{'fraction_non_alphanumeric': 0.04015582858855259, 'fraction_numerical': 0.027270002996703627, 'mean_word_length': 4.804347826086956, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
A complementary chemical probe approach towards customized studies of G-quadruplex DNA structures in live cells

G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform to generate complementary chemical probes centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design and substantial synthetic devlopments to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. We next used this reactive handle to conjugate a BODIPY fluorophore to Phen-DC3. This generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effect that revealed the localization and function of Phen-DC3 in human cells. To increase cellular uptake, a second chemical probe with a conjugated cell-penetrating peptide was prepared using the same amine-substituted Phen-DC3 derivative. The cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this method to modulate and direct cellular uptake e.g. as delivery vehicles. The applied approach to generate multiple tailored biochemical tools based on the same core structure can thus be used to advance the studies of G4 biology to uncover molecular details and therapeutic approaches.


Taq Polymerase STOP ASSAY
Taq Polymerase STOP assays were performed as previously described. 1 Briefly, 40 nM of annealed templates (Table S1)

PROTEIN EXTRACTION AND IMMUNOBLOT
HeLa cells were seeded on 10 cm dishes the day before treatment in order to have 80-90% confluency the day after. Cells were treated for 12 h with the indicated concentrations.
Nuclear histones-bound protein fractions were extracted as previously described. Tween 20 (T-TBS) buffer. Antibodies were diluted in 5% non-fat milk. Chemiluminescent detection was performed using ECL western blotting substrates (ThermoScientific) and ChemiDoc Touch Imaging System (Bio-Rad). Signal quantification was performed using ImageQuant TL Software (GE Healthcare Life Sciences).

CIRCULAR DICHROISM SPECTROSCOPY
5 μM of Pu24T G4-DNA was folded in 10 mM K-phosphate buffer (pH 7.4), with 5 mM KCl by heating for 5 min at 95 °C and then allowed for cooling to room temperature. A quartz cuvette with a path length of 1 mm was used for the measurements by JASCO-720 spectropolarimeter (Jasco Internatiol Co. Ltd.). CD spectra were recorded at 25 °C over λ = 230-350 nm with an interval of 0.2 nm and a scan rate of 100 nm/min.

FLUORESCENCE RESONANCE ENERGY TRANSFER ASSAY
The fluorescence resonance energy transfer (FRET) occurs between two dyes (5'-FAM as donor and 3'-TAMRA as acceptor) linked at both extremities of a DNA oligonucleotide.
When the oligonucleotides are folded into G4 structures, the donor and acceptor are in close proximity, which results in an energy transfer from the donor to the acceptor. This process can be detected by a reduction in the fluorescence emission of the donor.
Fluorescence emission of the donor is recovered when the temperature increment triggers the thermal denaturation of the G4 structure. The experiments were performed in a Bio-rad CFX96 real-time PCR device at temperatures from 10 to 95 °C at 1.5 °C/min heating rate using a 492-nm excitation wavelength and a 516-nm detection wavelength in 96-well plates. Each condition was tested in duplicate and analysis of the data was carried out by

General experimental
All reagents and solvents were used as received from commercial suppliers unless stated otherwise. TLC was performed on aluminum backed silica gel plates (median pore size 60 Å, fluorescent indicator 254 nm) and detected with UV light. Flash column chromatography was performed using silica gel with an average particle diameter of 50 μm (range 40−65 μm, pore diameter 53 Å), eluents are given in brackets. DMF, THF and DCM were dried in a solvent drying system (THF and DCM drying agent: neutral alumina; DMF drying agent: activated molecular sieves, also equipped with an isocyanate scrubber) and were collected fresh prior to every reaction. 1   Procedure for preparation of 2,9-dimethyl-5-nitro-1,10-phenanthroline (2) The cool mixture of nitric acid (HNO3) (5 ml) and fumed sulfuric acid (H2SO4) (10 ml) was added drop-wise to 2,9-dimethyl-1,10-phenanthroline (5 g, 24 mmol) in fumed sulfuric acid (5 mL) at 0 °C. Then, the reaction mixture was stirred at 120 °C for 3 h. Procedure for preparation 5-nitro-1,10-phenanthroline-2,9-dicarbaldehyde (3) To a solution of 2,9-dimethyl-5-nitro-1,10-phenanthroline 2 (5 g, 19.7 mmol) in 0.4% H2O and 1,4-dioxane (20 mL) was added SeO2 (5 g, 45.06 mmol) at room temperature under nitrogen atmosphere. The reaction mixture was stirred at reflux for 3 h. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was filtered through the celite pad and the filtrate was concentrated under reduced pressure. The crude compound was used for the next step without purification.
Water was added to the crude and the resulting slurry was filtered through sintered funnel.
Diethyl ether was added to the obtained solid in flask and filtered through sintered funnel and this process was continued for three times. The solid was washed with DCM and diethyl ether then dried under vacuum to give 5-(2-azidoacetamido)-N 2 ,N 9 -di(quinolin-3yl)-1,10-phen-anthroline-2,9-dicarboxamide 9 in 70% yield as brown solid.

Synthesis of Peptide (PP) (21)
Solid phase synthesis of the peptide was carried out on SyroI peptide synthesiser, starting on 4 μmol Rink Amide Fmoc-D-Arg (Pbf) tenta gel resin using NFmoc-D-Arg (Pbf), NFmoc-Cha-OH amino acids, HBTU and HOBT as the coupling reagent, DIPEA/NMP and piperidine as bases (Merck Chemicals). Following chain assembly of the Fmoc protected amino acids, the final Fmoc was removed from the peptidylresin. Next, the peptidyl-resin was added to 10 ml of trifluoroacetic acid (TFA), triisopropyl silane (TIPS) and H2O (15:1:1). After 3 h, the resin was removed by filtration and the peptide was precipitated with ice cold diethyl ether. The peptide was isolated by centrifugation, then dissolved in H2O and freeze-dried overnight. MS (ES mass): m/z 551.6 (M+1); HPLC integration analysis: 96% pure.