Thermodynamics of Duplex Formation and Mismatch Discrimination

Chapter 13

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 18, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch013

Thermodynamics of Duplex Formation and Mismatch Discrimination on Photolithographically Synthesized Oligonucleotide Arrays Jonathan E. Forman, Ian D. Walton, David Stern, Richard P. Rava, and Mark O. Trulson 1

Affymetrix, 3380 Central Expressway, Santa Clara, CA 95051

Oligonucleotide probes immobilized on solid supports are finding increasingly widespread application in genetic analysis. We seek to provide a better understanding of the fundamental thermodynamic aspects of duplex formation in these systems. Equilibrium melting curves for the adsorption of 10-, 20-, 30-, and 40-base oligonucleotides to 10-, 12-, 14-, 16-, 18-, and 20-base probe sites on an Affymetrix GeneChip array have been investigated. The melt curves are depressed and broadened relative to corresponding solution phase species. Melt curves show multistep behavior above a threshold target concentration that decreases for longer target DNA. Melting temperatures (T ) are weakly dependent on probe length. Mismatches introduce T depressions that are nearly normal at 10-mer probe sites but decrease with increasing probe length. Saturating adsorption densities at perfect match probe sites are less than 10% of the probe density and exceed the corresponding mismatch densities in all cases. The anomalies in the equilibrium data cannot be fully attributed to the presence of truncated probes, and implicate target DNA binding to multiple probes ("probe bridging") and probe-probe interactions that compete with 1:1 probe:target binding. ®

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Recognition of D N A or R N A from solution using arrays of immobilized nucleic acid probes (Figure 1) is finding increased application in a variety of areas, including polymorphism detection, gene expression, and sequence analysis. ' A number of embodiments have been described in which the D N A probes are immobilized onto appropriate support materials (usually glass or polymer surfaces); these arrays provide a rapid means by which nucleic acid sequences can be analyzed using a minimum amount of sample. ' Our photolithographic synthesis methodology allows probe arrays to be prepared with as many as 132,000 synthesis sites (35 μπι x 35 μιη feature size), capable of interrogating every position on a target of up to 33 kb (16.5 kb if both sense and antisense strands are being screened). Duplex formation in solution has been extensively studied, and the kinetics and thermodynamics of such interactions for short oligonucleotides can be described by a model involving nucleation followed by helix zipping (Figure 2a). In immobilized 2 3

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'Corresponding author

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© 1998 American Chemical Society

Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

Duplex Formation and Mismatch Discrimination

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13. FORMAN ET AL.

Figure 1. (a) Recognition of a solution-borne nucleic acid (target) by a nucleic acid probe immobilized on a surface, (b) Fluorescence microscope image of a 1.28 cm x 1.28 cm probe array with 200 μτη x 200 μπι synthesis sites on which labeled target oligonucleotides have adsorbed.


MOLECULAR MODELING OF NUCLEIC ACIDS

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probe systems, the behavior may be more complex due to the combined influences of bulk diffusion, surface diffusion, and small intermolecular distances typically encountered in these systems (Figure 2b). The small intermolecular distances on the probe array raise the potential for interactions of the target with multiple probes, requiring an annealing step to occur in order to form the optimized duplex structure. Indeed, the equilibrium state may not be made up entirely of ideal duplexes. Additionally, probe-probe interactions may limit the number of sites available for target binding and/or reduce the stability of the probe:target duplexes relative to solution. The stability of the surface bound duplexes may be further affected by differences in dielectric environment, ionic strength, or pH relative to the solution from which the target adsorbs. We are interested in physically characterizing the factors that govern the adsorption of complex solution-borne nucleic acid targets to surface bound oligonucleotide probes. This paper represents our first efforts toward understanding the thermodynamics of oligonucleotide recognition by immobilized probe arrays.


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Materials and Methods Probe arrays and DNA target. The studies described here have been carried out with arrays of 10-, 12-, 14-, 16-, 18-, and 20-base oligonucleotide probe synthesis sites 200 μιτι x 200 μιη in size. The probes are covalently attached to a siloxane derivatized 15 mm x 15 mm x 0.7 mm borosilicate glass substrate and were synthesized using photolithographic techniques that have been described elsewhere. J Target oligonucleotides were labeled at the 5' end with fluorescein; samples were obtained from GENSET (HPLC purified) and are estimated to have 99% purity. Table I lists the sequences of the probes and the 20 base oligonucleotide target used in these studies. This particular target sequence was chosen to minimize the chance of intra- and intermolecular secondary structure and has a GC content of 50%, yielding a fairly average stability for a duplex of this length. A l l studies were carried out using 6X SSPE buffer (1 M NaCl, 0.07 M N a H P 0 , 0.07 M EDTA) at pH 7.8, stabilized with 0.005% Triton-X. The probe arrays were pretreated with this buffer for 30-60 minutes before target adsorption studies were undertaken. 2i

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Table I. Sequences of oligonucleotides employed in these studies, fl = Fluores cein, su = surface. Target 10-mer probe 12-mer probe 14-mer probe 16-mer probe 18-mer probe 20-mer probe

Position | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 5'-fl- C T G A A C G G T A G C A T C T T G A C -3* su-3'G C C A T C G T A G -5' su-3'-

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Figure 1 shows a fluorescent microscope image of the probe array with adsorbed fluorescein labeled target. Each of the bright cells is a 200 μηη x 200 μπι synthesis site containing probes of a given sequence. The layout of the probe array includes nine repeats of a six-block set of probe synthesis sites (Figures 1 and 3); each


13.

FORMAN E T A L .


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(b) Adsorption to probes at an interface Figure 2. Oligonucleotide interactions in solution (a) and at an interface with surface bound probes (b). While the mechanism of the solution phase process is well understood, duplex formation at an interface is complicated by a number of processes resulting from adsorption mechanisms and probe distribution.


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Figure 3. Probe array features examined in this study, (a) Fluorescence microscope image and map of probe types and base substitution sequences, (b) Illustration of the tiling for the 10 sequence positions within a given set of probes. We have used the 10-mer probes for illustration; matched positions are in bold-face type.


13. FORMAN ET AL.


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of these six blocks represent the six 10-20 base probes with substitution at the 6-15th positions of each of the four possible naturally occurring bases (Table I). As shown in Figure 3, each block of probe sites of a given length contains 10 matched sequence cells (the brightest within each column of four cells) and 30 single base mismatches, making it possible to read the sequence of the target by simply identifying the brightest cells across the 1 0 x 4 block of probe sites. The blank lanes between the 1 0 x 4 blocks were used as background for the signals measured on the probe array surface. Instrumentation and procedures. The adsorption measurements were performed with an in-house built confocal laser scanning fluorescence microscope employing a 50 mW A r laser (488 nm excitation). Probe arrays were mounted to an anodized aluminum flow cell equipped with water jacket for controlling temperature by means of a V W R 1167 Forma bath. Target solution was continuously pumped through the system using a peristaltic pump (VWR Variable Flow Tubing Pump, using 3/16 OD, flow rate -1.5 ml/minute). The temperature of the target solution was controlled through a sample reservoir equilibrated with a water bath whose temperature was maintained by the same Forma bath used to control the flow cell temperature. Additionally, the fluidic lines from the target reservoir were routed through the water flow lines of the Forma bath to provide better pre-equilibration of sample temperature. Solution phase melting studies were performed by measuring absorption hypochromism '° n a HP8453 UV/Visible spectrometer at 260 nm using a minimum of three concentrations of unlabeled oligonucleotides with total oligonucleotide concentration ranging from 5 χ 10" to 3 χ ΙΟ" M (1:1 ratio of probe:target sequences) in 6X SSPE. The solution measurements were performed for the 20-mer targe^20-mer probe and the 20-mer target/10-mer probe perfect match duplexes, as well as single base substitution at the central point of the duplex, van't Hoff plots were used to determine thermodynamic parameters to allow extrapolation of T 's for solution phase duplexes to sub μΜ concentrations. > A l l experiments were performed with the probe array continually exposed to a constant concentration of target solution. The flow cell sample chamber volume is 250 μΐ; however total volume of target solution in the flow cell and target reservoir was maintained at ~6 ml, to ensure there was no depletion of target. Kinetic experiments were carried out under constant temperature conditions, with initial focusing of the scanner, and fluorescence images taken at appropriate time intervals. For variable temperature equilibrium experiments, a 30 minute equilibration time was allowed at each temperature point, the scanner was focused and four scans taken. The fluorescence intensities reported at a given feature site for a given temperature point are the average of the four scans. Equilibrium experiments were run in both the high-low and low-high temperature directions to check for hysteresis. The experimentally accessible target concentration range for equilibrium measurements was 1 nM to 500 nM. The low concentration limit was dictated by the adsorption kinetics, which have been observed to be proportional to target concentration. The high concentration limit was set by the ability of the confocal imaging system to reject the luminescence of the bulk fluorophore. The conversion from fluorescence intensity (counts/pixel) to adsorbed target density (molecules/pm or pmol/cm ) was accomplished by measuring the system gain of the optical scanner. The system gain was determined from the derivative of the axial dependence of the signal from a solution of fluorophore labeled oligonucleotide of known quantum yield and concentration. The measured adsorbed target densities are estimated to be accurate to ± 0.2 pmol/cm .


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Adsorption Kinetics Initial inspection of the adsorption time course suggests that rates of adsorption for all probe lengths level off within an hour of exposure for a 10 nM target concentration, with longer probe lengths adsorbing a greater amount of target (Figure 4a). Observed


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13. FORMAN ET AL.

adsorption rate constants are comparable to duplex formation rate constants in solution (~1(K- 10 M sec ) ; however, variations in adsorption rates have been observed with different mixing schemes, and our measured rate constants therefore should be regarded as a lower limit. The initial adsorption rates for mismatched probe sites were within 5% of those to the matched sites, but at all times the densities of adsorbed target were lower (Figure 5). Experiments carried out beyond the 1 hour time course showed a continued change in adsorbed density. At 25°C, the adsorbed density for longer probe sites leveled off after several hours, but within short probe sites adsorption was still increasing after 30 hours! (Figure 4b). The increase in adsorbed material from the initial hour to 30 hours is greatest for the shorter probe sites, with the 10-mer sites showing 175% and the 18-mer sites showing 108% of the density at the 1 hour time point. The 20-mer probe sites, while following the trend of 10-mer > 12-mer > 14-mer > 16-mer > 18-mer > 20-mer adsorbate change over time, differ from the other probe sites by decreasing in adsorbate density relative to the 1 hour time point (90% of one-hour signal at 30 hours). After equilibration, the 20-mer probe sites show the lowest adsorption levels of all the probes (Figure 6). These long term changes in adsorbed density are strongly indicative of some type of structural reorganization within the probe array. This apparent annealing process has been found to be driven by the adsorption of target and is not a consequence of structural rearrangements following hydration of the array: The association kinetics are essentially unchanged by 72 hours of soaking in buffer prior to the start of the kinetic experiment. 5



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Adsorption Equilibrium Probe length dependence. The isotherms shown in Figure 6 represent the concentration range from 10-500 nM and it can be seen that at all target concentrations (except 150 nM) the adsorption densities follow the order: 18-mer > 16-mer > 14-mer > 12-mer > 10-mer > 20-mer. An unusual aspect of these studies is the shape of the isotherms. At concentrations as high as 100 nM, the adsorption isotherms appear to be made up of of two components with binding constants of

Thermodynamics of Duplex Formation and Mismatch Discrimination

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