DNA surface hybridization regimes

Abstract

Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.biosensor ͉ ferrocene ͉ ionic strength ͉ microarray ͉ probe coverage S olid-phase or ''surface'' hybridization is the foundation of modern microarray and biosensing technologies widely used in applied genomics for genotyping, drug discovery, gene expression profiling, and related applications based on measurement of genomic information (1, 2). These tools function through detection of interactions between nucleic acids immobilized on a solid support, or ''probes,'' with analyte ''target'' nucleic acids present in solution. Binding, or hybridization, between probes and targets to form an immobilized duplex takes place based on the degree of complementarity between the probe and target base sequences. The tremendous growth in applications of surface hybridization has been mirrorred by increased emphasis on experimental and fundamental aspects of the assays that directly impact measurement and interpretation of data (3, 4).As summarized in several recent reviews (5-8), physical studies under simplified experimental conditions have begun to unravel the rich phenomenology that occurs in diagnostic assays. Applications typically operate away from equilibrium and are faced with a highly diverse pool of target sequences competing for the probe sites. A variety of aids to enhance hybridization performance is used, including surfactants and blocking agents to control nonspecific adsorp...

Scaffolded biosensors with designed DNA nanostructures

“…33 The DNA probe density is a controlling factor for the efficiency of the target capture, as well as for the kinetics of the target/probe hybridization. 14, 15 Gong and Levicky 34 studied the hybridization behavior as a function of the ionic strength (C B ) and probe coverage (S P ) in the form of a diagram of hybridization regimens 34 ( Figure 3). They predicted that DNA hybridization is most favorable in the 'Langmuir' (L) regimen, which exists in the limits of sparse films where probes are so far apart that they do not interact.…”

Entropy-driven collective interactions in DNA brushes on a biochip

“…In the past two decades, DNA brushes have become useful in a range of applications such as next-generation sequencing (8,9), hybridization arrays (10)(11)(12)(13)(14), protein biosynthesis compartments (15)(16)(17)(18), and coated particle assembly (19,20). The utility of the diverse DNA-based reactions carried out in such brushes requires an in-depth understanding of their basic materials properties.…”

Nucleic acid electrochemical and electromechanical biosensors: a review of techniques and developments

“…(Peterson et al 2001, Gong and Levicky 2008, Springer et al 2010. The general strategy for detection of target nucleic acids with biosensors is nearly always the same.…”

“…When Π > 2.2, the osmotic pressure from the high surface concentration of cations relative to the bulk inhibits hybridization. This figure was reproduced with permission from Gong and Levicky (2008).…”

“…Cations from solution balance the phosphate charges, and if this local cation concentration (C c,s ) is much more than the bulk concentration (C c,b ), a strong thermodynamic barrier inhibits binding of target ssDNA or ssRNA. More cations would move to surface to balance the negative charges, acting against the concentration gradient (Gong and Levicky 2008). Hence, we define a parameter, Π, given in equation (1) for classifying hybridizing conditions into three regimes: pseudo-Langmuir, suppressed hybridization, and nonhybridizing.…”