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

Abstract

BackgroundDNA is a carrier of biological information. The hybridization process, the formation of the DNA double-helix from single-strands with complementary sequences, is important for all living cells. DNA microarrays, among other biotechnologies such as PCR, rely on DNA hybridization. However, to date the thermodynamics of hybridization is only partly understood. Here we address, experimentally and theoretically, the hybridization of oligonucleotide strands of unequal lengths, which form a bulged loop upon hybridization. For our study we use in-house synthesized DNA microarrays.ResultsWe 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.ConclusionsWe 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.

Probing hybridization parameters from microarray experiments: nearest-neighbor model and beyond

“…There has been considerable attention in recent years [9,22,[24][25][26][27][28][29][30][31][32][33][34] in understanding the fundamentals of hybridization in DNA microarrays and its impact in data analysis. Here, we have shown that microarrays are a reliable and high-throughput tool to gain insight on DNA hybridization thermodynamics.…”

Stochastic Ratchet Mechanisms for Replacement of Proteins Bound to DNA

“…A more general (and precise) model would describe the invading protein through a binding energy ε ′ different from the binding energy ε for the bound protein in case of heterogeneous replacement, as well through its number of bound units, rather than the on/off description used here. Binding/unbinding of small DNA fragments (oligonucleotide) on a DNA under force [11] and exchange of DNA-binding oligonucleotides in DNA hybridization assays [9, 13, 14] are likely described by the Z-S-SB model. Including sequence specificity (dependence of ε on the sites) could help in modeling such experiments [12].…”

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

“…To get a better insight into the hybridization process of two strands of unequal length, Trapp et al considered a DNA microarray with additional bases in the probe sequence motifs (54). The target solution contains only one target species of a specific length.…”

“…Taking into account unavoidable sequence defects, the model can reproduce our data well. Details can be found in (54).

Figure 6.Averaged signal intensity over loop length (top) and loop position (bottom).

Figure 7.Comparison between the experimental data and the model.

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