Three-Dimensional Structure and Thermal Stability Studies of DNA Nanostructures by Energy Transfer Spectroscopy

Authors


Abstract

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Structural changes and stability of DNA nanoarchitectures including Y-shaped DNA (see picture), dendrimer-like DNA, and DNA hydrogels are investigated. The results demonstrate the feasibility and flexibility of FRET and NSET (Förster resonance/ nanometal surface- energy transfer) in determining difficult-to-obtain 3D structures and characterizing the thermal responses of DNA nanoarchitectures in real time.

DNA building blocks have attracted much attention because of their well-controlled nanostructural features,1 leading to the synthesis of a variety of branched DNA junctions that have enabled bottom-up assembly of DNA materials.2 Numerous characterization techniques, including atomic force microscopy,3 optical tweezers,4 and crystallography,5 have been successfully used to study the properties of DNA junctions. Despite these achievements, it still remains a challenge to determine the three-dimensional (3D) structure in aqueous solution because the aforementioned methods generally limit configurational freedom due to surface immobilization, trapping, or crystallization. In addition, these methods typically study the behavior of the entire molecule, but do not specifically isolate the effects of individual junctions within complex DNA nanostructures. Overcoming these challenges requires a method that can provide precise spatial and stability data in solution, in real time, and in a subunit-specific way. Recently, Niemeyer and co-workers reported real-time monitoring of the self-assembly of DNA tiles by Förster resonance energy transfer (FRET).6 Herein, we employ FRET and nanometal surface energy transfer (NSET)7 to study the melting behavior of diverse DNA nanostructures and the effects of a specific junction on the overall stability of the molecule. These approaches are powerful in obtaining nanometer or sub-nanometer resolution of 3D structures that will aid in the rational design of DNA nanomaterials, yet they are also flexible in extending to macromolecules beyond DNA as long as fluorescence conjugation is attainable.

Based on our recent progress in the assembly of DNA nanostructures8 (dendrimer-like DNA, or DL-DNA, as a model) and DNA hydrogel,9 3D structures of Y-shaped DNA and the thermal stability of individual junctions within a complex DNA architecture were investigated. We first labeled individual arms of Y-shaped DNA with FRET pairs [a donor, Alexa 488, and an acceptor, 4-{[4-(dimethylamino)phenyl]azo}benzoic acid (DABCYL)] at different locations (Figure S1 A in the Supporting Information). We then measured the fluorescence intensity and constructed a separation distance curve by converting intensity to distance. More specifically, the relative fluorescence intensities of the donor in the absence (FD) and presence (FDA) of the acceptor were converted into the absolute distance between the two bases (Scheme 1 and the Supporting Information). The initial distance (RI) between the donor and acceptor pair before heating was then subtracted from the calculated distance (R) between the pair to obtain the separation distance (RS) resulting from thermal denaturation (see the Supporting Information). Since these measurements were carried out in bulk solution, our distance measurements reflect the ensemble average from individual nanostructures. Distances at the single molecule level may deviate from this average due to thermodynamic fluctuations and orientation effects.

Scheme 1.

Illustration of the conversion from a melting transition curve to separation distance of a DNA nanoarchitecture with a FRET pair. The distance is converted by the equation, R=R0[1/(FD/FDA−1)]1/6, where the fluorescence intensities of donor in the absence (FD) and presence (FDA) of the acceptor are experimentally determined from the melting transition curve. When an increase in temperature no longer results in an increase in fluorescence, the fluorescence intensity at the lowest temperature can be taken as FD (bold dot) because the separation distance between donor and acceptor prevents any further energy transfer. The fluorescence intensity from when an increase in temperature begins to result in an increase in fluorescence to right before FD can be taken as an FDA.

Using FRET, the structure of Y-shaped DNA with 39 total base pairs, Y-DNA-39, was determined as shown in Figure 1 A, revealing a 3D, rather than planar, structure. Next, by conjugating FRET pairs on each arm separately, we observed similar melting transition curves for each arm during a steady temperature increase from 25°C to 55°C (Figure S1 B in the Supporting Information). Moreover, fluorescence intensity from each of the separately conjugated Y-DNA-39 reached a plateau at the same temperature, 45.3°C, suggesting that the Y-DNA-39 was fully denatured to three single oligonucleotides regardless of the conjugation position (Figure 1 B and Figure S1 B in the Supporting Information). To confirm that the plateau was caused by denaturation rather than by experimental limitations of FRET (which only allows for energy transfer up to 100 Å), melting transition curves of NSET (with energy transfer up to 250 Å) and FRET were compared. Melting transitions by both methods began to plateau at the same temperature, ensuring the validity of FD (Figure S2 in the Supporting Information). These results showed that FRET was sufficient for our studies.

Figure 1.

Real-time structural change monitoring of DNA nanoarchitectures. a) Schematic drawing of three-dimensional Y-DNA-39, which is determined by converting the fluorescence intensity into absolute distances. b) Separation distances of Y-DNA-39 at three different ends. c) Separation distance of Y-DNA-39 at junction (▴) and end (•) based on temperature. The arrows in the Y-DNA-39 indicate the direction of denaturation (inset). d) Separation distance of X-DNA-72 at the junction (▪), end (•), and middle of the arm (▴) based on temperature. The arrows in the X-DNA-72 indicate the direction of denaturation (inset).

In order to determine the melting behavior at the DNA junctions, we then conjugated a FRET pair on the junction (instead of the ends) of Y- and X-shaped DNA (Figures S3 A, B in the Supporting Information). The results showed that, in both Y- and X-DNA, the separation distance of the junction- and end-conjugated pairs matched very closely (Figures 1 C, D ). In the case of X-DNA-72, the middle of the arm denatured at the temperature of 40.5 °C, which was approximately 10 °C higher than either the junction or the ends (Figure 1 D). This suggests that denaturation of X-DNA begins at both the junction and ends and then propagates towards the middle of each arm until complete denaturation occurs. These phenomena, hereafter referred to as the junction-destabilization and end-destabilization, serve as destabilization factors to the overall structure. We believe that this is due to the lack of neighboring interactions at the ends and junctions, such as base-pair hydrogen bonding and pi-stacking.10 To the best of our knowledge, this is the first report in which FRET data was converted to dynamic distance information to provide temperature-dependent structural profiles for branched DNA nanoarchitectures.

To further explore the structural stability of more complex DNA nanostructures, we designed DL-DNA which was constructed from four units of Y-DNA-39. We then labeled the DL-DNA with FRET pairs at four different locations: end (E), outer junction (OJ), middle (M), and inner junction (IJ) [Figure 2 A]. These differential conjugations enabled us to map the sequential denaturation of DL-DNA at different positions (Figure S4 in the Supporting Information). We found that the ends of DL-DNA denatured at a much lower temperature (30 °C gap) than other locations, suggesting that the outermost arms of DL-DNA were denatured first at 45.0 °C. Interestingly, the separation distance curve of DL-DNA(E) was nearly identical to that observed for individual Y-DNA-39 (Figures 1 B, 2 B), further suggesting that there are limited interactions between the outer arms and the inner stems of the DL-DNA (Figure 2 C). This is consistent with the end destabilization that we had observed previously with Y- and X-DNA. When the temperature was higher than the denaturing temperature (45.0 °C), the regions between E and OJ remained as single-stranded overhangs. Further denaturation did not occur until 60.0 °C. At 60.0 °C, positions at the junctions (OJ and IJ) began to denature before the middle stem (M), displaying the same propagation trend (i.e. junction destabilization) as X-DNA. Between 60.0 °C and 69.0 °C, we observed a shoulder curve at the OJ site, which was attributed to the destabilizing effect of the dangling single strands. After further increasing the temperature to 70.0 °C, the middle stem (M) of DL-DNA finally began to denature, and complete denaturation occurred at 76 °C. Using the corresponding separation distance curves, we made the accompanying movie (Movie 1 in the Supporting Information), illustrating the ensemble average denaturation. The 3D DL-DNA model (also shown in Figure 2 C and Movie 1 in the Supporting Information) was constructed using open-source 3D modeling software (Blender, http://www.blender.org) based on the structural data obtained from FRET for Y-DNA-39. These results reveal the similarity between end-destabilization and the junction-destabilization on a much more complicated DNA structure.

Figure 2.

Structural changes of DL-DNA at four different positions. a) Four DL-DNA separately conjugated with a FRET pair at end (E), outer junction (OJ), middle of stem (M), and inner junction (IJ). b) Separation distance curves of each DL-DNA at end (▪), outer junction (▴), middle of stem (⧫), and inner junction (•). c) The denaturation process of DL-DNA, based on the observed separation distances.

The above results suggested that the overall stability of a DNA nanostructure can be rationally tuned. To demonstrate this tunability, we first measured the melting temperature of individual X-DNAs. X-DNA with 72 base pairs, X-DNA-72, was synthesized (Figure 3 A). Each arm of X-DNA-72 consisted of 18 base pairs plus extra four-base overhangs with a FRET pair conjugated at the junction. Upon adding ligase, X-DNA-72 formed a bulk DNA hydrogel. We predicted that this DNA gel would have the same melting temperature as a larger, individual X-DNA whose arm length was equivalent to the number of base pairs between junctions within a gel (Figure 3 A). In this case with X-DNA-72 gel, the length of double stranded DNA between junctions was 40 bases (i.e. 40=2×18+4). We then synthesized a larger X-DNA with 160 base pairs, X-DNA-160, whose arms consisted of exactly 40 base pairs. The melting temperature of the X-DNA-72 gel was then measured to be 74.5 °C, found by taking the derivative of the melting transition (inset in Figure 3 B). As predicted, the melting temperature of the larger X-DNA-160 was almost identical to that of X-DNA-72 gel (74.5 °C). In addition to melting temperature, we also compared the complete denaturation temperatures between subunits and their corresponding bulk structures (Figure 3 B). As expected, they behaved similarly: the denaturing temperature of X-DNA-160 was 77.3 °C, whereas the X-DNA-72 gel was 77.6 °C. These data demonstrate that the thermal stability of more complex structures built from simple branched junctions can be predicted from individual subunits with regard to both end- and junction-destabilizations as well as the length between junctions.

Figure 3.

Demonstration of stability tunability. a) As shown in the animation (Supporting Information, Movie 1), X-DNA-72 are ligated to form DNA hydrogel. The DNA gel possesses the form of X-DNA-160 in between four junctions. b) A comparison of melting transition curves of DNA hydrogel (▴) and individual X-DNA-160 (▪) reveals a similar denaturation transition. The melting temperatures are determined from the derivative curves of DNA hydrogel and X-DNA-160 (inset). Fluorescence image of DNA hydrogel before heating showed relatively weak fluorescence due to the high efficiency of FRET (left inset). However, the entire solution shows bright fluorescence after complete thermal denaturation of the entire structure at about 77.6 °C (right inset).

In summary, using FRET and NSET methods we have systematically monitored thermal behavior of branched DNA subunits and their corresponding bulk structures, revealing the effects of ends, junctions, and length on structural stability. We revealed that junctions destabilized DNA nanostructures in a fashion similar to the ends. In addition, with FRET we have successfully determined the 3D structure of Y-DNA with nanometer resolution. Furthermore, we demonstrated that the thermal behavior of bulk and more complicated DNA structures could be predicted from that of single subunits. By construction of a relatively simple library of subunits with known stabilities, we will be able to rationally design and precisely control the properties of future DNA materials.

Experimental Section

DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). The Alexa 488- and DABCYL-modified DNA oligonucleotides were purchased with high-pressure liquid chromatography (HPLC) purification; the remaining oligonucleotides were purchased with polyacrylamide gel electrophoresis (PAGE) purification. Monomaleimido-modified 1.4 nm gold nanoparticles (AuNPs) were purchased from Nanoprobes (Yaphank, NY). Y- and X-shaped DNA were synthesized according to previously published methods from our group.8b, 9a

In order to conjugate thiol-modified DNA nanostructures with 1.4 nm AuNPs, thiol-modified oligonucleotide strands were first deprotected with dithiothreitol (DTT) according to literature procedures.11 The deprotected strands were then assembled with Alexa 488-tagged strands to form NSET pair-tagged Y- and X-shaped DNA. Monomaleimido-modified 1.4 nm AuNPs were incubated with the deprotected thiol-modified Y- or X-shaped DNA overnight at 4 °C. DL-DNA was assembled from four Y-shaped DNA with complementary sticky ends using T4 DNA ligase (Promega, Madison, WI) according to literature procedures.8b

The positions of FRET pairs on the DL-DNA were determined by controlling sticky ends and the position of FRET pairs on Y-DNA-39. DL-DNA was separately labeled with a FRET pair at one of four different positions: end (E), outer junction (OJ), inner junction (IJ), and the middle (M). Similar to DL-DNA, DNA hydrogel was enzymatically assembled from X-DNA-72 by T4 ligation of four palindromic sticky ends.9a A single FRET pair was conjugated at the junction of each X-shaped DNA. Thermal denaturation transitions were then monitored to evaluate the stability of the DNA nanoarchitectures using a SLM model 8000 fluorescence spectrometer (SLM Instruments, Inc.). Each FRET or NSET pair-conjugated DNA nanostructure was denatured by heating at a rate of 0.5 °C/30 seconds. For Alexa 488, an excitation wavelength of 488 nm and an emission wavelength of 530 nm were used.

Acknowledgements

This work was partially supported by the USDA NRI and the NSF CAREER award, and performed in part at the Nanobiotechnology Center and Cornell Center for Materials Research, which are supported by the National Science Foundation, Cornell University, and industrial affiliates.