DNA microarrays constitute an in vitro example system of a highly crowded molecular recognition environment. Although they are widely applied in many biological applications, some of the basic mechanisms of the hybridization processes of DNA remain poorly understood. On a microarray, cross-hybridization arises from similarities of sequences that may introduce errors during the transmission of information. Experimentally, we determine an appropriate distance, called minimum Hamming distance, in which the sequences of a set differ. By applying an algorithm based on a graph-theoretical method, we find large orthogonal sets of sequences that are sufficiently different not to exhibit any cross-hybridization. To create such a set, we first derive an analytical solution for the number of sequences that include at least four guanines in a row for a given sequence length and eliminate them from the list of candidate sequences. We experimentally confirm the orthogonality of the largest possible set with a size of 23 for the length of 7. We anticipate our work to be a starting point toward the study of signal propagation in highly competitive environments, besides its obvious application in DNA high throughput experiments.
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.
Binding of two complementary DNA single strands to a double-helix, DNA hybridization, is a sequence specific molecular recognition process that plays important roles in biology and biotechnological applications. In the past much work has been devoted to understand double helix formation, however, DNA binding in complex situations often remains difficult to deal with. Here we use fluorescence anisotropy to assess the binding affinities of DNA oligonucleotide strands that compete for hybridization to the same probe molecule in thermal equilibrium. We find that the ratio of the binding constants in competition can change substantially compared to pairwise assessments. This is a signature of non-trivial interaction among the competitors: the binding microstates of each strand are affected by the presence of the other, but to a different degree. To our knowledge this type of phenomenon is not included in current equilibrium models of oligonucleotide binding. We suggest interactions beyond double helix conformations to cause the observed cooperative behavior. The cooperativity could produce more complex binding phenomena than previously thought.
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