Methods for determining the binding affinity of a probe to a target or targets in a polynucleotide composite using an automated system. Such methods determine relative binding sites based on thermodynamic principles, using a thermodynamic alphabet and a thermodynamic scoring matrix, with appropriate computer software, such as BLASTP. The invention also provides methods for designing polynucleotide probes to be used in hybridization assays, and minimizing the occurrence of cross-hybridization.