Conformation of ring single-stranded DNA measured by DNA origami structures.

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a complex challenge that has been addressed lately by different methods. We measured the persistence length of ring ssDNA using a combination of a special DNA origami structure, a self-avoiding ring polymer simulation model, and nonparametric estimation statistics. The method overcomes the complexities set forth by previously used methods. We designed the DNA origami nano structures and measured the ring ssDNA polymer conformations using atomic force microscopy. We then calculated their radius of gyration, which was used as a fitting parameter for finding the persistence length. As there is no simple formulation for the radius of gyration distribution, we developed a simulation program consisting of a self-avoiding ring polymer to fit the persistence length to the experimental data. ssDNA naturally forms stem-loops, which should be taken into account in fitting a model to the experimental measurement. To overcome that hurdle, we found the possible loops using minimal energy considerations and used them in our fitting procedure of the persistence length. Due to the statistical nature of the loops formation, we calculated the persistence length for different percentages of loops that are formed. In the range of 25-75% loop formation, we found the persistence length to be 1.9-4.4 nm, and for 50% loop formation we get a persistence length of 2.83 ± 0.63 nm. This estimation narrows the previously known persistence length and provides tools for finding the conformations of ssDNA.

Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure.

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a challenge that has been addressed by different methods lately. The short persistence length, fragile structure, and the appearance of stem loops complicate the measurement, and this leads to a large variability in the measured values. Here we describe an innovative method based on DNA origami for studying the biophysical properties of ssDNA. By synthesizing a DNA origami structure that consists of two rigid rods with an ssDNA segment between them, we developed a method to characterize the effective persistence length of a random-sequence ssDNA while allowing the formation of stem loops. We imaged the structure with an atomic force microscope (AFM); the rigid rods provide a means for the exact identification of the ssDNA ends. This leads to an accurate determination of the end-to-end distance of each ssDNA segment, and by fitting the measured distribution to the ideal chain polymer model we measured an effective persistence length of 1.98 ± 0.72 nm. This method enables one to measure short or long strands of ssDNA, and it can cope with the formation of stem loops that are often formed along ssDNA. We envision that this method can be used for measuring stem loops for determining the effect of repetitive nucleotide sequences and environmental conditions on the mechanical properties of ssDNA and the effect of interacting proteins with ssDNA. We further noted that the method can be extended to nanoprobes for measuring the interactions of specific DNA sequences, because the DNA origami rods (or similar structures) can hold multiple fluorescent probes that can be easily detected.