Preliminary Studies On Fabrication Of Single-Molecule DNA Chip

Conventional single-molecule DNA micro-/nano-arrays for optical detection systemsare mainly fabricated through random deposition of DNA molecules on a surface. Unevendistribution of individual molecules on certain areas of the surface through these methodscan lead to either overlapping or too-far-away spacing between DNA molecules. Theformer issue will exceed the resolution limit of a microscope, and the latter insufficientloading of DNA molecules. Both of the cases will prohibit maximization of signalcollection in a limited area of the surface. To circumvent one of the problemsmentionedabove, overlapping between DNA molecules, we have developed a new method in whichnanobeads are used as single DNA carrier and spacer for DNA delivery onto achemically-modified surface. First,3’-biotinylated single-standed DNA(ssDNA) ishybridized with the complementary5’ overhang of a hairpin DNA with an amine group inits loop, followed by the biotinylated end of the DNA hybrid bound ontostreptavidin-coated nanobeads in a pre-set high beads to DNA ratio in order to obtainsparse DNA molecule on each bead. This will assure that, when complex is loaded ontothe surface modified with succinimidyl groups, only one or no amine group in the DNA loop will be able to be bound with the succinimidyl groups on the surfacebecause ofsparse distribution of DNA molecules on the bead surface, short DNA length,and specialoccupation of beads with large diameter compared with DNA length. After binding ofamine group in the DNA loop with succinimidyl group on the glass surface,beads withbiotinylated ssDNA were washed away with basic solution, leaving single-moleculehairpin DNA on the surface. Single-molecule DNA chip with pixel pitch of no less thanabout700nm has been fabricated when using1bead to6DNA ratio,940nmof beadsdiameter, and around15nm DNA length. Experimental results show that about4%of itshighest theoretical density of DNA molecules can be achieved after ten times ofdeposition as indicated by microarray image of confocal microscopy. The new DNAnan oarray fabrication technology paves the way for further researches at the singlemolecule.In another approach to manipulate single DNA molecules, streptavidin is substitutedfor nanobead. Streptavidin tetramer, which can respectively bind with0,1,2,3or4biotin-modified DNA molecules to form a mixture depending upon their molar ratio andreaction dynamics, in combination with electrophoresis, has led to the conception that it ispossible for one to separate the conjugate of one tetramer combined with only one DNAmolecule from the mixture. Through the adjustment of the length of DNA andelectrophoresis of the complex, a method has been basically established to obtainone-bead-one-DNA complex, providing a novel way for manipulation of single-moleculeDNA, and even other biomolecules, such RNA and protein.