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The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins.
During various biological processes, such as transcription, replication, and packaging, DNA undergoes substantial structural changes. Conformational changes of the DNA duplex have been identified in these processes and are well-known [1, 2, 3]. At the same time, these processes are accompanied by the transient formation of single-stranded DNA (ssDNA) segments, which coexist with DNA duplex conformations [4, 5]. The mechanical properties of both types of DNA are dramatically different. A DNA duplex is a very stiff polymer with a persistence length as large as 150 bp [6], whereas ssDNA is much softer with a persistence length as small as 2–5 residues [7, 8].
DNA transcription, replication, repair, and recombination are accomplished by specialized protein machines which produce variations of the DNA duplex structure, such as twisting and bending. Several enzyme families (topoisomerases, gyrases, and helicases) participate in the generation and relaxation of torsional mechanical stress on DNA [9, 10]. However, mechanical stress, due to the large persistence length of the DNA duplex, covers large DNA segments. Neighboring soft ssDNA segments can absorb mechanical stress, decreasing the tensions within the DNA duplex. The importance of mechanical stress is clear; DNA polymerases have been shown to display elevated activity at low force [11], while the binding of single-stranded binding proteins such as RPA display force-dependent activity [12]. Moreover, CRISPR/Cas9 off-target activity is increased due to DNA stretching [13]. This interplay in the mechanical properties of ssDNA and the duplex, however, has not been well studied.
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Here, we describe an approach that allowed us to demonstrate how the high stiffness of the DNA duplex induces a mechanical strain on the ssDNA segment. In our approach, a DNA duplex with the size comparable to the persistence length is linked by a ssDNA with 30 residues and connected into a circle, which we refer to as a nanoring. We used atomic force microscopy (AFM) to visualize such hybrid DNA constructs and characterize their structural features. AFM is a powerful single-molecule technique capable of providing conformational details of individual biomolecules with nanometer resolution, as demonstrated by us (e.g., [14] or [15]) and others (reviewed in [16]). Here, we show that tension within the duplex stretches the ssDNA connector, so its end-to-end distance is increased by 16% on average. Time-lapse AFM imaging revealed broad dynamics of the overall shape of such DNA nanorings, capable of significantly extended end-to-end distances, working as unfolding nanotweezers. Theoretical estimates of ssDNA stretching, within the nanoring construct, corroborate the stretching observed in experiments.
The nanorings were obtained using a multistep process, schematically illustrated in Figure 1. In step one, 190 nt ssDNA was ligated into a ring using a template strand complementary to the ends of the DNA molecule. In step two, the ring was annealed to a 160 nt DNA complement, resulting in the formation of a hybrid DNA circle with 160 bp dsDNA segment connected through a 30 nt ssDNA connector. The final products were analyzed by AFM.
DNA nanoring samples were deposited on mica functionalized with 1-(3-aminopropyl)- silatrane (APS) [17], incubated for two minutes, dried with a gentle flow of argon, and imaged in air as described in the Methods section. Representative images of these assemblies can be seen in Figure 2A. DNA nanorings appear as circular “c”-shaped molecules in the images. DNA duplex appeared with a high contrast, while ssDNA segment does not show such a high contrast, which is typical for AFM images of ssDNA. A few zoomed-in images of the nanorings are shown in Figure 2A(i, ii). The yield of fully intact DNA nanorings, assessed by the circularity of the molecules, was 82%.
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As a control, we assembled linear DNA constructs, with identical sequence, in which 80 bp DNA duplexes are flanking the same 30 nt ssDNA molecule; we named this construct gap DNA. AFM images of these constructs are shown in Figure 2B. Fully intact gap DNA constructs, possessing both the double-stranded and single-stranded DNA regions, is the dominant species of nanostructures, as seen in Figure 2B. ssDNA segments are clearly seen in the zoomed-in images shown in Figure 2B(i, ii). Gap DNA nanostructures take on a variety of conformations, from those with almost no discernible ssDNA to those with extended ssDNA flanked by duplex DNA oriented at different angles.
To improve the accuracy of the DNA length measurements and eliminate potential effect of the sample drying process, we imaged nanorings and gap DNA samples in aqueous solution. Representative images of the DNA nanoring construct and gap DNA construct in aqueous buffer are shown in Figure 3. The blue lines in the traces below the zoomed-in images represent the contours of the dsDNA, while the red lines represent the end-to-end (ETE) distance of the ssDNA. Note that, in the images, we are not able to visualize the path of single-stranded DNA regions primarily due to the low height of ssDNA and its high flexibility.
Quantitative analysis of the ETE distance for the nanorings was performed. Figure 4 (bottom) shows the histogram of nanoring ETE distances. Similar measurements were completed for gap DNA constructs and a corresponding histogram is shown in Figure 4 (top). The data show that the mean ETE distance of the nanoring constructs (11.1 nm ± 0.2 SEM, n = 265) was 16% larger than the ETE distance of the non-circular gap DNA constructs (9.6 nm ± 0.2 SEM, n = 207), indicating a statistically significant stretching effect for ssDNA (p < 0.001).
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Time-lapse AFM imaging of the DNA nanorings allowed us to track the dynamics of individual particles over time. Selective frames from 35 frames of the time-lapse AFM experiment with three individual nanorings are presented in Figure 5A. The full set of the frames is shown in the supplement (Figure 2). Green, red, and blue arrows in each frame point to selected nanorings. The nanoring indicated by the green arrow is shown to occupy roughly the same position across frames. However, a clear change in orientation happens between frames 16 and 22. The single-stranded region, on the right side of the nanoring in frames6–16, is shown to suddenly face left by either “turning” or “flipping” in frame 22. The nanoring indicated by the blue arrow shows fluctuations in end-to-end dsDNA distance before leaving the frame in frame 17, indicative of the freedom of movement of the DNA on the functionalized surface. The nanoring indicated by the red arrow shows the most interesting dynamics; in frame 6, it is shown in a standard circularized shape. However, in frame 9, the nanoring adopts a transient folded shape, similar in shape to a “3”. This shape rapidly changes to a figure “8”
As a control, we assembled linear DNA constructs, with identical sequence, in which 80 bp DNA duplexes are flanking the same 30 nt ssDNA molecule; we named this construct gap DNA. AFM images of these constructs are shown in Figure 2B. Fully intact gap DNA constructs, possessing both the double-stranded and single-stranded DNA regions, is the dominant species of nanostructures, as seen in Figure 2B. ssDNA segments are clearly seen in the zoomed-in images shown in Figure 2B(i, ii). Gap DNA nanostructures take on a variety of conformations, from those with almost no discernible ssDNA to those with extended ssDNA flanked by duplex DNA oriented at different angles.
To improve the accuracy of the DNA length measurements and eliminate potential effect of the sample drying process, we imaged nanorings and gap DNA samples in aqueous solution. Representative images of the DNA nanoring construct and gap DNA construct in aqueous buffer are shown in Figure 3. The blue lines in the traces below the zoomed-in images represent the contours of the dsDNA, while the red lines represent the end-to-end (ETE) distance of the ssDNA. Note that, in the images, we are not able to visualize the path of single-stranded DNA regions primarily due to the low height of ssDNA and its high flexibility.
Quantitative analysis of the ETE distance for the nanorings was performed. Figure 4 (bottom) shows the histogram of nanoring ETE distances. Similar measurements were completed for gap DNA constructs and a corresponding histogram is shown in Figure 4 (top). The data show that the mean ETE distance of the nanoring constructs (11.1 nm ± 0.2 SEM, n = 265) was 16% larger than the ETE distance of the non-circular gap DNA constructs (9.6 nm ± 0.2 SEM, n = 207), indicating a statistically significant stretching effect for ssDNA (p < 0.001).
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Time-lapse AFM imaging of the DNA nanorings allowed us to track the dynamics of individual particles over time. Selective frames from 35 frames of the time-lapse AFM experiment with three individual nanorings are presented in Figure 5A. The full set of the frames is shown in the supplement (Figure 2). Green, red, and blue arrows in each frame point to selected nanorings. The nanoring indicated by the green arrow is shown to occupy roughly the same position across frames. However, a clear change in orientation happens between frames 16 and 22. The single-stranded region, on the right side of the nanoring in frames6–16, is shown to suddenly face left by either “turning” or “flipping” in frame 22. The nanoring indicated by the blue arrow shows fluctuations in end-to-end dsDNA distance before leaving the frame in frame 17, indicative of the freedom of movement of the DNA on the functionalized surface. The nanoring indicated by the red arrow shows the most interesting dynamics; in frame 6, it is shown in a standard circularized shape. However, in frame 9, the nanoring adopts a transient folded shape, similar in shape to a “3”. This shape rapidly changes to a figure “8”
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