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智能文字提取功能测试中
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
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