Supporting Online Material
DNA Functionalized Nanotube Membranes with Single-Base Mismatch Selectivity
Punit Kohli, C. Chad Harrell, Zehui Cao, Rahela Gasparac, Weihong Tan and Charles R. Martin*
Materials and Methods:
DNA functionalization of the Au nanotube membranes. Functionalization with the oligonucleotide thiols (alphaDNA, Inc., HPLC purified) was accomplished by immersing the membrane for four days at room temperature into a 100 µM solution of the desired thiol. The thiol was dissolved in pH = 7.2, ionic strength ~0.2 M, phosphate buffer. The functionalized membrane was then thoroughly rinsed by immersion in buffer for 4 hours.
Determination of the quantity of thiolated DNA transporter immobilized within the nanotubes. As discussed in detail in reference 10 of the published paper (reference S1 here), the electroless plating method yields the Au nanotubes lining the pores in the membrane and Au surface films covering both faces of the membrane. The Au surface films are so thin that they do not block the mouths of the nanotubes at the membrane faces (S1). However, we are specifically interested in knowing how much thiolated DNA transporter (either linear or hairpin) is present along the nanotube walls within the membrane. Therefore, prior to the analysis discussed here, the Au surface films were removed as described in detail in reference S2.
µM solution (buffered as per Part 1, above) of FAM-labeled perfect-complement (PC) DNA (see Table 1 of the published paper) for 24 hrs at room temperature. The membranes were then immersed into fresh buffer solution for 2 hrs at room temperature to remove any unhybridized FAM-labeled PC-DNA. The membrane was then placed into a quartz cuvette containing 1 mL of 9 µM unlabelled PC-DNA solution dissolved in the same buffer.
As we have shown in Figure 3 of the published paper, the excess unlabeled PC-DNA in the solution displaces the FAM-labeled PC-PC-DNA that is hybridized to the PC-DNA transporter attached to the nanotube walls. If we assume that each immobilized DNA-transporter molecule was hybridized with a FAM-labeled PC-DNA, then the amount of released FAM-labeled PC DNA is equivalent to the amount of immobilized DNA transporter. The amount of released FAM-labeled PC-DNA was quantified via the fluorescence of the FAM label after release of all of the labeled PC-DNA into the solution (λex = 495 nm and λem = 520 nm).
Because it is easier to obtain a good approximation of the dimensions of the DNA transporter (S3, S4), we begin with the data obtained for this thiolated linear-DNA transporter. The fluorescence intensity of the released FAM-labeled PC-linear-DNA from the membrane indicates that 0.006 nmoles of linear-DNA transporter was immobilized per cm2 of geometric surface area of the membrane. The question then becomes- what
surface roughness of unity, and the actual surface roughness inside the Au nanotubes might be higher. However, evaluating the roughness within these nanoscopic cylinders would be experimentally quite challenging.
The immobilized linear-DNA transporter is a 30-mer, and the radius of gyration for this 30-mer can be calculated via the standard methods (S3, S4). A radius of gyration of ~2.6 nm is obtained from this calculation. Hence, to a first approximation, the footprint of an immobilized linear-DNA transporter corresponds to a circle that is 5.2 nm in diameter. If we assume that a complete monolayer corresponds to hexagonal packing of these 5.2-nm diameter circles across all of the available Au surface area within the nanotubes, the amount of immobilized linear-DNA transporter corresponds to 61% of a monolayer. In fact, this is an underestimate because the radius of gyration of the immobilized linear-DNA thiol is undoubtedly bigger than the radius of gyration of a 30-mer dissolved in solution. However, this underestimation of the % monolayer coverage will be offset by the surface roughness effect discussed above.
diameter of 6.5 nm. This indicates that the immobilized hairpin-DNA transporter corresponds to 55% of a monolayer.
Details of the restriction-enzyme experiments. The objective of these experiments was to show that the permeating PC-DNA does, indeed, hybridize with the membrane-bound hairpin-DNA transporter. The general strategy was to expose the membrane with the immobilized hairpin-DNA transporter to a solution of the PC-DNA and then to a solution of the restriction enzyme. If hybridization between the PC-DNA and the hairpin transporter occurred this enzyme will cut the resulting double-stranded DNA such that the last five bases of the binding loop, and all of the stem-forming region at the 3’ end of the hairpin, are removed. This reaction will substantially damage the binding site, and based on our prior work (S5, S7), we would predict that if this membrane is subsequently used in a permeation experiment, a lower PC-DNA flux should be obtained. The restriction enzyme Sfc I was purchased from New England Biolabs (www.neb.com). Based on data obtained from the supplier, we estimate that the diameter of this protein is < 3 nm, much smaller than the 12 nm-diameter of the Au nanotubes. Hence the enzyme has no difficulty accessing the DNA immobilized within the nanotubes.
serum albumin. The membrane was immersed into this solution for 18 hours, at room temperature, and in the dark. The membranes were then rinsed by immersion for 2 hours in phosphate buffer, 15 mins in deionized water, and again in phosphate buffer for 10 min.
Figure S1 shows the results of this experiment. As per our prediction, the flux is less after treatment with the restriction enzyme indicating that hybridization does occur within the nanotubes. The damaged hairpin DNA transporter was then removed from the nanotubes by exposure of both faces of the membrane to UV light (S6) for 9 hours followed by immersion sequentially in phosphate buffer (2 hours), deionized water (15 min) and phosphate buffer again (10 min). Fresh hairpin-DNA transporter was then applied and the transport experiment was repeated. The transport data are super-imposable with the data obtained before treatment. This shows that the nanotubes were not blocked by the restriction-enzyme treatment. These data also show how reproducible the transition time is.
However, Figure 3 of the published paper shows that dehybridization occurs spontaneously, and for this reason, the FAM label will be released into the solution, even if the duplex is not clipped. Hence we must demonstrate that the time course of release is different when the membrane is exposed to the solution containing the restriction enzyme vs. an identical solution but without the restriction enzyme. The composition of the solution was described above.
Figure S2 shows the results of these experiments. The blue curve shows release into the solution devoid of the restriction enzyme, and the red curve shows release into the solution with this enzyme. At times less than ~10 hours the data are essentially identical, indicating no influence of the restriction enzyme on release; i.e., the release is due to spontaneous dehybridization as per analogous data in the published paper (lower, red curve in Figure 3). For the experiment with the restriction enzyme, this induction period is associated with transport of the restriction enzyme into the nanotubes and the time required for the enzymatic reaction to yield an appreciable concentration of the clipped FAM tag. At times greater than ~10 hours the rate of release of FAM is higher when the enzyme is present. This higher rate of release results because the enzyme clips the FAM tag from the duplex providing a second (and faster) mechanism for release of FAM from the membrane.
Supporting Figures:
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Supplement References and notes:
S1. C. R. Martin, M. Nishizawa, K. Jirage, M. Kang, J. Phys. Chem. 105, 1925-1934 (2001).
S2. V. P. Menon, C. R. Martin, Anal. Chem. 67, 1920-1928 (1995). S3. S. S. Sorlie, R. A. Pecora, Macromolecules 23, 487-497 (1990).
S4. M. C. Olmstead, C. F. Anderson, M. T. Record, Biopolymers 31, 1593-1604 (1991). S5. S. B. Lee et al., Science 296, 2198-2200 (2002).
S6. Y. Zhang, R. H. Terrill, T. A. Tanzer, P. W. Bohn J. Am. Chem. Soc. 120, 2654-2655 (1998).
S7. B. B. Lakshmi, C. R. Martin, Nature (London) 388, 758-760 (1997).
Supporting Online Material www.sciencemag.org
