets, we constructed a series of DNA tetrahedral bioprobes (tetra A26, tetra A36 and tetra A50) by altering the edge length or inserting stem-loop domain in one edge of DNA tetrahedral bioprobes (figure 2a), through which, we can investigate the influence of the configuration of nanostructure on target-binding affinity. As demonstrated, three DNA tetrahedral bioprobes were rationally constructed with different configurations. Based on our previous design principle34, no undesirable secondary structures were present at edges. Each DNA tetrahedral bioprobe contains a pendent probe at one vertex and three thiol moieties at the other three vertices for surface immobilization. We characterized the successful assembly (yield: >80%) of these DNA tetrahedral bioprobes by native polyacrylamide gel electrophoresis (PAGE, figure S1). To determine the binding affinity of these DNA tetrahedral bioprobes for their targets, we then integrated the DNA tetrahedral bioprobes into a well-established sandwich type assay. We first immobilized our DNA tetrahedral bioprobes on planar gold surface via the well established goldsulfur bond. The quantification of surface density of DNA tetrahedral bioprobes indicated that surface density of these designed bioprobes was similar (figure S2). Upon the target binding, the signaling probe modified with biotin label was attached on the gold surface. After the conjugation with avidin-HRP (avidin-horse radish peroxidase), the electrocatalytic signal can be readily detected by electrochemical method (figure 2b, typical i-t curves of electrochemical detection). With our optimized conditions (figure S3), we obtained the dose-response curves of DNA tetrahedral bioprobes with different configurations (figure 2c, figure S4). As we hypothesized, the binding affinity varied with different DNA tetrahedral bioprobes (figure 2d).


7


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 8 of 21

Our results indicated that the binding affinities of DNA tetrahedral bioprobes for their targets have a strong dependence on the different configurations (The increase of negatively charged DNA sequences in the edge of DNA tetrahedral bioprobe led to a decrease of binding affinity). The free energy calculation also revealed that the target binding tended to unfavorable direction along with the increase of the local charges of DNA tetrahedral bioprobes (figure 2d). The detection limits were efficiently modulated from 100 pM to 1 pM with various configurations of DNA tetrahedral bioprobes (figure 2e, 2f and 2g). By understanding the influence of the configuration of DNA tetrahedral bioprobes on the binding affinity to their targets, we were inspired to allosterically modulate the binding affinity of DNA tetrahedral bioprobes (figure 3). To achieve this goal, we first immobilized DNA tetrahedral bioprobe (Tetra A26) on gold surface and investigated the influence of the effector binding to remote site on the target binding to active site. With our reconfiguration method, the inserting of the effector on remote site would reconfigure the DNA tetrahedral bioprobe (Tetra A26) to Tetra A50 (figure S5). Consequently, the in-situ configuration change of DNA tetrahedral bioprobe induced an obvious modulation in binding affinity (figure 3a), resulting a 10-fold narrowed dynamic range. The Kd value was successfully modulated from 2.7 µM to 8.6 µM. We also observed that the slope of the binding curve increased, which indicated that the sensitivity was improved for ~1.4-fold. Furthermore, we designed another effector and insert it into tetra A26 to reconfigure DNA tetrahedral bioprobe from Tetra A26 to Tetra E (figure S6). As expected, the dynamic range was successfully narrowed for 5-fold. The Kd value was modulated from 2.7 µM to 6.7 µM (figure 3b). To test the generalizability of our strategy, we devised a new DNA tetrahedral bioprobe by embedding an i-motif domain35,36 into one edge of the DNA tetrahedral bioprobe, which can


8

Page 9 of 21

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


form 3-dimensional tetraplex structure at pH 5 (figure 4a). The tetraplex i-motif structure can be reconfigured by hybridizing it with its complementary sequence (effector), which was identified by gel results (figure S7). Interestingly, we found that the binding affinity of DNA tetrahedral bioprobe was modulated by the distinct structure reconfiguration of the DNA tetrahedral bioprobe (figure 4b, c, d, e). As a result, the dynamic range was remarkably narrowed for 100fold (from ~3-order of magnitude to ~1-order of magnitude) along with the reconfiguration of tetrahedral bioprobe. The kd value was modulated from 2.3 µM to 5.4µM, while, the sensitivity was improved for ~ 3-fold. Both the modulation of kd and sensitivity contributed to the 100-fold modulation of dynamic range. With the successful configuration control of DNA tetrahedron and the binding affinity modulating of the surface confined DNA tetrahedral bioprobes, we are capable to realize the controllable capture and release of target molecules that mimics the function of hemoglobin to load and deliver the oxygen molecules19,22,37 (figure 5a). In natural system, original state of hemoglobin binds oxygen tightly, which is difficult to deliver the oxygen to tissues. Some metabolic stimuli (ATP, CO2 and H+) aid the unloading of oxygen by shifting the binding curves of hemoglobin to oxygen. Here, we performed the target capture by using the surface immobilized tetra A26 (figure 5a). To make the target capture detectable, we still employed electrochemical sandwich assay to produce electrochemical signal. With 5 nM of target DNA, the electrochemical signal was ~ 9700 nA. After adding the effector into the system, we observed a remarkable signal decrease (~47%), which indicated that some targets were released during the reconfiguration of the bioprobe from tetra A26 to tetra A50. Interestingly, we can quantitatively estimate the signal decrease through the shifting of the dynamic range (figure 5b), which is in consistent with our experimental result.


9


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

In order to extend the dynamic range, we can design composite biosensing interface

Page 10 of 21

11,21,38

with

high dynamic range by mixing these DNA tetrahedral bioprobes. Interestingly, we observed that the binding constants of our designed five types of DNA tetrahedral bioprobes (tetra A26, tetra A36, tetra A50, tetra E and tetra C) were shifting to the high target concentration (figure S8, figure S9). This provided an opportunity to allow the accurate determination of target concentration over a large continuous dynamic range by constructing a composite biosensing interface. Within 1% accuracy, we obtained an improved dynamic range with 10-fold increase by mixing these bioprobes in equal molar proportions (figure S9). The dynamic range of biosensors plays important role in various real applications. As we mentioned, the increasing demand for viral load monitoring at regular intervals could prevent the spread of drug resistance. The RNA detection or DNA detection based viral load monitoring requires the biosensor with large dynamic range (from 50-106 molecules /ml for HIV monitoring). Hence, our design to modulate the dynamic range using allosteric DNA tetrahedral nanostructures could provide a flexible and programmable method in this application. In addition, for the applications with requirement of narrow dynamic range such as the monitoring of therapeutic drugs, which is critically important to achieve beneficial effect and minimize adverse effect, we devised the i-motif based DNA tetrahedral nanostructure as a model, which demonstrated extremely narrow dynamic range (1-order of magnitude). The design of i-motif based dynamic DNA tetrahedral nanostructure provide the basis of narrowing the dynamic range of biosensors for DNA detection, however, it could be translated to monitor the therapeutic drugs by replacing the capture probe with aptamers. The dynamic 3-D DNA nanostructures are important in the design of modulating the binding affinity and dynamic range of biosensors. Firstly, the 3-D DNA structures could be readily


10

Page 11 of 21

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


designed to dynamic 3-D DNA structures by embedding responsive DNA domain in the nanostructures. Secondly, the various types of responsive DNA domain (e.g., stem-loop structure, i-motif, and target-responsive aptamers) could be used to reconfigure the 3-D DNA structures, which is flexible in design. Last, the conformational change of the dynamic 3-D DNA structures is highly predictable, which could be used to predict the modulating functions of the DNA nanostructures. Conclusion Here, we demonstrated that our reconfigurable DNA tetrahedral bioprobes offer the possibility of creating novel allosteric bioprobes with biotechnological value (tunable binding affinity and controlled target capture and release). The advantages of our system include: firstly, based on the highly programmable and predictable nature of DNA nanotechnology, a simple allosteric system that can form the basis for the construction of the allosteric biosensing interface. Secondly, the DNA nanostructure allows the accurate design of geometric separated sites and predictable conformational change for allosteric effect. Thirdly, our reconfiguration technology provides a simple and efficient way that can reconfigure our tetrahedral bioprobes and allosterically modulate the binding affinity for target.


11


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 12 of 21

Figure 1. (a and b) A typical allosteric effect is demonstrated. A target molecule can bind its probe (e.g., allosteric protein) with a characteristic binding affinity. The binding affinity can be allosterically modulated with the effector binding to the remote site of the probe. (c) We designed allosteric DNA tetrahedral bioprobe by prearranging a stem-loop domain as remote site at one edge of DNA tetrahedral bioprobe. A DNA recognition probe was designed on the top vertex of DNA tetrahedron as the active site to capture target DNA molecules. The other three vertices of DNA tetrahedron were modified with thiols for surface immobilization. We could reconfigure the surface confined DNA tetrahedron by inserting a DNA sequence (effector) into the DNA tetrahedron. The binding of the effector to remote site of the DNA tetrahedral bioprobe could be employed to reconfigure the DNA tetrahedral bioprobe and allosterically modulate the binding affinity of the DNA of active site.


12

Page 13 of 21

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


Figure 2. (a) We designed three types of DNA tetrahedral bioprobes, which contain a stem-loop domain on one edge. We systematically increased the charges on these edges by inserting more nucleotides. (b) The representative i-t curves of electrochemical detection with sandwich type assay. (c) The dose-response curves of three types of DNA tetrahedral bioprobes. (d) The Kd and the Gibbs free energy increased with the increase of total charges of DNA tetrahedral bioprobes. (e-f) The detection limits of DNA biosensors based on tetra A26, tetra A36 and tetra A50 was identified by 3σ standard. The detection limits were 1 pM, 10 pM and 100 pM for tera A26, tetra A36 and tetra A50, respectively.


13


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 14 of 21

Figure 3. (a) A stem-loop DNA (effector) was inserted into Tetra A26 to reconfigure Tetra A26 to Tetra A50. As a result, the binding affinity and dynamic range were allosterically engineered. (b) To generalize our design, another DNA (effector) was inserted into Tetra A26 to reconfigure Tetra A26 to Tetra E. We observed the allosterical engineering of the binding affinity and dynamic range.


14

Page 15 of 21

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


Figure 4. (a) With our rational design, an i-motif sequence, which formed tetraplex structure at pH 5, was inserted into one edge of DNA tetrahedral bioprobe. By hybridizing effector, the DNA tetrahedral bioprobe was reconfigured. The binding curve (b and c), kd (d) and sensitivity (e) was modulated along with the reconfiguration of the tetrahedral bioprobe. The kd value was modulated from 2.3 µM to 5.4µM, while, the sensitivity was improved for ~ 3-fold (from 0.14µM-1 to 0.44µM-1).


15


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 16 of 21

Figure 5. (a) Tetra A26 was employed as bioprobe to capture the target DNA molecules with relatively higher binding affinity. After inserting an effector, the tetra A26 was reconfigured to tetra A50 with decreased binding affinity. Consequently, some target DNA molecules that were captured on the surface were released into solution phase. (b) The prediction from the binding curves of tetra A26 and tetra A 50 indicated that ~42.4 % of target DNA molecules would be released when the target concentration was 5 nM. (c) Our experimental results demonstrated that the signal decreased ~47% with the allosterical modulation, which is consistent with the prediction of binding curves. As contrast, without adding effector, the signal changed negligibly.


16

Page 17 of 21

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


ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was financially supported by the National Basic Research Program (973 Program grant numbers 2012CB932600), NSFC (grant numbers 21422508, 31470960), and the Chinese Academy of Sciences. Ali Aldalbahi acknowledges the support by the Deanship of Scientific Research, College of Science Research Center at King Saud University. Supporting Information. Supporting figures (figure S1-S9 including electrophoresis analysis, optimization of experimental conditions, electrochemical detection), materials and experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

REFERENCES (1)

Rizk, S. S.; Paduch, M.; Heithaus, J. H.; Duguid, E. M.; Sandstrom, A.;

Kossiakoff, A. A. Nat. Struct. Mol. Biol. 2011, 18, 437-442. (2)

Seo, M. H.; Park, J.; Kim, E.; Hohng, S.; Kim, H. S. Nat. Commun. 2014, 5, 3724.


17


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

(3)

Page 18 of 21

Simon, A. J.; Vallee-Belisle, A.; Ricci, F.; Watkins, H. M.; Plaxco, K. W. Angew.

Chem. Int. Ed. 2014, 53, 9471-9475. (4)

Vallee-Belisle, A.; Ricci, F.; Plaxco, K. W. Proc. Natl. Acad. Sci. U. S. A. 2009,

106, 13802-13807. (5)

Kang, D.; Vallee-Belisle, A.; Porchetta, A.; Plaxco, K. W.; Ricci, F. Angew.

Chem. Int. Ed. 2012, 51, 6717-6721. (6)

Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642-6643.

(7)

Nesterova, I. V.; Nesterov, E. E. J. Am. Chem. Soc. 2014, 136, 8843-8846.

(8)

Porchetta, A.; Vallee-Belisle, A.; Plaxco, K. W.; Ricci, F. J. Am. Chem. Soc.

2012, 134, 20601-20604. (9)

Porchetta, A.; Vallee-Belisle, A.; Plaxco, K. W.; Ricci, F. J. Am. Chem. Soc.

2013, 135, 13238-13241. (10)

Ricci, F.; Vallee-Belisle, A.; Porchetta, A.; Plaxco, K. W. J. Am. Chem. Soc.

2012, 134, 15177-15180. (11)

Vallee-Belisle, A.; Ricci, F.; Plaxco, K. W. J. Am. Chem. Soc. 2012, 134, 2876-

(12)

Xiang, Y.; Tong, A.; Lu, Y. J. Am. Chem. Soc. 2009, 131, 15352-15357.

(13)

Simon, A. J.; Vallee-Belisle, A.; Ricci, F.; Plaxco, K. W. Proc. Natl. Acad. Sci. U.

2879.

S. A. 2014, 111, 15048-15053. (14)

Mulvaney, S. P.; Sheehan, P. E. ACS nano 2014, 8, 9729-9732.

(15)

Drabovich, A. P.; Okhonin, V.; Berezovski, M.; Krylov, S. N. J. Am. Chem. Soc.

2007, 129, 7260-7261. (16)

Boehr, D. D.; Nussinov, R.; Wright, P. E. Nat. Chem. Biol. 2009, 5, 789-796.


18

Page 19 of 21

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


(17)

Christopoulos, A. Nat. Rev. Drug. Discov. 2002, 1, 198-210.

(18)

Conn, P. J.; Lindsley, C. W.; Meiler, J.; Niswender, C. M. Nat. Rev. Drug.

Discov. 2014, 13, 692-708. (19)

Komiyama, N. H.; Miyazaki, G.; Tame, J.; Nagai, K. Nature 1995, 373, 244-246.

(20)

Raman, S.; Taylor, N.; Genuth, N.; Fields, S.; Church, G. M. Trends Genet. 2014,

30, 521-528. (21)

Marvin, J. S.; Corcoran, E. E.; Hattangadi, N. A.; Zhang, J. V.; Gere, S. A.;

Hellinga, H. W. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, 4366-4371. (22)

Marinelli, F.; Kuhlmann, S. I.; Grell, E.; Kunte, H. J.; Ziegler, C.; Faraldo-

Gomez, J. D. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1285-1292. (23)

Lefstin, J. A.; Yamamoto, K. R. Nature 1998, 392, 885-888.

(24)

Omabegho, T.; Sha, R.; Seeman, N. C. Science 2009, 324, 67-71.

(25)

Ke, Y.; Ong, L. L.; Shih, W. M.; Yin, P. Science 2012, 338, 1177-1183.

(26)

Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Science 2015, 347, 1260901.

(27)

Han, D.; Pal, S.; Yang, Y.; Jiang, S.; Nangreave, J.; Liu, Y.; Yan, H. Science

2013, 339, 1412-1415. (28)

Goodman, R. P.; Heilemann, M.; Doose, S.; Erben, C. M.; Kapanidis, A. N.;

Turberfield, A. J. Nat. Nanotechnol. 2008, 3, 93-96. (29)

Zhang, F.; Jiang, S.; Wu, S.; Li, Y.; Mao, C.; Liu, Y.; Yan, H. Nature

nanotechnology 2015, 10, 779-784. (30)

Fu, J.; Yang, Y. R.; Johnson-Buck, A.; Liu, M.; Liu, Y.; Walter, N. G.;

Woodbury, N. W.; Yan, H. Nature nanotechnology 2014, 9, 531-536.


19


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

(31)

Page 20 of 21

Lu, N.; Pei, H.; Ge, Z.; Simmons, C. R.; Yan, H.; Fan, C. J. Am. Chem. Soc. 2012,

134, 13148-13151. (32)

Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C. Angew. Chem. Int. Ed.

2012, 51, 9020-9024. (33)

Pei, H.; Lu, N.; Wen, Y.; Song, S.; Liu, Y.; Yan, H.; Fan, C. Adv. Mater. 2010,

22, 4754-4758. (34)

Lin, M.; Wang, J.; Zhou, G.; Wang, J.; Wu, N.; Lu, J.; Gao, J.; Chen, X.; Shi, J.;

Zuo, X.; Fan, C. Angew. Chem. 2015, 54, 2151-2155. (35)

Dong, Y.; Yang, Z.; Liu, D. Accounts of chemical research 2014, 47, 1853-1860.

(36)

Liu, H.; Xu, Y.; Li, F.; Yang, Y.; Wang, W.; Song, Y.; Liu, D. Angewandte

Chemie 2007, 46, 2515-2517. (37)

Zhong, Z.; Anslyn, E. V. Angew. Chem. Int. Ed. 2003, 42, 3005-3008.

(38)

Yamazaki, T.; Kojima, K.; Sode, K. Anal. Chem. 2000, 72, 4689-4693.


20

Page 21 of 21

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


For TOC only


21

Dynamic Modulation of DNA Hybridization Using Allosteric DNA

Recommend Documents