Conformations of DNA Hairpin Loops under Crowded Conditions

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Luminescence, UV & Microplate Readers
Conformations of DNA Hairpin Loops under Crowded Conditions Olivia Stiehl, Kathrin Weidner-Hertrampf, and Matthias Weiss, Experimental Physics I, University of Bayreuth, Universitätsstr. 30, D-95440 Bayreuth, Germany

Motivation
Hairpin loops of oligonucleotides are of great interest since hairpin-like confi gurations can form in any biological process that involves (single-stranded) DNA/RNA, e.g. during gene duplication or transcription. Artifi cially manufactured hairpin loops basically consist of a single-stranded piece of DNA that can be dissected into a stem and a loop (see Figure1).
In the stem, complementary bases provide a means for transient association via hydrogen bonds whereas the loop acts as a polymeric spacer between the stem regions. Due to thermal energy, a hairpin stochastically undergoes a transition between the open and the closed state. Hence, at low temperatures the majority of hairpins can be expected to be in a closed confi guration whereas an open confi guration is preferred at suffi ciently high temperatures. So far, the conformation of DNA hairpin loops has been extensively studied in a variety of aqueous solutions [1]. However, given that the intracellular concentration of macromolecules is in the range of 400 g/l, it is of great interest to study the DNA hairpin confi guration in artifi cially crowded fl uids that mimic intracellular conditions. Indeed, theoretical predictions and fl uorescence experiments have indicated that macromolecular crowding leads to an enhanced fraction of closed DNA hairpins at physiological temperatures [2]. To gain deeper insights into this, we have used the SPECORD® PLUS (Figure 2).

Materials and Methods
DNA constructs with fi ve cognate bases in the stem region (CCCAA-X-TTGGG) and a poly-T confi guration in the loop (X=21T, 30T, or 50T) were purchased from metabion (Martinsried, Germany). As crowding agents we have used polyethylene glycol (10kDa; Sigma Aldrich) and sucrose (Roth). Crowders and DNA were dissolved in TE buffer (1 mM EDTA, 100 mM NaCl, 10 mM Tris at pH 7.5). In order to determine the DNA’s confi guration, we exploited DNA hyperchromicity, i.e. in the open state DNA shows an enhanced absorption in the wavelength range 250-270 nm. For quantitative results, we recorded melting curves of our samples using a SPECORD® PLUS equipped with temperature-regulated sample holders. Thanks to the dual-chamber option of the SPECORD® PLUS, we could record in parallel a sample fl uid with DNA and the same fluid without DNA (‘control’). Hence, we directly obtained the difference in UV absorption between sample and control. Measurements were conducted in accordance with the settings reported in application note UV_PLUS_08_12_d |06/2012|AK. Owing to the very exact and fast temperature regulation, it was possible to start our experiments near freezing point and heat up sequentially to 90°C. The availability of low temperatures enabled us to also measure DNA hairpins at high loop lengths, e.g. constructs with 50 thymine bases. Besides the effective and fast cooling, we also appreciated the precise heating. Having increased the temperature in steps of one degree, a waiting time of 30 s
guaranteed a temperature uncertainty of less than 0.3°C within the sample. For simplicity, we compared the absorbance at 20°C with that obtained in the saturation regime of the melting curve. This ratio reports on the fraction of open DNA hairpins at room temperature.

Results and Discussion
Conformational changes of three DNA strands in sucrose and PEG solutions (10% and 30% weight per volume) were evaluated as described above (Figure 3). As a result, we found that the probability for DNA hairpins to be in the open state slightly increases with loop length for each condition. Furthermore,
in sucrose solution the open probability is largely independent of the additive’s concentration (Figure 3, grey bars) whereas we observed signifi cantly lower probabilities for the open confi guration in PEG-crowded fl uids (Figure 3, red bars). The tendency for closed hairpins increased with the concentration of PEG, hence confi rming the hypothesis that crowded fl uids feature a signifi cantly increased probability of the associated/closed state of the DNA hairpin. These data highlight that crowded fluids may not only affect the opening-closing kinetics by slowing down association and dissociation. Rather, the steady-state fraction of closed hairpins is signifi cantly enhanced. A ssDNA hairpin can be dissected into a loop region (shown as full line) that connects two complementary sequences of bases that form the stem region (explicit bases are shown). Due to thermal energy, the hairpin stochastically switches between an open and a closed state. The fraction of open DNA hairpin loops, i.e. the probability of being in the open state, increases for increasing loop lengths irrespective of the additive (sucrose, PEG) in the solution. Yet, a signifi cant drop in the fraction of open hairpins is observed in PEG-crowded solutions (red bars) is compared to sucrose solutions (grey bars). Notably, the probability of being in the open state is basically independent of the amount of sucrose but it signifi cantly decreases with increasing PEG concentrations.

Acknowledgements
Analytik Jena would like to thank the University of Bayreuth for performing the application and providing all research and test data for this DNA applications with the SPECORD® PLUS by Analytik Jena.

References
[1] G. Bonnet, O. Krichevsky, and A. Libchaber, Proc. Natl. Acad. Sci. USA 95, 8602 (1998).
[2] O. Stiehl, K. Weidner-Hertrampf, and M. Weiss, New J. Phys. 15, 113010 (2013).
Using UV Vis absorption spectroscopy on a SPECORD® PLUS, we have monitored the conformation of various ssDNA hairpin loops in crowded fl uids that mimic
intracellular conditions. As a result, we have found that macromolecular crowding signifi cantly alters the steady-state fraction of closed ssDNA hairpins.
Figure 1: Scheme of a single-stranded DNA hairpin loop.
Figure 2: SPECORD® PLUS
Figure 3: Open probability of DNA hairpin loops.
Analytik Spotlight Article.indd 1 01/07/2015 12:18
Luminescence, UV & Microplate Readers
Conformations of DNA Hairpin Loops under Crowded Conditions
Olivia Stiehl, Kathrin Weidner-Hertrampf, and Matthias Weiss,
Experimental Physics I, University of Bayreuth, Universitätsstr. 30, D-95440 Bayreuth, Germany