通过QCM-D检测磷脂双分子层与特异性DNA杂交

APPLICATIONNOTEQS 405-04-1 www.q-sense.com SPECIFICITY OF DNA HYBRIDISATIONON A FUNCTIONALISEDLIPID BILAYER INTRODUCTION We have investigated the possi-bility of using the QCM-D tech-nique and surface immobilizedsynthetic peptide nucleic acid(PNA) and DNA， as selectiveprobes for DNA (Hook F et al.).Streptavidin was immobilized ona fluid biotin-containing phos-pholipid bilayer supported on aSiO2 surface in order to allowoligonucleotide attachment andminimize unspecific binding dur-ing subsequent DNA hybridisa-tion. RESULTS The initial rapid increase in Dfollowed by a slower decreaseduring the continuous binding(indicated by a decrease in f) ofstreptavidin indicates transfor-LIalimation from a non-rigid to a rigidlayer of attached proteins (Fig-ure 1). This is interpreted as adirect measure of protein-2D-crystal growthi，， as also sup-ported by， e.g.， atomic forcemicroscopy images of similarprotein-lipid systems [Reviakine，l.， et al. J. Structural Biol， vol.121 p.356 (1998)1. Mixed-sequence 15-mer biotin-PNAand DNA，， respectively，with identicalbase-pair se-quences， were immobilised ontop of the streptavidin layer. Thesamples weressubsequentlyexposed to 1 mM of comple-mentary DNA at 24℃ (see Fig-ure 2 below). The binding rate of complemen-tary DNA was different to theimmobilised PNAand DNAstrands， respectively， indicating a difference in the associationrate at 24°℃(not shown). Also various mismatched DNAwere investigated. Only the fullycomplementary and singly mis-matched DNA oligomers hybrid-ized with the immobilised PNAand DNA， demonstrating a highselectivity with no influence fromunspecific binding. In the caseof single mismatch the bindingwas weaker and reversiblecompared to the fully comple-mentary strand， which can beseen in Figure 2. It should be noted that the largerratio between AD and Af (i.e.dissipation per bound mass) forthe PNA-DNA compared to theDNA-DNA hybrids (see the ADvs Af plot， Figure 3) indicatesthat the PNA-DNA hybrid has amore flexibleand elongatedstructure.，whichnprovesttheQCM-D technique capable of detecting structural differencesbetween various oligonucleotidestrands. CONCLUSIONS By using an inert backgroundconsisting of functionalised lipidbilayer， QCM-D shows remark-able specificity valuable for bio-molecular interaction research.During DNAhybridisation，， casingle mismatch inaa15-merstrand results in less adsorptionand reversible instead of irre-versible binding， compared tothe fully complementary strand. REFERENCES Characterisation of PNAcandDNA immobilisation and subse-quent ihybridisationwithDNAusing acoustic-shear-wave aat-fartenuation measurements. Langmuir 2001，17， p.8305-8312 Hook， F， Ray A， Krave U， Nor-den， B and Kasemo， B Figure 1 - Formation of a biotinylated lipidbilayer followed by immobilization of a strepta-vidin protein layer. Af (Hz) Figure 3 - Structural differences betweenhybridized single strands of PNA-DNA andDNA-DNA. Figure 2 - Binding of single stranded bioti-nylated PNA to Streptavidin followed by spe-cific hybridization of complementary and sin-gle mismatch DNA strands， respectively. ( H eadquarters： Q-Sense AB， R e degatan 13 ， S E - 4 26 7 7 V a stra F r ol u nda，Sweden， P h o n e +46 31 7 6 9 769 0，F a x +4 6 31 6 9 80 4 0 ， info @ q-sen s e.com North America： Q-Sense I n c， + 1 (949) 250 0273， sales@q - se n se . com S candinavia： Q - S en s e A B， + 46 31 769 769 0， s ales@q- s en s e.com U K/IE： Scientific & Medical Products Ltd， +44 1 6 1 491 3068， scimed@dircon.co.u k Germany/CH/AT： LOT-Oriel G m bH，+49 6151/8 8 06- 4 4， q sense@lot - oriel.de France： LOT - Ori e l FR， + 3 3 1 691 9 494 9 ， t c herbak@lot-oriel.fr I t aly： LOT-Oriel IT， +3 9 2 2 6 822104， doriano@lot-o r iel.it Benelux： LOT-O r iel Bene l ux， + 31 1 0 2859 511， bene l ux@lot- o r i el . com Japan： Meiwafosis Co Lt d ， + 81 3 5379 0051，i n uzuka@meiwanet.co. j p ) Korea： DI Biotech Ltd， +82 2 511 4811， inform@dibiotech.com Australia： ATA Scientific Ltd， +61 295430477， enquiries@atascientific.com.au