Tuning of Oxidation Potential of Ferrocene for Ratiometric Redox Labeling and Coding of Nucleotides and DNA

Abstract Three sets of 7‐deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcXTPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox‐labelled DNA probes. Square‐wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc‐labelled A (dAFc) and FcPa‐labelled C (dCFcPa) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox‐labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA.


Synthesis of ferrocene-labeled dNTPs
Synthesis of modified nucleoside triphosphates -Sonogashira cross-coupling Method A: A 1:1 mixture of H2O-CH3CN (2 mL) was added through a septum to an argonpurged flask containing a halogenated nucleotide dN I TP (1 equiv.), FcX (1.5 equiv.), CuI (10 mol %), Et3N (10 equiv.), PPh3 (2 mol %) and [Pd(PPh3)2Cl2] (5 mol %). The flask was evacuated and purged with argon, and then the reaction mixture was stirred at 60 ºC for 1 h until complete consumption of the starting material and then evaporated under vacuum. Product was isolated from the crude reaction mixture by HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB (triethylammonium bicarbonate) in H2O to 0.1 M TEAB in H2O-MeOH (1:1) as eluent. Several co-distillations with water followed by freeze-drying from water gave a solid product.

Synthesis of modified nucleoside triphosphates -triphosphorylation
Method B: POCl3 (2.5 equiv) in PO(OMe)3 (1 ml) was added through a septum to an argonpurged flask containing modified nucleosides dN EPT (1 equiv.), Reaction mixture was then stirred at 0 ºC for 12 h until complete consumption of the starting material. Then an ice-cooled solution of (NHBu3)2H2P2O7 (5 equiv) and Bu3N (4.2 equiv) in dry DMF (2 ml) was added and the mixture was stirred at 0 °C for another 1.5 h. The reaction was quenched by addition of 2 M aqueous TEAB (triethylammonium bicarbonate) (2 ml) and the solvents were evaporated under vacuum and the residue was co-distilled with water three times. The product was isolated by HPLC on a C18 column with the use of a linear gradient of 0.1 M TEAB in H2O to 0.1 M TEAB in H2O-MeOH (1:1) as eluent. Several co-distillations with water followed by freeze-drying from water gave solid product.

General remarks
All gels were analysed by fluorescence imaging using Typhoon FLA 9500 (GE Healthcare).
In the template ONs the segments forming duplex with the primer are underlined. Samples were analysed with a 12.5 % denaturing polyacrylamide gel electrophoresis (1 h, 50 °C) and visualised using fluorescence imaging ( Figure S1).

Single incorporation (templates temp
Method B: PEX reactions with temp C were performed in the same way as described for temp A except either dCTP or dC FcX TP (4 mM, 1μl) were used ( Figure S1).

Kinetics of incorporation of modified dNTPs (templates temp termA and temp termC )
The simplified kinetics studies in the presence of KOD XL DNA polymerase were performed to examine the incorporation efficiency of modified dN FcX TPs by PEX experiments in comparison with natural dNTPs (Figures S3-S6). The rates of the PEX with natural dNTPs or modified nucleotides were revealed using templates temp termA or temp termC and primer rnd . The   (50 μL) was then added and the sample was denatured for 2 min at 70 °C and 1000 rpm. The beads were collected on a magnet and the solution was transferred to a clean vial. The product was analysed using MALDI-TOF mass spectrometry (Table S2).

MALDI-TOF measurements
The MALDI-TOF spectra were measured with 1 kHz smartbeam II laser technology. The measurements were carried out in reflectron and linear mode using the droplet technique, with the mass range up to 30 kDa. The matrix consisted of 3-hydroxypicolinic acid (HPA)/picolinic acid (PA)/ ammonium tartrate in ratio 9/1/1. The matrix (1 μL) was applied to the target (ground steel) and dried down at room temperature. The sample (1 μL) and matrix (1 μL) were mixed and added on the top of the dried matrix preparation spot and dried at room temperature.

Multiple incorporation (template temp rnd16-bio )
The   After the incubation, the magnetic beads were collected on a magnet (DynaMag-2, Invitrogen) and the solution was discarded. The beads were washed successively with Wash buffer TEN 500 (10 mM Tris, 1 mM EDTA, 500 mM NaCl, pH 7.5; 3 × 500 μL), and Milli-Q water (4 × 300 μL). Water (100 μL) was then added and the sample was denatured for 2 min at 72 °C and 900 rpm. The beads were collected on a magnet and the solution was transferred to a clean vial.
After magnetoseparation, small aliquots (2 μL Visualisation was performed using fluorescence imaging ( Figure S9). All single-stranded oligonucleotide products were lyophilized, and before use were dissolved in 10 μL of Milli-Q water.

Polymerase chain reaction (PCR) with modified dNTPs
The PCR was performed with a C1000Touch thermal cycler (Biorad) using following cycling protocol: 95 °C for 3 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, followed by a final elongation step at 72 °C for 5 min.

Electrode fabrication and functionalisation
Gold sputtered electrode arrays consisting of 3 groups of 3 circular working electrodes (1 mm 2 ), were fabricated on a glass substrate as described previously. 3 Prior to functionalisation, the electrodes were cleaned by sonicating for 5 minutes in acetone, followed by 5 minutes in isopropanol and were then rinsed with Milli-Q water and dried with N2. Subsequently, the electrodes were electrochemically cleaned in KOH (0.1 M) using cyclic voltammetry with a scan rate of 100 mV/s, from 0 to -1.2 V vs Ag/AgCl, for 10 scans. The electrodes were then rinsed with Milli-Q water, dried with N2, and again electrochemically cleaned, this time in H2SO4 (0.1 M), with a scan rate of 100 mV/s from 0 to 1.6 V vs Ag/AgCl, again for 10 cycles, before a final rinse with Milli-Q water and drying with N2.
A capture probe cocktail solution was prepared to contain 1 µM capture probe, 100 µM of mercaptohexanol and 1 M KH2PO4. One microlitre of the capture probe cocktail was dropcast on to each working electrode and the array was then incubated for 3 h at 22 ºC in a humidity saturated chamber. Following functionalisation, the electrodes were rinsed with Milli-Q and then dried with N2. A 7 µL volume chamber was created to host the PEX purified products for hybridisation and electrochemical detection, by placing a PMMA cover over a patterned double adhesive gasket. Once assembled, the microfluidic chambers were washed with 20 µL of PBS Tween-20, then 200 µL of milli-Q and finally dried them with N2 prior to use.

Agarose gel electrophoresis
PEX products were visualised using agarose gel electrophoresis, which was prepared with ultra low pure agarose (4 % w/v) in 1× Tris-Borate-EDTA buffer (TBE) and stained with GelRed™nucleic acid stain. PEX product (3 µL) was added to 3 µL of loading buffer 2X (stock buffer 6X contained glycerol 30 %, bromphenol blue 0,25 %) per well, and electrophoresis was carried out with an applied voltage of 100 V for 40 min. The DNA was imaged using a UV transilluminator (λ = 254 nm) ( Figure S16). Figure S16. Agarose gel electrophoresis of PEX products using temp 8A,2C , temp 8C,2A or temp 4C,4A templates and primer 8/2_4/4 obtained in the presence of (a) all four natural dNTPs (+), or combination of dA Fc TP, dC FcPa TP with TTP and GTP (*Fc). P/T corresponds to primer hybridised to template in the absence of polymerase.

Electrochemical measurements of PEX products
Square wave voltammetry was performed using a potentiostat/galvanostat PBSTAT 12 Autolab controlled with Nova 2.1.3 software. Following hybridisation, measurements were carried out in 0.1 M Ca(NO3)2 electrolyte solution using an external Ag/AgCl reference electrode and the internal counter and working gold electrodes on the electrode array, with a step potential from 0 V to 0.6 V using a 1 mV step, 20 mV modulation amplitude and 50 Hz of frequency.

Hybridisation for liquid phase PEX products
PEX product (7 µL) was added to each electrode array chamber (3 working electrodes/chamber,