Studies On Preparation Of Protein-DNA Conjugates Using Movalent Streptavidin

Fabrication of chips carrying patterned high-density single-molecule DNA nanoarray is one of critical steps in fluorescence-based sequencing of single-molecule DNA by synthesis.An ideal chip would be one on which a molecule-to-molecule distance nearly approaches resolution limit of the optical detector in order to maximaze data output per unit area of the chip.In addition to maximization of throuphput,it will substantially reduce cost,which is one of the bottlenecks challenging market share of the existing fluorescence-based sequencing by synthesis(SBS).However,there are great difficulties in fabrication of such chips,prohibiting their application in SBS.Single-molecule DNA chips previously reported were fabricated either by spreading highly-diluted DNA solution onto the surface of a substrate or by using dip pen lithography with the aid of the tip of an atomic force microscop(AFM).Yet,the disadvantage of the former is that the resultant chip has a random distribution of DNA molecules with uncotrllable didtance between adjacent molecules,disenabling maximization of data output.The latter is slow and cannot assure that no more than one DNA molecule is deposited on each sample site although precisely control of distance between adjacent molecules promises patterned high molecule density.Therefore,neither of these methods can meet the demand for high-density single-moecle DNA chip.Another patterned high-density single-molecule DNA chip was fabricated using DNA nanoballs.But each nanoball contains multiple copies of a target DNA fragment due to rolling circle amplification,and is,therefore,not a single molecule in biological sense.Here,we propose a novel method to fabricate high-density single-molecule DNA chip through immobilization of a single DNA molecule on each spot over a patterned nanospot array.Thie method is mainly comprised of two components.The firse is substrate preparation,on which spot nanoarray is patterned through nanofabrication method.On activation,each nanospot of the array will be able to capture the protein side of a protein-DNA conjugate.The second part is to prepare massive amount of single-protein-single-DNA conjugate.Here we plan to use streptavidin(STV)protein and biotinylated double-stranded DNA(biotin-DNA)to obtain 1STV-1DNA conjugate.Then,1STV-1DNA conjugate will be immobilized onto nanospot array.The reason for use of STV as a DNA vehicle is that the minimal size(tens of nanometers)of a nanospot fabricated by current electron beam lighography is comparable to that of a protein,much smaller than DNA diameter.Thus,preparation of massive amount of 1STV-1DNA with high purity conjugate is one of prerequisites to fabricate such a high-density single-molecule DNA chip.Wild type STV was previously used to prepare 1STV-1DNA conjugate in our laboratory.After incubation of STV with biotin-DNA,the product is electrophoretically separated,followed by recovery of the conjugate.This process is time-consuming and not highly efficient.For this reason,we have developed a new method,in which monovalent STV is used to conjugate with biotin-DNA to achieve 1STV-1DNA complex.This procedure includes three parts.The first is preparation of monovalent STV.That is,denatured both of mutant STV with inactivated biotin-binding site and wild type STV is mixed at a ratio of 3:1,refolded,then,the product is run through a nickel-nitrilotriacetic acid(Ni-NTA)column to collect tetramers with a single active biotin-binding site,i.e.mutant monovalent STV.The second part is to prepare biotin-DNA.And the third is to mix monovalent STV and biotin-DNA at different mole ratios.Results from agarose gel electrophoresis and AFM observation shows that this new method is feasible,and more convenient and efficient than the previous one,providing a facile tool not only for fabrication of single-molecule DNA chips,but also for studies of DNA manipulation and DNA-protein interactions at the single molecule level.