Baricitinib phosphate We have previously designed an azobenz
We have previously designed an azobenzene-based molecule, Razo, which could switch the conformations of G-quadruplex structures by photoregulation. 12, 13, 14, 15 However, it lacks selectivity to different G-quadruplexes. Therefore, we want to find a way to modify this molecule so that it could be used to stabilize specific G-quadruplex structures of interest as we expected. The ideal method is the use of in situ hybridization (ISH) techniques that use a complementary DNA strand to localize the probe to a specific DNA sequence. 16, 17 Enlightened by the powerful ISH technique, we decided to add a DNA anti-tail sequence complementary to a tail sequence that is adjacent to the G-rich sequence of interest to the stabilizer. We expect it will be a versatile method to turn universal compounds that stabilize G-quadruplex structures into specific ones.
Results and discussion To test the application of this ISH technique, (TTAGGG)-repeating sequences located in the human telomeric DNA, were chosen as the targets. These sequences can fold into G-quadruplex structures, and induce telomere end protection and telomerase inhibition.  As shown in Fig. 1A, we expect that the anti-tail will bring the stabilizer and the defined G-rich sequence together to enable its stabilization through binding to the G-quadruplex structure. Herein, we designed and synthesized a Razo-DNA probe, containing a Razo stabilizer, and an oligonucleotide as the director. In particular, an azido side chain was introduced into the Razo moiety to create the new molecule, Razo-ba (Fig. 1B). In this way, we can conjugate the Razo with commercially purchased alkyne-modified oligonucleotides conveniently by a click reaction, without affecting its stabilization ability (Fig. 1C). The Razo-ba was prepared following the protocol depicted in Fig. 2. Compound Razo-ba was synthesized by treating 4,4-dihydroxyazobenzene with 1-(2-chloroethyl)piperidine hydrochloride according to literature  to obtain 1,2-bis(4-(2-(piperidin-1-yl)ethoxy)phenyl)diazene, which was subsequently transformed to the final product. The Baricitinib phosphate Razo-ba was fully characterized by NMR spectroscopy and HRMS methods (SI). The ability of Razo-ba to stabilize the G-quadruplex structure was first evaluated by circular dichroism (CD), which is a reliable method for distinguishing DNA secondary structures (Fig. 3). As shown in Figure 3, Razo-ba can stabilize the G-quadruplex structure effectively. Upon the addition of Razo-ba, the CD spectrum of the G-rich sequence, HT, appears as a parallel G-quadruplex structure, which can be identified by a positive peak at 265 nm and negative peak at 240 nm. This result is consistent with the previously reported Razo without modification. 12, 13, 14, 15 In particular, the stabilization ability reached the peak when the ratio of Razo-ba and G-quadruplex was 7:1. The result indicated Razo-ba was able to induce the formation of parallel G-quadruplex structure without the addition of any metal cations. Therefore, the planar azobezene could still stabilize the G-quadruplex structure after the introduction of azido side chain. To investigate the binding property of the Razo-ba, UV–visible titrations were also carried out to determine the binding constant of the Razo-ba towards the G-quadruplex DNA. The binding constant was calculated to be 2.1 × 105 M−1 (Fig. S7). The Polymerase Stop Assay was introduced to identify the utility of the Razo-DNA probe, because the CD spectra of double stranded DNA formed by the tail and anti-tail sequences are overlapped with the CD spectra of G-quadruplex DNA. This assay has been used to evaluate the stability of G-quadruplex in the presence of the probe which is known to interact with DNA. Fig. 4 shows that without the G-quadruplex stabilizer Razo-ba, in the presence of K+, the DNA extension pauses at the G-rich site. Fig. 4 also shows that only the extension of the sequence with a 2 nt separation is blocked. It indicates that only having the Razo-ba located at an appropriate distance from the G-quadruplex structure can result in an effective block of the DNA polymerase. The separation of 2 nt is the best distance between the tail sequence and G-quadruplex forming sequence. In contrast, the Razo-ba alone even with high concentration cannot stabilize the G-quadruplex at the temperature of 55 °C. Therefore, the DNA anti-tail with proper length can bring the Razo closer to the G-quadruplex so that it will stabilize the G-quadruplex with high specificity. To further identify the selectivity of the Razo-DNA probe to the targeted G-quadruplex DNA against others, three mismatched G-quadruplex DNAs were used in the Polymerase Stop Assay. Razo itself without any modification was also used as a control here. The result showed that this probe cannot stabilize the mismatched G-quadruplex DNAs (Fig. S8). Additionally, three marker DNAs were used in the same assay precisely identify the DNA polymerase stop site. The result indicated that the primer extension stopped right before the (TTAGGG)-repeating (Fig. S9).