Burst experiments in a gel

In solution experiments (see chapter 4.2.4.1) showed that a pure two state system is not sufficient to describe the distributions obtained from single molecule burst analysis experiments. Additionally there are indications (see figure 4.15) that the observed intermediate pop- ulations are caused by both, systems that interchange fast as well as more long lived intermediate states. One method to gain further in- sight is to try a separation of possible subpopulations by running a gel and perform an in gel burst analysis experiment. These experi- ments were performed as described in chapter 4.2.2 collecting burst data in 6% Poly-acryl-amide gels at pH 8.0 and 400mM NaCl. The reproducability of the results was tested for each dye combination by runing triplicates on non consecutive days in independent gels using fresh buffers.

When analyzing FRET histograms obtained from solution and in gel measurements it is important to note that for a currently unknown reason the peak positions of identical samples do not exactly match. The most likely reason is that the local environment somehow influ-

80 New developments and applications

Atto 647N

Alexa 647

6

-T

a

m

ra

A

tto

5

3

2

A

le

x

a

5

3

2

σε

σε

σε

Proximity Ratio (ε)

Proximity Ratio (ε)

A B

C D

E F

Figure 4.15: σǫ/ǫdistributions of DNA hairpins for different dye combi-

nations are compared for data measured at 100µWlaser power with NaCl

concentrations of 160mM. The shot noise limitσǫSN as a function ofǫis

highlited (green line). Shown are burst analysis data for Atto532–Alexa647 (A), Atto532–Atto647N (B), Alexa532–Alexa647 (C), Alexa532–Atto647N (D), 6-Tamra–Alexa647 (E), and 6-Tamra–Atto647N (F).

4.2 Influence of dye selection on DNA hairpin dynamics 81

Figure 4.16: FRET distributions of DNA hairpins in a Poly-acryl-amide gel. In gel FRET efficiency distributions for different FRET pair combinations measured at 80 µWlaser power in presence of 400mM NaCl. Gels were

6%Poly-acryl-amide, running in 0.4%TBE (pH8, 400mMNaCl). Measure-

ment points were located above (blue), at the edge (red) and below (green) the maximum count rate in the respective gel as indicated (A). Shown are burst analysis data for (B) Atto532-Alexa647, (C) Atto532-Atto647N, (D) Alexa532-Alexa647, (E) Alexa532-Atto647N, (F) 6-Tamra-Alexa532 and (G) 6-Tamra-Atto647N. For all dye pairs, the sub-population that moves faster through the gel shows a significant increase in the population of the closed conformation. Note that these state distributions are stable for hours at room temperature.

82 New developments and applications

ences the dye properties hence biasing the Förster radius. Despite this differences between in solution and in gel measurements, assum- ing equal changes in the local environment for open and closed DNA hairpins it is however still possible to compare the general shape of the FRET distributions.

Before the discussion is focused on positions in the gel, lets first look at the overall features of the distributions obtained (see fig- ure 4.16).

All gels run at 0mM NaCl show only one low FRET population comparable to the in solution data at all measurement positions and is hence not discussed here (data not shown). The same was true for in gel burst data measured at 200mMNaCl.

At elevated salt concentrations (400 mM)6 almost no open hair- pins (i.e. no low FRET population) can be observed anymore (see figure 4.16). Instead now a distinct high FRET population as ex- pected for the fully closed hairpin and except for Atto532-Alexa647 (figure 4.16B) an additional intermediate state are detected.

Importantly all distinct populations observed in the gel (400mM) experiments are also clearly visible in solution (160 mM, 320 mM). This is true for for all the dye combinations under investigation except 6-Tamra-Atto647N (compare figure 4.16G and 4.13F) which shows a significant high FRET population in gel that does not appear in so- lution. Such a stabilization of the closed hairpin conformation as observed for 6-Tamra-Atto647N is most likely caused by the elevated salt concentrations and the higher pHof the in gel experiments.

The most important changes however can be seen when compar- ing data obtained for one and the same sample but at different mea- surement positions within the gel. In the following the measurement positions will be described as upper (blue), lower (green, leading edge) and right (red) which is always meant relative to the intensity maximum detected in the respective band (see figure 4.16A).

With respect to these measurement positions all dye pairs show a similar behavior namely a high share of conformations showing

6_{The different NaCl concentrations required for DNA hairpin closure in gel and in}

solution are most likely a result of the different buffer conditions (TE, TBE, see chap- ter 4.2.2) but the actual origin has not been investigated in detail.

4.2 Influence of dye selection on DNA hairpin dynamics 83

low to intermediate FRET (0−80% FRET efficiency) at the upper and right positions compared to an enrichment of high FRET molecules at the lower measurement position.

This data is in good agreement with the in gel distribution one would expect since the more compact and less flexible closed state (showing high FRET) can be mainly found at the leading edge while the slower diffusing more open DNA strands are found up-shifted. The only exception to this behavior was found for Cy3-Cy5 where only an intermediate FRET population could be observed at 400mM NaCl in gel (data not shown) and which required 600 mM NaCl to show first signs of a distinct closed conformation(see Appendix A.5 figure A.16).

The most important feature of this observation is the fact that this spacial separation is stable for hours. A possible explanation for this behavior can be found in the presence of the dye molecules. In prin- ciple one can think of several possible effects that might influence the hairpin stability.

One possibility is that the two dyes directly interact with each other. Since all dyes have an extended π system one could think of two dyesπ stacking which would cause a stabilization of the closed high FRET state. On the other hand, if the dyes are both positively or negatively charged they would repel each other causing a destabi- lization of the closed DNA hairpin.

A similar effect should be observed for a stacking of one or both dyes to a base of the stem sequence hence causing a hindrance of closure. In the two latter cases highly dynamic bursts originating from molecules attempting to close should be observed while in the first case only a stable high FRET population should be visible (For a more extensive discussion of these possibilities see chapter 4.2.5).