In search of visible-light photoresponsive peptide nucleic acids (PNAs) for reversible control of DNA hybridization

Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particular, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modification delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science.


Circular Dichroism
CD measurements were performed on a Jasco J-810 Spectropolarimeter equipped with a Jasco CDF-426S peltier controller and a Thermo Haake WKL 26 water recirculator at 20 °C using a CD cuvette (Hellma Analytics; QS 0.100). The settings were: continuous scanning mode, range from 360-220 nm, speed of 100 nm/min, response of 0.25 s, band width of 2 nm, data pitch of 0.2 nm and 5-10 accumulations per sample with standard sensitivity. The represented measurements were always buffer subtracted.

carbonyl]amino}ethyl) glycinate
The synthesis was followed the procedure of Bondebjerg et. al. [6]   The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was suspended in acetone (50 mL) and added into n-pentane (200 mL). The white precipitate was collected and washed with n-pentane (500 mL

Solid-phase synthesis
The peptide nucleic acids (PNAs) were synthesized manually according the procedures of Fmoc-based solid-phase peptide synthesis.

General protocol
The synthesis was performed in a 2 mL polypropylene reactor with plunger and frit (25 μm poresize, Multi Syn Tech GmbH). TentaGel ® S RAM resin (0.19 mmol/g, RAPP Polymere, Germany) was used as solid support. The amounts of reagents of the following synthesis protocol correspond to 1-5 μmol scale.

Swelling:
The resin was swollen in 2 mL DMF for 30 min.

Cleavage:
The dried resin was treated with a solution of TFA/m-cresol/H2O (90:5:5, v/v/v, 0.5 mL for 1 µmol scale and proportionally for bigger scales). After 2 h, the resin was washed with TFA and the combined eluates were added to dry ice-cold Et2O (1 mL of Et2O for each 100 μL of cleavage mixture). Centrifugation at 4 °C yield a precipitate which was with cold Et2O.
The residue was dried under reduced pressure.

Photoisomerization of photoswitchable PNAs
For the UV-vis characterization studies, solutions of corresponding PNA-photoswitch conjugates were prepared in phosphate buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) with a final concentration of 20 μM, the samples were placed in a 1400 μL quartz cuvette (Hellma Analytics (104F-QS)) with a pathlength of 1 cm. The cuvette was irradiated at the respective wavelengths, placed in the dark at room temperature or incubated in a 37 °C water bath for the indicated times, followed by measuring the absorbance spectra at room temperature. All determinations of each compound were repeated two times independently i.e. from two different stock solutions. For all spectra, the corresponding background signal was subtracted.
Afterwards, the sample was irradiated at the time intervals from 1 s to 30 s (1, 2, 5, 10, 15, 20, 30 s) at 405 nm ( Figure S24B). As shown in Figure S24C, after 120 s irradiation at 520 nm, maximal cis-isomer ratio was reached. The maximal conversion from cis to trans-isomer was achieved after only 2 s.

Thermal stability
A 2 μM sample containing 3 in phosphate buffer was irradiated at 405 nm for 2 min to obtain the maximal trans-isomer, the absorbance spectrum was measured once, and again after 24 h.
As shown in Figure S26, there was no back-isomerization. The sample was irradiated at 520 nm for 5 min to obtain maximal cis-isomer, the absorbance was measured after the irradiation and after the incubation at room temperature as well as at 37 °C for 24 h each. No back-isomerization was observed neither.

UV-vis characterization of PNA12(HTI) conjugate 4 Isomerization
The isomerization of HTI could be achieved under the irradiation at 405 nm for the trans-isomer, the cis-isomer could be obtained either upon irradiation at 520 nm or when heated. [7][8] A 20 µM solution of compound 4 in phosphate buffer was irradiated for 2 min at 405 nm to ensure maximal conversion to the trans state and then irradiated at 520 nm for different time intervals from 10 s to 30 min (10, 20, 30 s, 1, 2, 3, 4, 5, 7, 10, 15, 20, 30 min) ( Figure S30A).
Afterwards the sample was irradiated at different time intervals from 1 s to 1 min (1, 2, 5, 10, 20, 30, 60 s) at 405 nm ( Figure S30B). As shown in Figure S30C, after 10 min irradiation at 520 nm, maximal cis-isomer ratio was reached. The maximal conversion from cis-to transisomer was achieved after only 30 s upon irradiation at 405 nm.

Stability
The cis-isomer of HTI is thermally more stable, the trans-isomer could reconvert at room temperature to the cis-isomer within hours. [9] A 20 µM sample of compound 4 in phosphate buffer was irradiated at 405 nm for 2 min to obtain the maximum trans-isomer, absorbance spectra were measured every 15 min at room temperature on the time scale of 10 hours. As shown in Figure S32, the significant absorbance at 442 nm of the cis-isomer was increasing with time and reached maximum after 6 h.

UV-vis characterization of PNA15(Azo) conjugate 14
Thermal stability The stability was tested at different temperatures to ensure that no degradation of compound 14 occurrs during the measurements of the melting temperature. 25 μM solutions of the compound 14 in phosphate buffer were first irradiated at 365 nm for 1 h to obtain the maximum cis-form and at 430 nm for 30 min to obtain the maximum trans-form, then the samples were incubated for 10 min at various temperatures from 20 °C to 90 °C, the UV-vis spectra were recorded to detect changes.  Figure   S36 shows the RP-HPLC chromatograms of compound 14 with trans/cis-ratios after irradiation, tR = 16.77 min corresponds to the cis-isomer and 17.97 min to the trans-isomer (Gradient I). Where n represents the frequency of occurrence of the bases in the conjugate.
The concentration of photoswitch modified PNAs were determined by applying the following molar extinction coefficients with Lambert-Beer law. All melting curves were measured in phosphate buffer (10 mM NaH2PO4, 150 mM NaCl, pH = 7.4) at a heating rate of 2 °C/min. In order to obtain maximal isomer, the melting temperature PNA(oF4Azo) was measured without pre-hybridization due to the difficult photoisomerization of PNA(oF4Azo)/DNA duplex. As the cis-isomer of PNA(Azo) is not thermally stable, its photoisomerization was performed after the hybridization, Sample solutions of compounds 6-11 (cis/trans)-isomer conversion was achieved by irradiation of the sample at room temperature with 405 nm/520 nm LED for oF4Azo (cis/trans) and HTI Trans-isomers of compounds 12-17 were prepared by using the method described above.
Cis-isomers of compounds 12-17 were prepared by mixing respective PNAs and the complementary DNA (5'-GTG AGC CAA GAA ACA CTG CCT-3'), heating the sample to 90 °C and keeping them at this temperature for 15 min before cooling to room temperature during 3 h. Isomer conversion was achieved by irradiation of the hybridized sample at room temperature at 365 nm for 1 h for Azo (cis). Likewise the UV melting curve was measured from 20 °C to 95 °C at 260 nm in 2 °C/min steps.
All melting temperatures were determined from the first derivative of the absorbance temperature trace using OriginPro 2016 software. All determinations were repeated two times independently i.e. from two different stock solutions. For all data, the corresponding background signal was subtracted, and the melting curves were normalized.
The sequences of all employed PNAs and DNAs are summarized in Table S2.  The absorbance differentials at 260 nm were plotted against temperature and normalized.
Obtained UV melting curves and calculated first derivative curves for the studied compounds are given in Figures S37 to Figure S45.  Table S3 compares isomer differences between those obtained in the melting temperature experiments and fluorescence displacement assays for the PNA probes 6-9.

CD Experiments
For the characterization of the binding between our probes and DNA the concentrations of the PNAs 9, 18 and 19, compound 1 as well as complementary ssDNA (5'-GTG AGC CAA GAA ACA CTG CCT-3') were determined as described in the UV melting curves section. Solutions of 5 µM compound, ssDNA or 1:1 ratio of PNA and ssDNA in phosphate buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.4) were prepared in microcentrifuge tubes from 100 mM stocks in phosphate buffer, transferred to a CD cuvette. Isomer conversion was achieved by irradiation of the sample at room temperature at 405 nm for 5 min for trans-or for 10 min at 520 nm for the cis-isomer. Samples were kept in the dark and the measurements were performed in the absence of light. Spectra were buffer subtracted and display the mean out of two independent measurements. The final spectra were smoothed with a fourth order Savitsky-Golay smoothing algorithm [13] using a smoothing window of 25 data points. Figure   S52 shows an example of the dataset for the 1:1 ratio of PNA 18 and complementary DNA before and after smoothing.