Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer.

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.

[1]  J Wang,et al.  Mismatch-sensitive hybridization detection by peptide nucleic acids immobilized on a quartz crystal microbalance. , 1997, Analytical chemistry.

[2]  Y. Rogers,et al.  Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms. , 1994, Nucleic acids research.

[3]  G. Ramsay.  DNA chips: State-of-the art , 1998, Nature Biotechnology.

[4]  R. Karlsson,et al.  Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. , 1991, BioTechniques.

[5]  R. Pecora,et al.  Effect of electrostatic interactions on the structure and dynamics of a model polyelectrolyte. I. Diffusion , 1998 .

[6]  C M Niemeyer,et al.  Hybridization characteristics of biomolecular adaptors, covalent DNA--streptavidin conjugates. , 1998, Bioconjugate chemistry.

[7]  M. Thompson,et al.  Hybridization of complementary strand and single-base mutated oligonucleotides detected with an on-line acoustic wave sensor. , 1998, The Analyst.

[8]  K U Mir,et al.  Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids. , 1994, Nucleic acids research.

[9]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[10]  J. Remacle,et al.  Comparison between microwell and bead supports for the detection of human cytomegalovirus amplicons by sandwich hybridization. , 1997, Analytical biochemistry.

[11]  N. Thompson,et al.  Surface binding kinetics of prothrombin fragment 1 on planar membranes measured by total internal reflection fluorescence microscopy. , 1992, Biochemistry.

[12]  Richard G. Griskey.  Chemical engineering for chemists , 1979 .

[13]  R. Weiss,et al.  Binding of biotinylated DNA to streptavidin-coated polystyrene latex: effects of chain length and particle size. , 1996, Analytical biochemistry.

[14]  A. Thiel,et al.  Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. , 1994, Nucleic acids research.

[15]  C. Urbaniczky,et al.  Integrated fluid handling system for biomolecular interaction analysis. , 1991, Analytical chemistry.

[16]  L. Kricka.  Nonisotopic DNA probe techniques , 1992 .

[17]  H. Hakala,et al.  Time-resolved fluorescence detection of oligonucleotide hybridization on a single microparticle: covalent immobilization of oligonucleotides and quantitation of a model system. , 1997, Bioconjugate chemistry.

[18]  R W Glaser,et al.  Antigen-antibody binding and mass transport by convection and diffusion to a surface: a two-dimensional computer model of binding and dissociation kinetics. , 1993, Analytical biochemistry.

[19]  G. López,et al.  Ligand Receptor Dynamics at Streptavidin-Coated Particle Surfaces: A Flow Cytometric and Spectrofluorimetric Study , 1999 .

[20]  H. Nygren,et al.  Kinetics of antigen-antibody reactions at solid-liquid interfaces. , 1988, Journal of immunological methods.

[21]  B. Nordén,et al.  Hybridization of peptide nucleic acid. , 1998, Biochemistry.

[22]  K. M. Parkhurst,et al.  Kinetic studies by fluorescence resonance energy transfer employing a double-labeled oligonucleotide: hybridization to the oligonucleotide complement and to single-stranded DNA. , 1995, Biochemistry.

[23]  Chad A. Mirkin,et al.  One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes , 1998 .

[24]  John Crank,et al.  The Mathematics Of Diffusion , 1956 .

[25]  H. Gruber,et al.  Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin. , 1997, Bioconjugate chemistry.

[26]  Graham Ramsay,et al.  Commercial biosensors : applications to clinical, bioprocess, and environmental samples , 1998 .

[27]  D. Walt,et al.  A fiber-optic DNA biosensor microarray for the analysis of gene expression , 1996, Nature Biotechnology.

[28]  R J Leatherbarrow,et al.  Kinetics of protein-protein interactions at the surface of an optical biosensor. , 1995, Analytical biochemistry.

[29]  S C Huang,et al.  Binding of biotinylated DNA to streptavidin-coated polystyrene latex. , 1994, Analytical biochemistry.

[30]  M. Walker,et al.  Immobilization of polynucleotides on magnetic particles. Factors influencing hybridization efficiency. , 1991, The Biochemical journal.

[31]  J Kolberg,et al.  A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. , 1997, Nucleic acids research.

[32]  J. Dougherty,et al.  Rapid hybridization kinetics of DNA attached to submicron latex particles. , 1987, Nucleic acids research.

[33]  I. Brooks,et al.  Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods. , 1993, Analytical biochemistry.

[34]  D. Stimpson,et al.  Parallel production of oligonucleotide arrays using membranes and reagent jet printing. , 1998, BioTechniques.

[35]  D. Kelso,et al.  DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants. , 1999, Nucleic acids research.

[36]  J. Covino,et al.  Comparison of the Syva MicroTrak enzyme immunoassay and Gen-Probe PACE 2 with cell culture for diagnosis of cervical Chlamydia trachomatis infection in a high-prevalence female population , 1993, Journal of clinical microbiology.

[37]  B. Valeur,et al.  Fluorescence energy transfer as a probe for nucleic acid structures and sequences. , 1994, Nucleic acids research.

[38]  J. Storhoff,et al.  Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. , 1997, Science.

[39]  G. Hausdorf,et al.  Binding kinetics of an antibody against HIV p24 core protein measured with real-time biomolecular interaction analysis suggest a slow conformational change in antigen p24. , 1996, Journal of immunological methods.

[40]  L. Bachas,et al.  Enhancement of the emission intensity of fluorophore-labeled avidin by biotin and biotin derivatives. Evaluation of different fluorophores for improved sensitivity. , 1993, Talanta.

[41]  E. Southern,et al.  Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. , 1997, Nucleic acids research.

[42]  L. Stols,et al.  Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. , 1989, Analytical biochemistry.

[43]  D. Graves,et al.  The biophysics of DNA hybridization with immobilized oligonucleotide probes. , 1995, Biophysical journal.

[44]  P. Edwards,et al.  Determination of association rate constants by an optical biosensor using initial rate analysis. , 1997, Analytical biochemistry.

[45]  F. Crick,et al.  Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid , 1953, Nature.

[46]  Mikael Kubista,et al.  Experimental correction for the inner-filter effect in fluorescence spectra , 1994 .

[47]  H. Nygren,et al.  Calibration by ellipsometry of the enzyme-linked immunosorbent assay. , 1985, Journal of immunological methods.

[48]  B. Nordén,et al.  Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique. , 1997, Biochemistry.

[49]  P. Schuck,et al.  Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. , 1997, Annual review of biophysics and biomolecular structure.

[50]  T. R. Gingeras,et al.  Hybridization properties of immobilized nucleic acids , 1987, Nucleic Acids Res..

[51]  A. Mirzabekov,et al.  Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips. , 1998, Nucleic acids research.

[52]  Running Ja,et al.  A procedure for productive coupling of synthetic oligonucleotides to polystyrene microtiter wells for hybridization capture. , 1990 .

[53]  M Ronaghi,et al.  Detection of single-base changes using a bioluminometric primer extension assay. , 1997, Analytical biochemistry.