Stability of double-stranded oligonucleotide DNA with a bulged loop: a microarray study

We synthesize a microarray with additional thymine bases in the probe sequence motifs so that bulged loops occur upon target hybridization. We observe a monotonic decrease of the fluorescence signal of the hybridized strands with increasing length of the bulged loop. This corresponds to a decrease in duplex binding affinity within the considered loop lengths of one to thirteen bases. By varying the position of the bulged loop along the DNA duplex, we observe a symmetric signal variation with respect to the center of the strand. We reproduce the experimental results well using a molecular zipper model at thermal equilibrium. However, binding states between both strands, which emerge through duplex opening at the position of the bulged loop, need to be taken into account.We show that stable DNA duplexes with a bulged loop can form from short strands of unequal length and they contribute substantially to the fluorescence intensity from the hybridized strands on a microarray. In order to reproduce the result with the help of equilibrium thermodynamics, it is essential (and to a good approximation sufficient) to consider duplex opening not only at the ends but also at the position of the bulged loop. Although the thermodynamic parameters used in this study are taken from hybridization experiments in solution, these parameters fit our DNA microarray data well.The hybridization process - the formation of the well-known double-helix structure from two complementary nucleic acid strands (such that A · T and C · G base pairs are formed) - is pivotal to the living organism. Among other important biotechnological methods, PCR or DNA microarray technology rely on it.DNA microarrays consist of regular spaced domains of surface-attached probe sequences, which act as binding sites for their complementary fluorescently-labeled target sequences in solution. The probe sequence and position of each domain on the surface is known and the amount of bound target DNA can be determined quan