Article pubs.acs.org/JPCB
Single-Molecule FRET Studies of the Hybridization Mechanism during Noncovalent Adsorption and Desorption of DNA on Graphene Oxide Tapas Paul, Subhas Chandra Bera, Nidhi Agnihotri, and Padmaja P. Mishra* Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064, India S Supporting Information *
ABSTRACT: Remarkable observations on the adsorption and desorption mechanisms of single-stranded oligonucleotides and the hybridization of double-stranded DNA (ds-DNA) on a graphene oxide (GO) surface have been made using ensemble and single-molecule fluorescence methods. Probe and target DNAs labeled individually with fluorescence resonance energy transfer (FRET) pairs and having similar adsorption affinities toward the GO surface are used to provide detailed insights into the hybridization mechanism. Single-molecule FRET results reveal an “in situ” DNA hybridization mechanism, i.e., hybridization between the probe and target DNAs to form a dsDNA, and simultaneous desorption from the GO surface thereafter. These results also demonstrate that the electrostatic interaction between DNA and GO is of little importance to the overall theory of interaction and the largest effects are from solvation forces, specifically the hydrophobic effect. This investigation improves the fundamental understanding of the DNA hybridization dynamics on the GO surface, opening new windows in the field of biophysics as well as in sensing and therapeutic applications.
1. INTRODUCTION Large multiprotein machines are known to typically use cooperative interactions to promote and modulate DNA transactions that are responsible for a variety of fundamental procedures.1 Besides DNA bending and/or wrapping, hybridization of single-stranded DNA (ss-DNA) to double-stranded DNA (ds-DNA) and dissociation back to ss-DNA plays an important role in several life processes.2 For a long time, cooperative hydrogen bonding-based DNA hybridization assays of base pairs have been used in medicine, molecular biology, and other DNA-related technologies.3,4 Sensing of DNA is a tedious process that requires multistep amplification due to the negligible concentration of DNA under physiological conditions.5,6 Traditionally, DNA is detected by exploitation of nucleic acid probes grafted on a transducer surface that is used as a molecular recognition element,5 by targeting through Watson−Crick interactions. This type of DNA sensor needs to be precise enough in terms of its manifestation to distinguish between ss-DNA and ds-DNA at the interface, and this can be achieved either using hybridization indicators or through changes in the physicochemical behavior of the sensing layer.7−10 Hence, it is very important to understand the behavior of DNA at the interfaces, as the interactions between the substrate with DNA and the environment have substantial contributions in tuning the hybridization processes at the interface.2,11,12 Moreover, the structural and functional stabilities of the biomolecules in the environment play a crucial role in the practical viability of the biomolecule−material hybrid. It is known that noncovalent © 2016 American Chemical Society
interactions of biochemical functional groups with a mesoporous structure can significantly disrupt the biomolecular structure and function and hence affect nucleotide bioavailability in solution.13 Thus, insight into the detailed mechanism of this interaction is required to monitor it as well as the structural deformation that the biomolecules undergo during such an interaction.14 Since its discovery in 2004, graphene15 and its derivatives,16 especially graphene oxide (GO), have been used as an immobilization platform for biosensors due to their unique electronic, mechanical, and optical properties as well as their high surface area and fluorescence quenching ability.17−19 Being a low-dimensional system and due to its increased hydrophobicity and processability, the study of the interaction of GO with DNA has accumulated great research interest in the recent past.20−23 Irrespective of the negative charge on both DNA and GO, DNA still adsorb reversibly onto the surface of GO in the solution phase, and this can be tuned by the concentration of salts in the medium.24,25 It has also been seen that the adsorption affinity of purine bases or purine-based DNA toward a GO surface is stronger than that of pyrimidine bases or pyrimidine-based DNA.26 The driving force for the adsorption could be an effective combination of either two or all three phenomena, namely, the shielded electrostatic force, the dehydration effect, and the intermolecular hydrogen bonding. Received: June 15, 2016 Revised: October 14, 2016 Published: October 17, 2016 11628
DOI: 10.1021/acs.jpcb.6b06017 J. Phys. Chem. B 2016, 120, 11628−11636
Article
The Journal of Physical Chemistry B
Scheme 1. Three Possible Reaction Mechanisms for Hybridization of Surface-Adsorbed Bimolecules: (A) Displacement Mechanism, (B) Eley−Rideal Mechanism, and (C) Langmuir−Hinshelwood Mechanisma
a
In all of these cases, the probe DNA (yellow) is initially adsorbed onto the GO surface and complementary target DNA (cyan) is added to it later. (A) Displacement mechanism: displacement, followed by hybridization; (B) Eley−Rideal mechanism: direct hybridization; (C) Langmuir− Hinshelwood mechanism: hybridization on the surface, followed by diffusion. The surface functional groups and conjugated bonds of GO have been excluded for simplicity.
Table 1. Sequence of the Oligonucleotides with Modification
As GO is also a competent fluorescence quencher,21,27 fluorophore-labeled DNA and aptamers have been extensively coupled with GO as an analytical tool for nucleic acid detection;28 sensing of metal ions,29 small molecules,18,30,31 proteins,32−34 viruses,35−37 cells,18,38 and drug delivery.39,40 The dynamic nature of GO, to be used as a sensor, is based on its ability to bind to ss-DNA with a greater affinity compared to that of binding to ds-DNA or well-folded ss-DNA.21 Despite the valuable functionalities of GO-loaded ss-DNAs in DNA biosensors,41 little information is available about the role of GO in the mechanistic pathway of the hybridization of its DNA cargo. The majority of work carried out so far in this field is based on mixing of a fluorophore-labeled ss-DNA with GO, leading to DNA adsorption onto the GO surface, with successive quenching of the fluorescence. Upon addition of target DNA, the probe DNA desorbs from the GO surface and hybridizes to form a ds-DNA in solution, restoring the fluorescence. Two possible mechanisms have been proposed earlier by Liu et al. and Kim et al. to explain these phenomena,
as represented in Scheme 1. Out of the three possible surface reaction mechanisms (Scheme 1), namely, (A) displacement,26 (B) the Eley−Rideal mechanism, and (C) the Langmuir− Hinshelwood mechanism,42 Liu et al. proposed that hybridization follows nonspecific probe displacement in the solution phase, i.e., the displacement mechanism.26 On the other hand, according to Kim et al., desorption of the DNA adsorbed onto GO is followed by hybridization with the complementary DNA (cDNA) on the GO surface, i.e., the Langmuir−Hinshelwood mechanism.43 However, it has been quite challenging to define the analyte-induced desorption reaction or duplex formation mechanism on a molecular scale due to the lack of substantial experimental evidence. Our work demonstrates the mechanistic aspects underlying the adsorption of DNA onto or its desorption from the GO surface in aqueous solution using both ensemble and singlemolecule fluorescence resonance energy transfer (sm-FRET) methods.44 The experiments were carried out by fluorophorelabeling both of the probe and target DNAs with a donor and 11629
DOI: 10.1021/acs.jpcb.6b06017 J. Phys. Chem. B 2016, 120, 11628−11636
Article
The Journal of Physical Chemistry B
For adsorption of only D2 onto GO, the above-mentioned DNA/GO complex mixture was centrifuged at 13 000 rpm for 15 min and the free DNA was removed by discarding the supernatant, followed by washing with the same buffer. The washing was carried out two times, and this mixture was used in the sm-FRET experiment for further analysis. It was checked whether the adsorbed probe DNA was relatively stable without further addition of DNA and the background fluorescence remained unchanged, which are well supported by the previous report.26 Similarly, samples of unlabeled complementary (D3) and noncomplementary (D5) oligonucleotides adsorbed onto GO are prepared following the same protocol. 2.4. sm-FRET Measurements. We followed an already established procedure to execute the sm-FRET experiments.46 In brief, predrilled quartz microscope slides were cleaned thoroughly and poly (ethylene glycol) (PEG)/biotin−PEG was coated onto their surfaces. Cy3-tagged DNA oligonucleotides (D1) were then surface-immobilized onto the quartz slides through streptavidin−biotin chemistry. The preadsorbed and completely quenched Cy5-labeled oligonucleotides with the GO were real-time flown into the sample chamber containing the D1 substrate. Our home-built prism-type total internal reflection-based setup is developed on an inverted microscope (Olympus IX 71), as shown in Figure S2. A solid-state diode laser of 532 nm (Laser Quantum, U.K.) was used to excite Cy3 using the evanescent wave generated because of total internal reflection of the light beam by a pellin-broca prism. A 532 nm solid-state green laser is used to excite Cy3, and the emission signals from both Cy3 and Cy5 were simultaneously collected using a water immersion objective (60×, 1.2 NA; Olympus). After the signals pass through a long-pass filter (550 nm; Chroma), the fluorescences of Cy3 and Cy5 are separated using a dichroic mirror (640DCXR, Chroma). Individual signals were recorded simultaneously using an electron multiplying charge coupled device camera (emCCD, Ixon3 + 897; Andor Technologies), with a frame integration time of 30 ms. The Visual C++ (Microsoft, WA)-based data acquisition software used was a generous gift from T.J. Ha (University of Illinois) or Tae-Hee Lee (Pennsylvania State University). Each image frame, containing ∼500 molecules, was analyzed by program codes written on an Interactive Data Language platform, and the legitimate FRET traces were extracted. Cy3 emission bleeds though to the Cy5 channel (typically
