Basic knowledge of G-quadruplexes

5.1.1 Common structure of G-quadruplexes

Different to Watson-Crick bonds between complementary nucleic acid bases (A to T and C to G), a G-quartet plane is formed with four guanine bases which are connected to each other in a ring with a total of 8 hydrogen bonds (in the form of Hoogsteen bonds), as shown in Figure 5.1 a^{4, 5}. Two or more such G-quartet planes can be stacked together due to the hydrophobic and base stacking interaction. The typical distance of two adjacent stacked quartet planes is 0.34 nm, similar to the base distance in dsDNA, due to the preferred distance of best stacking interaction.

One-dimensional G-wires can be formed by binding together four individual strands with all guanine bases (with length up to micrometers). A single strand of DNA containing repeated guanine bases can fold to a quadruplex with other bases (T, A or

into quadruplex structures . A particular example is the human telomer sequence which contains repeated sequence of [-TTAGGG-] ⁸. In addition, potential G-quartets forming sequences have also been found to be enriched in promoters of proto-oncogenes⁹.

The G-quartet stacking can be further stabilized by cations (typically K⁺ or Na⁺) located in the center of a quartet plane or between two quartets. Because there are more overlapping π electrons between quartet planes and its structure is less flexible compared to dsDNA, it is proposed that DNA in the G-quadruplex structure may have better conductance ¹⁰. Furthermore, K⁺ or Na⁺ ions trapped in the quadruplex center may also improve the conductance because they provide hybridization with the base stack and an intrinsic doping factor, similar to electronic modifications introduced in dsDNA helices by metal cations inserted in the inner core ^11-14.

Figure 5.1: a, G-quartet plane with a cation in the center (green sphere); green dashed lines: hydrogen bonds; the scale bar represents 0.5nm. b, Structure of a parallel G-quadruplex with two cations of a 22mer oligomer (TG3[TTAGGG]3) obtained by x-ray diffraction⁸ (pdb entry: 1KF1); c, Structure of mixed parallel/anti-parallel with the same sequence obtained with NMR¹⁵ (pdb entry:2HY9).

For clarity only the guanine bases and the sugar-phosphate backbone (blue line) are shown. Distances between adjacent quartet planes are 0.34 nm.

5.1.2 Conformation variety of intramolecular G-quadruplexes

Dependent on the chain orientation (parallel or anti-parallel), G-quadruplexes, even with the same sequence, have different conformations^{16, 17}. For example, DNA oligomer with human telometric sequence (TG3[TTAG3]3) can adapt 28 conformations, in which four of them have been crystalized and their structures have been determined with x-ray diffraction or NMR ^{8, 18, 19}. When Na⁺ is present, it is most likely to form an antiparallel quadruplex. When K⁺ is present, either a parallel quadruplex (as shown in figure 5.1b) or mix parallel-antiparallel quadruplex (Figure 5.1c) can be formed.

(a) (b) (c)

Folding to G-quadruplex structure from a random coil happens in micro-seconds when Na+ or K+ ions are added to the molecule solution in pure water ^20. Upon a high temperature or upon external force (as in case of opening of MCBJ), the quadruplex can be melted or forced to unfold to single strands again ²¹. The temperature or the required unfolding force is dependent on several factors, such as the G-quadruplex conformation or the ion (K+ or Na+) concentration. The free energy change during unfolding of G-quadruplex is on the order of a few KJ/mol as has been measured with Differential Scanning of Calorimetry (DSC) 22, which is much smaller than the binding energy between thiol and gold (~70 kBT, 200 KJ/mol). So, where opening a MCBJ with a G-quadruplex bond between the electrodes, the G-quadruplex is unfolded first before it is detached from the electrodes. During this force-induced unfolding, the quartet planes are deformed and lose stacking between each others, which could have dramatic effect on the measured conductance signal.

5.1.3 Methods to measure conductance of G-quadruplexes with MCBJ

R

I R

I

Figure 5.2: Sketch to show the conductance measurement of G-quadruplex with MCB set-up: (left) A drop of G-quadruplex solution is deposited to a narrow (100 nm wide) metallic bridge which is bent by pushing the central support of the substrate.

(right) Measuring dc conductance in MCBJ with a single G-quadruplex between the gold electrodes. A parallel quadruplex structure is shown here.

The 23mer oligonucleotide (sequence: 5’-T*GGG[TTAGGG]3T*-3’, we named it as G1 in the following) was synthesized by standard automated DNA oligonucleotide synthesis ²³. T* are thymine bases that contain protected thiol groups on their 5 position, which directly connect to the π-system of the nucleobase to allow binding of the oligonucleotide to gold electrodes without alkyl spacing ²³. Mass spectroscopy has been applied to verify the integrity of the oligonucleotides. As

named it as C1 in following) was synthesized by the same method but all the guanine bases were replaced by cytosine. C1 will be in the single strand form unless it is in a low pH (~5) solution ²⁴. In our experiment, we used buffer solution with pH about 7.0 in which case most C1 molecules are in single-stranded form. The molecules were dissolved in pure water (100 μM) and kept in the refrigerator (-20°).

Before measurement, the oligomers were diluted with PBS buffer (40mM K2PO4

and 26 mM KH2PO4, pH 6.98) to a final concentration of 10 μM. In this solution (~100 mM K⁺), the 23mer G1 oligonucleotide would adapt a parallel or mixed parallel/anti-parallel quadruplex structure ²⁵ (see Figure 5.1 b and 5.1 c) while the C1 would still be in single-stranded conformation.

The transport experiments were conducted at room temperature with a MCBJ setup^26-29. Similar to the experiments shown in chapter 4, we pre-open the MCBJ, then a droplet of 10 μl G-quadruplex (or control sample) solution (10 μM) was positioned on top of the MCBJ. We monitored in real time the resistance between the electrodes continuously by maintaining a small bias voltage (100 mV). When the gap of the electrodes was around 0.6-1 nm, a voltage of -1V to1V was swept between the electrodes to attract molecules to the region between the electrodes. Thiol ends with protecting groups at both ends of the G1 or C1 samples allow their binding between two electrodes²³. We measured the conductance both in buffer solution and in vacuum, presented in section 5.2 and 5.3 respectively.

5.2 Conductance characterization of quadruplex in aqueous