Adding a new dimension to DNA melting curves

Abstract

Abstract. -Standard DNA melting curves record the separation of the two strands versus temperature, but they do not provide any information on the location of the opening. We introduce an experimental method which adds a new dimension to the melting curves of short DNA sequences by allowing us to record the degree of opening in several positions along the molecule all at once. This adds the spatial dimension to the melting curves and allows a precise investigation of the role of the base-pair sequence on the fluctuations and denaturation of the DNA double helix. We illustrate the power of the method by investigating the influence of an AT rich region on the fluctuations of neighboring domains.DNA melting, i.e. the separation of the two strands of the double helix, and its reverse process hybridisation are ubiquitous in biology, in vivo for instance for DNA transcription in the reading of the genetic code of a gene, as well as in vitro in biological laboratories for PCR (Polymerase Chain Reaction) or the use of DNA microarrays. This is why DNA melting has been extensively studied even in the early days of DNA structural studies [1]. An approximate understanding of the melting curves of long DNA segments, with thousands of base pairs, can be provided by simple statistical physics models, using empirical parameters because, at this large scale, the subtle effects of the base pair sequence are smoothed out. Understanding the fluctuations and melting of short DNA fragments of a few tens of base pairs with a high degree of heterogeneity is much more challenging. And it is also very important because this size is the scale at which the genetic code can be resolved. This would have some significant biological consequences to unravel the processes by which specific binding sites are recognised by proteins, drugs, mutagens and other molecules. This would also have a lot of practical importance in the design of the PCR primers which are used everyday in most of the biological laboratories [2].The two kind of base pairs which exist in the DNA double helix have different thermal stability, the AT pair, bound by two hydrogen bonds, being weaker than the GC pair bound by three hydrogen bonds. This explains why the melting curve of a heterogeneous DNA sequence, which shows the fraction of open pairs as a function of temperature, can exhibit complex features. Those curves are easy to record experimentally because the UV absorbance of a DNA solution increases drastically when the bases are unstacked, which is (a) Mailing

Effect of sequence-dependent rigidity on plectoneme localization in dsDNA

“…Whether nature utilizes such effects to facilitate the search of TFs for their DNA binding sites depends on whether promoter sites tend to be localized at the edges of plectonemes. Indeed, many bacterial promoters contain TATA-box sequences that are known for their lower rigidity and higher tendency to create DNA bubbles (kinks) (26)(27)(28)(29) and, ac-cording to our results, such sequences will tend to nucleate plectonemes and to be positioned at their edges. Finally, we would like to mention that although we are not aware of direct experimental proof of our proposed mechanism of plectoneme localization to low-rigidity DNA sequences, there has been a recent experimental study of the effects of torsional stress on stretched linear dsDNA in which repeated hopping of plectonemes between specific locations along DNA was reported.…”

“…While the distribution for the no-kink case is qualitatively similar to that for σ = 0 (Fig 4(a)), the peak of the distribution of a kink-containing segment is shifted towards contact. In principle, this prediction can be tested by FRET experiments by attaching donor and acceptor molecules to the ends of a short (of order l p ) sequence in a long circular dsDNA molecule, provided that one is able to design a sequence that has a high propensity for forming a kink (26)(27)(28)(29). In the absence of supercoiling, the donor and acceptor will be separated by tens of nanometers and there will be no FRET signal.…”

Experimental and theoretical studies of sequence effects on the fluctuation and melting of short DNA molecules

“…The AT-rich center is however interrupted by a few GC pairs, the largest consecutive span of AT base pairs containing only 8 pairs. According to our experiments [40] a few isolated GC pairs scattered in an AT domain are able to reduce significantly the fluctuations of this domain. This is certainly the case for sequence L60B36, which explains why the small GC rich domains at the ends of the sequence are sufficient to stabilize the molecule and prevent a full separation of the strands in spite of the large open domain.…”

“…From our experimental data on various sequences [40] and from the theoretical studies of several cases, we set k = 9, n 1 = 10, n 2 = 16, c 0 = 0.2, but these parameters could certainly be refined by more extensive experimental studies.…”

Denaturation patterns in heterogeneous DNA

“…In heterogeneous DNA, AT-rich portions of the molecule tend to open at lower temperatures than GCrich regions. However openings occurring at lower temperatures extend also well inside the GC domains indicating a role for nonlocal effects in shaping multistep denaturation patterns [64]. The sequence pattern is particularly relevant in relatively short segments made of a few tens of bps which is the relevant scale for those transcription starting domains where the genes are read.…”