Mechanisms of Surface-Mediated DNA Hybridization

Single-molecule total internal reflection fluorescence microscopy was employed in conjunction with resonance energy transfer (RET) to observe the dynamic behavior of donor-labeled ssDNA at the interface between aqueous solution and a solid surface decorated with complementary acceptor-labeled ssDNA. At least 100 000 molecular trajectories were determined for both complementary strands and negative control ssDNA. RET was used to identify trajectory segments corresponding to the hybridized state. The vast majority of molecules from solution adsorbed nonspecifically to the surface, where a brief two-dimensional search was performed with a 7% chance of hybridization. Successful hybridization events occurred with a characteristic search time of ∼0.1 s, and unsuccessful searches resulted in desorption from the surface, ultimately repeating the adsorption and search process. Hybridization was reversible, and two distinct modes of melting (i.e., dehybridization) were observed, corresponding to long-lived (∼15 s) and short-lived (∼1.4 s) hybridized time intervals. A strand that melted back onto the surface could rehybridize after a brief search or desorb from the interface. These mechanistic observations provide guidance for technologies that involve DNA interactions in the near-surface region, suggesting a need to design surfaces that both enhance the complex multidimensional search process and stabilize the hybridized state.


Sensitivity analysis of hybridization threshold
In the main text of the article the relative distance between donor and acceptor fluorophores was used to identify when hybridization events occurred. The selection criteria involved a cut-off where steps of a trajectory with d values less than the threshold were considered to be hybridized and values greater than the threshold were searching. It was important when selecting these criteria to choose a hybridization threshold that was low enough to avoid selecting noise or incidental RET events as hybridization events, while not being low enough to identify partial melting events or other fluctuations in RET signal as true dehybridization events. To insure that a sufficiently high hybridization threshold was selected, data was mined and a hybridization threshold sensitivity analysis was performed. This analysis extracted the number of observations of apparent "fluctuations" where a molecule rapidly crossed over the threshold and returned to the hybridized state. The threshold was systematically increased from d = 0-100 (E = 1 -10 -12 ) and the number of fluctuations calculated. The results of this sensitivity analysis are plotted in Figure S2. The fluctuations that exist at large threshold values are due Figure S2. Distribution of the number of fluctuations as a function of hybridization threshold (▪, left axis). Distribution of the number of hybridization events as a function of hybridization threshold (□, right axis) to the tracking of species that exhibit little RET, or d values approaching infinity, alternating with weak RET signals attributed to noise. In the range of d = 1.6 -2.0 (E = 0. 06 -0.02) fluctuations vary by less than 10% from the number of fluctuations at d ≥ 10 (E ≥ 10-6). This information combined with the results stated in the main text indicated that a hybridization threshold of d = 1.6 would reduce identification of false melting events, incidental RET, and RET signals due to noise.

Temperature dependence of fractional pathways to hybridization
Molecular trajectories were examined and then partitioned into "searching" and "hybridized" states based on RET efficiency. Transitions between states could then be counted and normalized by the destination or source state. These fractions were then assembled to provide a picture of the pathway to surface hybridization. This analysis was repeated for the temperature range of 293K -313K at 10 degree intervals; results are shown in Figure S3. The fractions were insensitive to temperature in this range. This observation was statically confirmed as described in the manuscript. Figure S3. Fractional pathway of DNA surface hybridization. Temperatures are read from top to bottom as decreasing temperatures 313K, 303K, and 293K.

Poly-AC and Poly-A State Time Distributions
Cumulative state time distributions were prepared by monitoring the dynamic behavior of singlemolecule RET trajectories. Using the relative distance between fluorophores "hybridized"-and "searching"-state time intervals were accumulated and used to create cumulative state time distributions. This process was repeated for both the poly-A and poly-AC oligonucleotides. The resulting distributions are compared in Figure S4.

Characteristic State Times as a Function of Temperature
State time distributions were prepared for all transitions at each measured temperature. These distributions were fit with a number of exponentials selected via the maximum entropy method as described in the text. Hybridization from the searching state had only a single characteristic search time,  s,on . While there were multiple characteristic state times for desorption from searching state, , desorption from hybridization,  des,off , and melting to the surface,  s,off . These characteristic residence times and their respective population fractions, f, are given in Table S1. As discussed in the text the majority of characteristic state times show little temperature dependence. However, the melting state times exhibit systematic variation. The longer hybridization state has a strong temperature dependence indicated by the decreasing characteristic state times from 15s to 8s over the range of 293K-313K. On the other hand, the shorter hybridization state demonstrates little temperature dependence. This difference is elaborated upon in the primary text.  Table S1. Characteristic state times, , and their associated population fractions, f, for the exponential fits of all state time distributions over the temperature range 293K -313K. The numbers in parentheses represent the uncertainty in the least significant digit.
The characteristic state times () and their associated population fractions were extracted from these fits. Hybridized state times correspond to the time spent hybridized before melting. The hybridization state times demonstrated strong temperature dependence. The characteristic times were inverted to represent an apparent rate of melting and then plotted on an Arrhenius plot ( Figure S5). The trend was fit using standard regression analysis; the results of these fits are reported in Figure

Structure of Target and Probe Oligonucleotides
The structures of both the poly-TG probe and poly-AC target oligonucleotides are shown in Figure S6.
The linker/fluorophore combinations are drawn explicitly and the DNA sequences are given by their letter code. The two sequences are arranged to resemble the structure of the molecules during hybridization. As discussed in the text the poly-AC target strand is labeled with a C6-Amino/Alexa Fluor 488, linker/fluorophore combination on the 5' end. The 3' end of the poly-AC molecule is unmodified.
The 5' end of the poly-TG complementary probe molecule was modified with a C6-Amino linker. A C7-Amino/Quasar 670, linker/fluorophore combination was attached to the 3' end. Figure S6. Structure of labeled probe and target ssDNA.

Optimization of GPTMS Monolayer Deposition Conditions
Vacuum assisted chemical vapor deposition was performed on silicon wafers (WRS). The deposition solution was a 1:2:20 solution by volume, of n-butylamine:3-glycidoxypropyltirmethoxysilane (GPTMS):toluene. The solution was placed in the bottom of a desiccator; silicon wafers were mounted in the desiccator via Teflon holder. The desiccator was sealed and evacuated via house vacuum and left to incubate for times ranging from 0-24 hours. Samples were extracted from the deposition chamber and immediately characterized using sessile drop experiments on a contact angle goniometer and variable angle spectroscopic ellipsometry. The results of these analyses are shown in Figure S7. Ellipsometry was used to measure film thickness. The measured film thickness was compared to the length of a fully extended GPTMS molecule film (dotted line, Figure S7); thicknesses equal to this value indicate the presence of a complete monolayer. Contact angle goniometry was used as a complementary technique.
The contact angle quantifies the wettability of the surface and is sensitive to different surface chemistries and can be used to characterize a complete monolayer. The combination of these two techniques allowed for the optimization of the deposition of high quality GPTMS monolayers.

Time (hours)
Ellipsometry Results Contact Angle Results Figure S7. Film thickness (Å), left axis, was measured using variable angle spectroscopic ellipsometry (◊). The dotted line indicates the length of a fully extended GPTMS molecule film. Contact angle (degrees, ■), right axis, quantifies the wettability of the surface.