Background: The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization -and in particular MM discrimination -are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations.

Background: The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.

Microarray technology uses the sequence dependent hybridization (binding) affinity of surface-bound oligonucleotide strands for the quantification of complex nucleic acid mixtures. In spite of its huge potential in life science and medicine, microarray oligonucleotide hybridization remains far from being understood. Taking advantage of microarray combinatorial possibilities we show that, although surface bound, the hybridization affinities of single-base mismatched oligonucleotides can be derived from first principles using parameters from bulk.

The manuscript has been published in Review of Scientific Instruments (http://rsi.aip.org/rsi/) Rev. Sci. Instrum. 77, 063711 (2006) We present a maskless microscope projection lithography system (MPLS), in which photomasks have been replaced by a Digital Micromirror Device type spatial light modulator (DMD TM , Texas Instruments). Employing video projector technology high resolution patterns, designed as bitmap images on the computer, are displayed using a micromirror array consisting of about 786000 tiny individually addressable tilting mirrors. The DMD, which is located in the image plane of an infinity corrected microscope, is projected onto a substrate placed in the focal plane of the microscope objective. With a 5× (0.25 NA) Fluar microscope objective, a fivefold reduction of the image to a total size of 9 mm 2 and a minimum feature size of 3.5 µm is achieved. The ultra high pressure (UHP) lamp of a video projector is a cheap, durable and powerful alternative to the mercury arc lamps commonly used in lithography applications. The MPLS may be employed in standard photolithography, we have successfully produced patterns in 40 µm films of SU-8 photoresist, with an aspect ratio of about 1:10. Our system can be used in the visible range as well as in the near UV (with a light intensity of up to 76 mW/cm 2 around the 365 nm Hg-line). We developed an inexpensive and simple method to enable exact focusing and controlling of the image quality of the projected patterns. Our MPLS has originally been designed for the light-directed in situ synthesis of DNA microarrays. One requirement is a high UV intensity to keep the fabrication process reasonably short. Another demand is a sufficient contrast ratio over small distances (of about 5 µm). This is necessary to achieve a high density of features (i.e. separated sites on the substrate at which different DNA sequences are synthesized in parallel fashion) while at the same time the number of stray light induced DNA sequence errors is kept reasonably small. We demonstrate the performance of the apparatus in light-directed DNA chip synthesis and discuss its advantages and limitations.

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The specificity of molecular recognition is important for molecular self-organization. A prominent example is the biological cell where a myriad of different molecular receptor pairs recognize their binding partners with astonishing accuracy within a highly crowded molecular environment. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide can prevail against a disproportionally more concentrated competitor with reduced affinity due to a mismatch. We investigate the situation using fluorescence-based techniques, among them Förster resonance energy transfer and total internal reflection fluorescence excitation. We find that the affinity of certain strands appears considerably reduced only as long as a better matching competitor is present. Compared to the simple Boltzmann picture above we observe increased specificity, up to several orders of magnitude. We interpret our observations based on an energy-barrier of entropic origin that occurs if two competing oligonucleotide strands occupy the same probe simultaneously. Due to their differences in binding microstate distributions, the barrier affects the binding affinities of the competitors differently. Based on a mean field description, we derive a resulting expression for the free energy landscape, a formal analogue to a Landau description of phase transitions reproducing the observations in quantitative agreement as a result of a cooperative transition. The advantage of improved molecular recognition comes at no energetic cost other than the design of the molecular ensemble and the presence of the competitor. As a possible application, binding assays for the detection of single nucleotide polymorphisms in DNA strands could be improved by adding competing strands. It will be interesting to see if mechanisms along similar lines as exposed here contribute to the molecular synergy that occurs in biological systems.

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