(84b) Sequence Selective Separation of DNA by Nanostructured Molecular Assembly

The recent great progress in the technologies of RNAi and nucleic acid aptamers suggests a necessity for the sequence-specific purification of nucleic acids. To date, a number of techniques have been proposed for the sequence-specific purification of short-stranded DNA and RNA. However, most of them are laboratory-scale and not appropriate for mass production, and they employ mainly poly(dT) as an affinity probe to recognize poly(A) of RNA. We propose the sequence-specific solvent extraction of DNA oligonucleotides using reverse micelles (water-in-oil microemulsions) with a DNA-surfactant. A reverse micelle, which is a nanoscale water pool surrounded by surfactant molecules in an organic solvent, is one of the most useful tools for extraction of water-soluble compounds from an aqueous solution into an organic solvent1-3). The advantages of reverse-micellar solvent extraction are i) the capability to scale-up the process, ii) the inhibition of microbial contamination in an organic solvent, thus avoiding biological decomposition of oligonucleotides, and iii) the inhibition of contamination with water-soluble biomacromolecules, due to the isolated and restricted nano-space of a reverse micelle. In this study we introduce the DNA-surfactant, which is composed of short- and single-stranded DNA with a hydrophobic moiety, as a molecular recognition agent in reverse-micellar solvent extraction4). Using this, we succeeded in the sequence-selective extraction of single-stranded DNA oligonucleotides from an aqueous phase to an organic solvent, driven by DNA hybridization5). A DNA-surfactant is short, single-stranded DNA conjugated with a hydrophobic moiety. Hybridization between a DNA-surfactant and a target DNA having a complementary sequence allows selective transport of the target DNA to an organic phase, from a mixture of DNA oligonucleotides. The extracted DNA is encapsulated in a water-pool of the reverse micelle, which is self-assembled in an organic solvent6). The present study reveals that precise molecular recognition at a liquid-liquid interface can be utilized as a technique for separation of biomolecules5). We investigated the sequence-selective extraction of a target DNA from a mixture of different oligonucleotides. DNA-surfactant 1, 2 or 3 was added to an aqueous phase containing Targets 1a, 2 and 3, followed by addition of an organic phase containing DLPC and 1-hexanol (Fig. 2a). Using DNA-surfactant 1, over 50% of Target 1a, which was complementary to DNA-surfactant 1, was extracted to the organic phase, while Targets 2 and 3 were scarcely extracted (less than 3%). Likewise, the use of DNA-surfactants 2 or 3 instead of DNA-surfactant 1 facilitated selective extraction of the target oligonucleotides (Targets 2 and 3) complementary to each DNA-surfactant. These results revealed that the present system affords sequence-selective extraction of DNA oligonucleotides from a mixture of oligonucleotides, to an organic phase. We also employed hairpin DNA conjugated to an oleoyl group as a DNA-surfactant. Hairpin DNA precisely recognizes its perfect complement, and not an oligonucleotide having a single-base mismatch. The DNA-surfactant having hairpin DNA (loop 16-mer, DNA-surfactant 4) was synthesized and applied to the liquid/liquid extraction of oligonucleotides (16-mer). The extraction of each target oligonucleotide was carried out independently (Fig. 2b). The DNA-surfactant 4 recognized the perfect complement (Target 4a) and over 60% of Target 4a was extracted to an organic phase, while the percent extractions of Target 4b (a single-base mismatch) and Target 4c (three-base mismatches) were only 6% and 2%, respectively.

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