DNA packing by low-intensity ultrasound

Abstract

Low-intensity ultrasound (hundreds of mW/cm2) has been used as an effective modality to accelerate the osteogenic differentiation response in fracture healing. Even small biological structures such as chromatin architecture of DNA may be influenced by low-intensity ultrasound. Accordingly, we hypothesized that ultrasound stimulation may cause the topological change of pDNA. In this study, we paid attention to the effect of ultrasound stimulation on topological change of pDNA. Two types of experiment, gel electrophoresis and atomic force microscope (AFM) imaging were designed with 4.7 kbp pDNA. First, the topological change of pDNA applied ultrasound was analyzed by electrophoresis which is physical method for determining the size of pDNA. Second, we utilized AFM imaging of pDNA to confirm and quantify topological change of pDNA. AFM imaging has been broadly employed for single molecule and nanoscale imaging of biological material such as pDNA. The topological change of pDNA by ultrasound sonication was compared with normal pDNA according to experimental conditions. Additionally, AFM image of pDNA was quantitatively analyzed by degree of bending using image processing approaches. The transducer which has 1.12MHz of center frequency and half inch diameter was used in the experiment. An acoustic pressure of 126kPa on the surface with a 1 % and 10 % duty cycle according to experimental conditions. The pulse repetition frequency was 100 Hz. The 5.4mW/cm2 and 53.6 mW/cm2 of intensity was applied as conditions of each experiment. The ultrasound sonication was applied for 30s in gel electrophoresis and for 30, 60, 120, and 300 sec in AFM imaging. We can observe the various types on topological change of pDNA which depended on applied ultrasound conditions through the AFM image. The normal pDNA seems to be relatively relaxable when it is compared to ultrasound stimulation groups. In contrast, ultrasound stimulation groups appear to be folding and packed in all conditions. The mean values of the folding index were 181.3, 262.63, 311.7, 494.1, and 640.7 in control, 30-second, 60-second, 120-second, and 300-second group. The folding index was increased in all sonication groups and the differences of folding index between groups were increased as increases of sonication time. The increased mean values were proportional to increased sonication time. Additionally, we evaluated how long will the topology of packed DNA by ultrasound be kept. After one week, the folding index in control group was not changed significantly when compared with one hour. However, the folding index in 30 sec sonication was significantly reduced after one week. This results mean that the changed topology by ultrasound sonication was returned back to original condition as time passed. This study indicates that low intensity ultrasound can change the DNA topology as compacted formation.