Tailoring DNA structure to increase target hybridization kinetics on surfaces

Abstract

We report a method for increasing the rate of target hybridization on DNA-functionalized surfaces using a short internal complement DNA (sicDNA) strand. The sicDNA causes up to a 5-fold increase in association rate by inducing a conformational change that extends the DNA away from the surface, making it more available to bind target nucleic acids. The sicDNA-induced kinetic enhancement is a general phenomenon that occurred with all sequences and surfaces investigated. Additionally, the process is selective and can be used in multicomponent systems to controllably and orthogonally "turn on" specific sequences by the addition of the appropriate sicDNA. Finally, we show that sicDNA is compatible with systems used in gene regulation, intracellular detection, and microarrays, suggesting several potential therapeutic, diagnostic, and bioinformatic applications.

Figure 1

sicDNA increases the rate of association of DNA–Au NPs to target DNA strands. (a) Scheme depicting the fluorescence-based measurement of a DNA–Au NP binding a target. The corresponding sequences are shown below. (b) Hybridization in the presence of different complements (ssDNA, sicDNA, secDNA, licDNA, and fcDNA). (c) Rate of binding of DNA–Au NPs to targets in the presence of increasing concentrations of sicDNA. (d) Comparison of ssDNA and sicDNA target binding in the absence of the NP. Inset: scheme of the experiment using a molecular quencher. Each plot represents the average of three independent experiments.

Figure 2

Effect of sicDNA on the bound strand and adjacent ssDNA sites. (a) Scheme of a nanoparticle containing a mixed monolayer of DNA. The different sequences can be orthogonally addressed by the corresponding sicDNA and target. This experiment was performed in the presence of both target-1 and target-2, which were distinguished by different fluorophore labels. (b) Plot of target-1 binding to DNA–Au NPs in the presence of sicDNA-1 and/or sicDNA-2. (c) Plot of target-2 binding to DNA–Au NPs in the presence of sicDNA-1 or sicDNA-2. Each plot represents the average of three independent experiments.

Figure 3

DNA conformation on the Au NP surface as a function of sicDNA concentration. (a) DLS measurements of the nanoparticle radii at different sicDNA concentrations. (b) Fluorescence spectra from DNA–Au NPs containing a distal fluorophore label. These spectra were taken before and after the addition of sicDNA. Each plot represents the average of three independent experiments. Each error bar represents the standard deviation of the three experiments.

Figure 4

MD simulation snapshots of ssDNA and sicDNA on flat gold surfaces. Seven strands were modeled on each surface. (a) ssDNA is shown with the final nine residues highlighted in light blue. (b) sicDNA is shown with the final nine residues highlighted in dark blue. (c) Normalized distribution of the distance (z) of the last residue of ssDNA (black) and sicDNA (red) from the surface. The average of z was 10.6 ± 0.9 nm for ssDNA and 11.8 ± 1.0 nm for sicDNA.

Figure 5

sicDNA increases the rate of target association on microarrays. (a) Scheme depicting the fluorescence-based detection of target binding to the microarray surface. (b) Fluorescence confocal microscopy images of representative spots after exposure to the labeled target. The reaction was stopped at different time points by washing away unbound target. (c) Quantification of the fluorescence experiments shown in (b). The initial rate of target association was determined by a linear fit of the data. Each error bar represents the standard deviation of four independent experiments.