Sequence- and Temperature-Dependent Intrastrand Photocrosslinking of 5-Fluoro-2’-O-methyl- 4-thiouridine Modified Oligonucleotides

doi:10.21203/rs.3.rs-3358580/v1

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Sequence- and Temperature-Dependent Intrastrand Photocrosslinking of 5-Fluoro-2’-O-methyl- 4-thiouridine Modified Oligonucleotides

https://doi.org/10.21203/rs.3.rs-3358580/v1

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DNA photocrosslinking reactions occur widely in biological systems and are often used as a valuable tool in molecular biology. With regard to the latter, highly efficient and selective photoactive DNA crosslinking agents are of particular interest. 4-Thiouridine is one of the most frequently used photoactivatable DNA crosslinking agents. Our previous studies revealed that it’s 5-halogeno derivatives, namely 5-fluoro- and 5-chloro-4-thiouridines (FSU and ClSU, respectively), incorporated into double stranded DNA oligonucleotide form highly fluorescent interstrand crosslink with thymidine in nearly quantitative yield. Here we reported the sequence- and temperature dependent formation of intrastrand crosslink products from the irradiation of single stranded oligodeoxynucleotides bearing FSU. Our results showed that two types of intrastrand crosslink products could be formed, namely the fluorescent one with T and nonfluorescent with C. Moreover, partial photooxidation of FSU residue to 5-fluorouridine was also observed. Our studies confirm the possibility of intrastrand photocrosslinking of nonadjacent bases in short oligodeoxynucleotides. The results were supported by molecular dynamics simulations and are valuable for the future designing of FSU-labeled probes for fluorescence-based detection of specific DNA sequences.

Physical sciences/Chemistry

Physical sciences/Chemistry/Biochemistry

Physical sciences/Chemistry/Chemical biology

Physical sciences/Chemistry/Organic chemistry

Physical sciences/Chemistry/Photochemistry

Physical sciences/Chemistry/Theoretical chemistry

The formation of crosslinks in DNA causes inhibition of transcription and replication as a result of covalently bonded DNA strands.^1,2 To understand the mechanism of crosslinks repair process and to learn crosslink structure, efforts are being made to efficiently synthesize short DNA oligonucleotides that can form crosslink, as a model compounds.^3–8 The formation of crosslinks can modulate nucleic acid conformation⁹ including aptamer,¹⁰ G-quadruplex^11,12 and i-motif structures.¹³ Moreover, crosslinking has the potential application for detection of specific DNA/RNA sequences ^14–20 and identifying targets for bioactive small molecules.²¹

Crosslinking of DNA can be induced by chemical reaction or activated by UV irradiation.²² Among the photoactivable crosslinking agents belonging to modified nucleobases, 4-thiouridine (4SU)^23–28 and 6-thioguanosine (6SG)^29,30 should be distinguished. We have previously described the UV induced interstrand crosslinking of 5-halogeno DNA duplexes labeled with derivatives of 4SU, namely 5-fluoro-2’-O-methyl-4-thiouridine (FSU)³¹ and 5-chloro-2’-deoxy-4-thiouridine (ClSdU).³² Similarly to the native 4SU, FSU and ClSU show a high photoreactivity towards thymidine. However, while 4SU forms predominantly the 6 − 4 and 5 − 4 pyrimidine-pyrimidone type adducts with thymidine,^33,34 FSU and ClSU undergo photocycloaddition with thymidine to produce unique pair of diastereomeric, highly fluorescent and thermally stable tricyclic photoproducts.³⁵ As we have already demonstrated the photoreaction occurs both under selective excitation monomeric FSU in the presence of thymidine,³⁵ as well as in double stranded oligonucleotides leading to fluorescent interstrand crosslink formation with nearly quantitative yield.^31,32 The interstrand DNA photocrosslink formation via [2 + 2] cycloaddition to the C5-C6 double bond of thymidine or other pyrimidine nucleosides was observed in the case of coumarin,^36–39 psolaren^40–42 and 3-cyanovinylcarbazole.^43,44 Recently the thymine base selectivity in photocrosslinking was reported for 4-methylpyranocarbazole modified DNA duplexes.¹⁸

The formation of fluorescent crosslink enables easy and quick monitoring of the reaction and has great potential for application for rapid, fluorescence based detection of specific DNA sequences. We have shown that DNA duplexes labeled with FSU are capable of photocrosslinking T and the process can be monitored by the increase in the fluorescence intensity during the formation of photoadduct between FSU and T.^31,45 For application purposes, it is essential that a DNA fluorescent probe sequence binds specifically to the target DNA. In the case of interstrand DNA photocrosslinking, Watson-Crick base pairing and strands complementarity meet this requirement. However, there is a risk that in the absence of the target sequence, a competitive reaction of intrastrand photocrosslinking will occur. For this reason, it is necessary to study possibility of intrastrand crosslinks formation because it can generate false detection signal (Fig. 1). In this report, we present the results of irradiation of synthetic, single stranded oligodeoxynucleotides labeled with FSU. Depending on the ODN sequence the formation of intrastrand crosslinks between FSU and T and FSU and C was observed. The influence of temperature of irradiated solutions on the distribution of photocrosslinks and other photoproducts will also be discussed.

Oligodeoxynucleotides modified with 5-fluoro-2’-O-methyl-4-thiouridine (FSU) (ODN 1–3) (Table 1) were synthesized using "ultra-mild" DNA synthesis, as described previously.⁴⁵

Table 1

Sequences of oligodeoxynucleotide (5’→3’) containing 5-fluoro-2’-O-methyl-4-thiouridine (FSU).
	1	2	3	4	5	6	7	8	9	10
ODN 1	C	G	A	T	A	C	G	A	FSU	A
ODN 2	A	FSU	A	G	C	A	T	A	G	C
ODN 3	A	T	A	G	C	A	FSU	A	G	C

ODN 1–3 have the same nucleosides composition. In ODN 1, FSU is located as 9th nucleoside in the sequence (near 3’ end of strand), while in ODN 2 (it’s reversed sequence), FSU is placed as 2nd nucleoside of oligonucleotide (near 5’ end of strand). ODN 3 has the same sequence as ODN 2 but positions of FSU and T are converted.

Aqueous solutions of ODN 1–3 in 0.1M phosphate buffer, pH 7.0, were irradiated at 5℃, 20℃ and 35℃ and the progress of reaction was monitored by HPLC (Fig. 2). Under these conditions (λ = 355 nm) selective excitation of FSU is achieved. For all oligonucleotides, after irradiation with 355 nm for 150s the formation of the same photoproducts (a-d) with appropriate nucleosides sequence 1–3 (corresponding to ODN 1–3) was observed but with different yields (Fig. 2A, 2B, 2C). Since ODNs 1–3 have the same nucleoside composition, photoproducts (a-d) obtained from ODNs 1–3, respectively, have similar UV absorption spectra (Fig. 1S). For ODN 1, fluorescent photoproducts 1a and 1b were formed as the main photoproducts (Fig. 2A), while for ODN 2, only trace amounts of photoproducts 2a and 2b were formed (Fig. 2B). For fluorescence spectra of 1a and 1b see Supplementary Fig. 2S. Non-fluorescent photoproduct 2c was observed in the mixture after irradiation of ODN 2 as the dominant one (Fig. 2B), while the product with the same spectroscopic properties was also detected in the case of ODN 1 (1c) but with significantly lower yield. Moreover, this type of product was not observed after ODN 3 irradiation (Fig. 2C). The formation of photoproduct 2d or 3d was noticed in the mixture after irradiation of ODN 2 or 3, respectively (Fig. 2B, 2C). However, it was not formed in the case of ODN 1.

In the case of ODN 3 (Fig. 2C) the formation of intermediate photoproducts with retention times between 15–17 minutes was observed. Their absorptions spectra are presented in Fig. 3S. After performing an additional experiment of irradiation of ODN 3 for 10 minutes at room temperature, we noticed the total conversion of starting ODN 3 and intermediate photoproducts to tree photoproducts 3a, 3b and 3d (Fig. 2D).

The main photoproducts after irradiation of ODN 1–3 were isolated and separated by HPLC and their UV absorption (Fig. 3), fluorescence (Fig. 2S) and MALDI TOF MS spectra (Table 1S, Fig. 4S-7S) were recorded. Photoproducts 1a, 1b, 2a, 2b, 3a, 3b have the same absorption and emission spectra characteristic for previously observed fluorescent photoadduct of FSU with T,³⁵ and were identified as two diastereomeric (a,b) intrastrand photocrosslinks of FSU with T (Fig. 3).

The UV absorption spectra of photoproducts 2d and 3d have a maximum at 260 nm and lack of the band with a maximum at 340 nm (characteristic for thiocarbonyl group) is observed (Fig. 1S). In MALDI TOF MS spectrum of the product 3d (Fig. 7S) the signal corresponding to a mass reduced by 16 compared to ODN 3 is present. This prompt us to claim that 3d and 2d are the products of photooxidation of FSU to 5-fluorouridine (Fig. 3). To identify photoproduct 2c, an additional experiment was performed by irradiating FSU in the presence of cytidine in a phosphate buffer, pH 7.0. The triple molar equivalent of cytidine was used to minimize the formation of photoadducts of FSU itself.⁴⁶ Comparing the absorption spectra of the products obtained after irradiation FSU + C, a similarity of the UV spectrum of one of the products to the spectrum of photoproduct 2c formed after irradiation of ODN 2 was noticed (Fig. 8S). This product was isolated from the reaction mixture and based on the result obtained from its ESI MS spectrum (Fig. 9S) and MALDI TOF MS spectrum of photoproduct 2c (Fig. 5S) we suggest that in the case of ODN 2 the intrastrand photocrosslink with C is formed (Fig. 3).

Numerous studies have shown that the formation of pyrimidine-pyrimidone photoadducts involves 2 + 2 photocycloaddition of C = S to C5 = C6 of another pyrimidine leading to the formation of a thermally unstable thietane in the first step.^33,34,47,48 We propose that this process in the case of reaction of FSU or ClSU with T is followed by thietane ring opening leaving the thiol on C6 of the pyrimidine part and ring closure with concomitant loss of HF to form tricyclic structure of diastereomeric photocrosslinks a,b (Fig. 10S). We were able to observe the thietane experimentally for duplexes labeled with ClSU.³² The photoadduct formation of 4SU with C observed in E. coli tRNA is well described.^33,34,47,48 We propose analogous pathway for the photoaddition of FSU to C with elimination of H₂S and formation of non-fluorescent 4–5 photoadduct c (Fig. 10S).

Since short single stranded oligonucleotides show very high flexibility, it is difficult to unequivocally determine from the obtained results which of the cytosines C1 or C6 and C5 or C10 for ODN1 and ODN2 respectively, participates in the photocrosslinking reaction with FSU. To assess the conformational flexibility of ODN 1 and ODN 2 explicit-solvent molecular dynamics simulations were performed. The simulations helped to investigate the possibility of interaction between FSU and cytosines present in the tested oligonucleotides. Both the systems were run for 1000 ns and distances between the thiocarbonyl group of the FSU and the cytosine C = C double bond were measured. The simulations showed that cytosines located at the ends of the tested oligonucleotides are able to come into closer contact with FSU compared to the cytosine in the middle of the sequence (Fig. 4) and most likely they react with FSU to form non-fluorescent adduct.

Since temperature is one of the factors affecting the conformational flexibility of a single-stranded DNA molecules, we irradiated ODN 1–3 under different conditions (at 5℃ and 35℃). HPLC analyses of these experiments are presented in Fig. 2. Different conversion of the starting ODN was observed (Fig. 2E) after the same irradiation time (150 s). For all oligonucleotides, an increase in conversion was observed with increasing irradiation temperature. The highest conversion value was obtained for ODN 1 at 35℃ (95%), while at low temperature, at 5℃ for ODN 2 (86%). At room temperature, the highest reactivity was also observed for ODN 1. In the case of ODN 3, a clear difference in reactivity was observed depending on the temperature (from 50% at 5℃ to 93% at 35℃). Taking into account the reactivity of FSU toward intrastrand crosslink formation with T, the highest selectivity was noticed in the case of irradiation of ODN 1 in 35℃. Also for others oligonucleotides an increase in temperature resulted in an increase in the photocrosslinking with T, while lowering the irradiation temperature favored the photocrosslinking reaction of FSU with C. In the case of ODN 3, competition between the photocrosslinking reaction of FSU with T and the photooxidation of FSU was observed. In this case, the increase in temperature also caused an increase in the photocrosslinking yield.

To conclude, the results showed that the intrastrand photocrosslinking reaction can occur in short DNA fragments between bases which are not in close proximity. Regardless of the fact that FSU shows much higher reactivity towards thymine than cytosine, the factor determining the direction of the intrastrand photocrosslinking reaction in short single-stranded DNA oligonucleotides is their sequence and the position of FSU in the chain. The obtained results are valuable for the future designing of FSU-labeled probes for fluorescence-based detection of specific DNA sequences. Since the formation of fluorescent photocrosslink is only possible with T, substitution of T by dU can be considered when designing a probe with FSU in order to avoid a false-positive detection signal in the case of intrastrand photocrosslinking.

Instruments

High-performance liquid chromatography (HPLC) was performed with an Agilent 1200 system with a binary gradient-forming module and diode-array UV–Vis and fluorescence detectors. Steady-state photochemical irradiation experiments were carried out in 1 cm × 1 cm rectangular fluorescence cell with stirring bar on a standard optical bench system equipped with an Coherent Genesis CX-355-100 cw laser and with the temperature-controlled cel holder (Quantum Northwest, model TC125). Absorption spectra were measured on a JASCO V750. The MS analyses were performed using the MALDI-TOF MS instrument model Autoflex II equipped with a reflectron (resolution about 5000 at m/z 1000), on a MALDI metal target plate (Bruker, Bremen, Germany). The instrument was equipped with a SmartBeam laser and operated under FlexControl. Spectra were calibrated in FlexAnalysis using the Protein Calibration Standard I from Bruker, 3-hydroxypicolinic acid was used as matrix. Fluorescence excitation and emission spectra were measured at room temperature using a JASCO Spectrofluorometer FP-8200. High resolution ESI mass spectra (HRMS) were obtained using Impact HD mass spectrometer (Q-TOF type instrument equipped with electrospray ion source; Bruker Daltonics, Germany). The sample solutions (DCM:MeOH) were infused into the ESI source by a syringe pump (direct inlet) at the flow rate of 3 µL/min. The instrument was operated under the following optimized settings: end plate voltage 500 V; capillary voltage 4.2 kV; nebulizer pressure 0.3 bar; dry gas (nitrogen) temperature 200℃; dry gas flow rate 4 L/min. The spectrometer was previously calibrated with the standard tune mixture.

Synthesis of oligonucleotides ODN 1–3

Synthesis was performed on DNA/RNA Synthesizer H-6 (K&A Laborgeraete) applying 0.2 µmol protocol and commercial CPG supports. Oligonucleotides were synthesized using previously published method.⁴⁵ Oligodeoxynucleotides were purified by reversed phase HPLC (Waters XBridge OST C18 Column, 2.5µm, 10 x 50 mm, 40℃ with mobile phases: A = 0.01M CH₃COONH₄, B = 0.01M CH₃COONH₄/ acetonitrile, 50/50, v/v, with a diode array detector monitoring at 260 nm) using the following solvent gradient: 0–10%B in 10 minutes, flow rate: 1 ml/min. The residue was evaporated and dissolved in 0.7 ml H2O and passed through a NAP 25 column to remove CH₃COONH₄. The column was washed with 0.1M phosphate buffer (pH = 7) and 7 fractions of 1.2 ml were collected and checked by UV. Fraction 3–5 containing oligomer were combined.

Irradiation experiments and isolation of photocrosslinks

Irradiations near UV light of ODN1-3 were carried out under aerobic conditions at 5℃, 20℃ and 35℃ in 0.1M phosphate buffer, pH 7.0, A₂₆₀ = 1, with 80mW optical laser power. The progress of the reaction was monitored by HPLC (analyses were performed on a Agilent Poroshell 120 EC-C18Column, 2.7µm, 4.6 x 150 mm, 40°C, eluted with 0.1M TEAA, using a linear gradient of 5%-15% of acetonitrile over 20 min, flow rate: 0.5 ml/min (Fig. 1A-C) and on Waters XBridge OST C18 Column, 2.5µm, 4.6 x 50 mm, 40℃, eluted with 0.1M TEAA, using a linear gradient of 7%-10% of acetonitrile over 10 min, flow rate: 1 ml/min (Fig. 1D)). The reaction mixture was concentrated to small volume and photocrosslinks were separated by reversed phase HPLC (Agilent Poroshell 120 EC-C18 Column, 2.7µm, 4.6 x 150 mm, 40°C) with mobile phases: A = 0.1M CH₃COONH₄/ acetonitrile, 95/5, v/v, B = 0.1M CH₃COONH₄/ acetonitrile, 50/50, v/v with a diode array detector monitoring at 260 nm using the following solvent gradient: 0–15%B in 25 minutes, flow rate: 0.5 ml/min. The residue was evaporated and passed through Waters XBridge OST C18 Column, 2.5µm, 10 x 50 mm, 40℃, with mobile phases: A = 0.01M phosphate buffer (pH = 7), B = acetonitrile/water, 80/20, v/v with a diode array detector monitoring at 260 nm using the following solvent gradient: 0–20%B in 12 minutes, flow rate: 1.5 ml/min to remove CH₃COONH₄. Irradiation of 5-fluoro-4-thiouridine (0.12mM solution in 0.1M phosphate buffer, pH 7.0) with triple molar equivalent of cytidine was carried out under anaerobic conditions at 20℃ with 50mW optical laser power over 30 minutes. The product was separated by HPLC (analyses were performed on a Agilent Poroshell 120 EC-C18Column, 2.7µm, 4.6 x 150 mm, 40℃, eluted with 0.1M TEAA, using a linear gradient of 5%-15% of acetonitrile over 20 min, flow rate: 0.5 ml/min).

MD simutions

The molecular dynamics simulations were run in explicit solvent using ParmBsc1⁴⁹ force field with OL15 modifications⁵⁰ implemented in Amber20 software. The single stranded ODN1 and ODN2 were generated using Nucgen software.⁵¹ The modified nucleotide was generated using ICM MolSoft program.⁵² Each system was then solvated in a cubic TIP3P waterbox whose dimension extended to 12 Å beyond the edge of the solute atoms. The final salt concentration was maintained at 0.15 M KCl. The simulations were run in 3 steps. Step 1 consisted of 5000 steps of conjugate gradient minimisation. This was followed by the equilibration step. Here, the restraints placed on the nucleotides were gradually reduced over 5 ns in an NPT ensemble. The water and ions were allowed to equilibrate with the nucleotides. The final production run consisted of 1000 ns of restraint-free simulation under NVT ensemble with a timestep of 4 fs. The simulations were run using Acemd molecular dynamics engine.⁵³ The post-simulation analysis was carried out using VMD software.⁵⁴

AUTHOR CONTRIBUTIONS STATEMENT

J.N-K., K.T-G., B.S. conceived the experiments, J.N-K. and K.T-G. conducted the experiments, S.H. conducted the MD simulations, J.N-K., K.T-G., B.S. and S.H. analysed the results. J.N-K. and B.S. prepared manuscript. All authors reviewed the manuscript.

SUPPLEMENTARY INFORMATION

Data generated or analyzed during this study are included in this published article and its supplementary information files.

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Sequence- and Temperature-Dependent Intrastrand Photocrosslinking of 5-Fluoro-2’-O-methyl- 4-thiouridine Modified Oligonucleotides

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INTRODUCTION

RESULTS AND DISCUSSION

METHODS

Instruments

Synthesis of oligonucleotides ODN 1–3

Irradiation experiments and isolation of photocrosslinks

MD simutions

Declarations

References

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Supplementary Files

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