Sonochemical Synthesis of DNA Nanospheres

Abstract

Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. DNA nanotechnology is a subfield of nanotechnology that seeks to use the unique molecular-recognition properties of DNA and other nucleic acids to create novel, controllable structures of DNA. Chemically, DNA consists of two long polymers. DNA is normally a linear molecule, in that its axis is unbranched. Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. The influence of ultrasonic waves on native DNA molecules has been previously reported. In those studies, it was shown that 2 min of ultrasonication of an aqueous solution of DNA splits the DNA helix into fragments; this makes ultrasonication a useful and convenient tool for obtaining DNA fragments on a preparative scale. Here, we show, for the first time, that ultrasonic waves can be used to convert native DNA molecules to extremely stable DNA nanoparticles (DNA nanospheres, DNs). In addition, the genetic information that was encoded in the DNA nanospheres was successfully delivered to competent cells and to human U2OS cancer cells, and expressed in competent (E. coli) cells. Our fundamental research on the synthesis and characterization of sonochemically produced DNA nanospheres provides an estimate of the efficiency of the sonochemical process in converting the native DNA molecules to biologically active DNA nanospheres. Ultrasonic emulsification is a well-known process that occurs in biphasic systems. Emulsification is necessary for microcapsule formation. Micrometer-sized gasor liquid-filled micro/ nanospheres can be produced from various kinds of proteins such as bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb), and from combination of proteins. The mechanism of the sonochemical formation of protein microspheres (PMs) has been discussed previously. The microspheres are formed by chemically crosslinking cysteine residues, which undergo oxidation by HO2 radicals formed around a micron-sized gas bubble or a nonaqueous droplet. The formation of S!S bonds is a direct result of the chemical effects of the ultrasound radiation on an aqueous medium. Our reaction involved the sonication of an aqueous solution of DNA and dodecane (or soya oil) in a 50 mL sonication cell for three minutes. Five kinds of DNA were used in this work: 1) genomic DNA extracted from cells, 2) genomic DNA extracted from leaves, 3) DNA plasmid, 4) linear DNA extracted from DNA plasmid, and 5) single-stranded DNA. For all five DNAs and for both organic solvents we obtained DNA nanospheres. No difference was found between DNA nanospheres filled with dodecane and those filled with soya oil. We further demonstrate that the denaturing conditions as well as denaturing agents, which are commonly used in DNA isolation, cannot destroy the dsDNA (double-stranded DNA) nanospheres of the four DNA’s, while the nanospheres obtained from ssDNA could disintegrate to re-form the individual starting molecules. The efficiency of the sonochemical method in converting native DNA to DNA nanospheres was analyzed by spectrophotometry (NanoDrop 1000 spectrophotometer). It was found that 73.6% of DNA was converted into nanospheres under air and 96% under argon. It is worth underlining that unlike PMs, which are formed only under air and not under argon, DNA nanospheres are formed under both. In order to be sure that the nanobubbles in the solution after sonication are DNA nanospheres and not a combination of fragmented DNA, EtBr dye solution (commonly used for DNA and RNA detection) was added to the product solution. Nanospheres were colored red; this means that the walls of the nanobubbles consisted of DNA molecules (see the Supporting Information). The morphology of the nanospheres in the solution was determined by using scanning environmental-electron microscopy (E-SEM) and light microscopy (Apo-Tome Zeiss1 microscope). For all five types of DNA, the same spherical structure was observed. In Figure 1A, an E-SEM image of the DNA nanospheres that were produced from DNA type I is presented. The spherical morphology of the nanospheres made of DNA is very similar to that of proteinaceous microspheres, but the diameter of the DNA nanospheres (280 nm) was much smaller than the 2500 nm seen for PMs. A large number of dsDNA (DNA type I) are presented in Figure 1B. The size distribution of three kinds of DN (from DNA types I, III, and V) was examined on a DLS apparatus. When a solution of native DNA molecules was sonicated, the DLS results yielded spheres with an average size varying from 290 to 486 nm. A table summarizing average sizes and electrical charges of DNA nanospheres versus DNA type is presented in the Supporting Information. The DNA nanospheres were found to have an electrical charge of !40.7 mV. The electrical charge (z-potential) of [a] Dr. U. Shimanovich, I. Vayman, Prof. Dr. A. Gedanken Department of Chemistry and Kanbar Laboratory for Nanomaterials Bar-Ilan University Center for Advanced Materials and Nanotechnology Bar-Ilan University Ramat-Gan 52900 (Israel) E-mail : gedanken@mail.biu.ac.il [b] D. Eliaz, A. Aizer, Prof. Dr. S. Michaeli, Dr. Y. Shav-Tal Mina and Everard Goodman Faculty of Life Sciences Bar-Ilan University Center for Advanced Materials and Nanotechnology Bar-Ilan University Ramat-Gan, 52900 (Israel) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201100009: Experimental preparation of DNs and the details of the instrumentation used in the current experiment are available in this section.