Stability of a Surface-Bound Oligonucleotide Duplex Inferred from Molecular Dynamics: A Study of Single Nucleotide Defects Using DNA Microarrays

Naiser, Thomas; Kayser, Jona; Mai, Timo; Michel, Wolfgang; Ott, Albrecht

doi:10.1103/physrevlett.102.218301

Cited by 18 publications

(43 citation statements)

References 25 publications

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“…Δ g init = -4.5 kcal/mol is the duplex initialization free energy [17]. For the binding constant K i ( P , L ), K i ( P , L ) = Z D ( P , L ) taking the totally denatured state, S 0 , as the reference state.…”

Section: Resultsmentioning

confidence: 99%

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

2011

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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.

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Section: Resultsmentioning

confidence: 99%

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

2011

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“…The general effect of incomplete probe synthesis on the hybridization of microarrays has been discussed in refs. [49] and [13].…”

Section: Discussionmentioning

confidence: 99%

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

2009

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BackgroundSingle nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.Methodology/Principal FindingsThe probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G's in their sequence.ConclusionsThe applied method of “triple-averaging” represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested.

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“…33 This might as well explain the known lower hybridization efficiency, but higher stability, of the duplex for surface tethered oligonucleotides. 31,34,35 Thus, the added strands, in a non-zipped configuration, imply a larger configurational entropy penalty, as the non-zipped strands have short persistence length and the increased number of molecules in the same surface area implies a larger surface stress upon the ssDNA polyelectrolyte swelling. Molecular dynamics simulations of DNA hybridization on surfaces have also shown that the cited non-zipped intermediate states occur during the hybridization process.…”

Section: Resultsmentioning

confidence: 99%

Hydration Induced Stress on DNA Monolayers Grafted on Microcantilevers

et al. 2014

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Surface tethered single-stranded DNA films are relevant biorecognition layers for oligonucleotide sequence identification. Also, hydration induced effects on these films have proven useful for the nanomechanical detection of DNA hybridization. Here, we apply nanomechanical sensors and atomic force microscopy to characterize in air and upon varying relative humidity conditions the swelling and deswelling of grafted single stranded and double stranded DNA films. The combination of these techniques validates a two-step hybridization process, where complementary strands first bind to the surface tethered single stranded DNA probes and then slowly proceed to a fully zipped configuration. Our results also demonstrate that, despite the slow hybridization kinetics observed for grafted DNA onto microcantilever surfaces, ex-situ sequence identification does not require hybridization times typically longer than 1 hour, while quantification is a major challenge.. 2

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Stability of a Surface-Bound Oligonucleotide Duplex Inferred from Molecular Dynamics: A Study of Single Nucleotide Defects Using DNA Microarrays

Cited by 18 publications

References 25 publications

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

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

Mismatch and G-Stack Modulated Probe Signals on SNP Microarrays

Hydration Induced Stress on DNA Monolayers Grafted on Microcantilevers

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