Intrastrand Photocrosslinking of 5-Fluoro-2’-O-Methyl-4-Thiouridine Modified Oligonucleotides and its Implication for Fluorescence-Based Detection of DNA Sequences

doi:10.21203/rs.3.rs-3358580/v2

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Intrastrand Photocrosslinking of 5-Fluoro-2’-O-Methyl-4-Thiouridine Modified Oligonucleotides and its Implication for Fluorescence-Based Detection of DNA Sequences

https://doi.org/10.21203/rs.3.rs-3358580/v2

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DNA photocrosslinking reactions occur widely in biological systems and are often used as a valuable tool in molecular biology. Herein we report the sequence and temperature-dependent intrastrand photocrosslinking of single-stranded oligodeoxynucleotides bearing 5-fluoro-4-thiouridine (FSU). Our research findings indicate that FSU can photoreact with non-adjacent bases, specifically, it can react with distant thymine and cytosine residues in the chain forming fluorescent and non-fluorescent intrastrand crosslinks, respectively. In addition, partial photooxidation of FSU residue to 5-fluorouridine was also observed. The results of the study are significant in terms of the use of FSU-labeled oligonucleotide probes in the fluorescence-based detection of specific DNA sequences because the creation of a fluorescent intrastrand crosslink could produce a false signal. To overcome this problem, replacing thymidine with deoxyuridine in FSU-labeled oligonucleotide probe is proposed and tested.

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

oligonucleotides

fluorescent probe

intrastrand photocrosslinking

5-fluoro-4-thiouridine

DNA crosslinking

Crosslinking of DNA can be induced by chemical reaction or triggered by exposure to UV irradiation.^[1] The formation of interstrand crosslinks in DNA causes inhibition of transcription and replication^[2] and can modulate nucleic acid conformation^[3] including aptamer,^[4]G-quadruplex^[5] and i-motif structures.^[6]Short DNA oligonucleotides capable of creating crosslinks are valuable model systems in the study of DNA damage repair mechanisms.^[7]Furthermore, crosslinking may have potential applications for detection of specific DNA/RNA sequences^[8] and identifying targets for bioactive small molecules.^[9]

Among the photoactivable crosslinking agents belonging to modified nucleobases, 4-thiouridine (4SU)^[10]and 6-thioguanosine (6SG)^[11]should be distinguished. We have previously described the UV induced interstrand crosslinking of DNA duplexes labeled with 5-halogeno analogs of 4SU, namely 5-fluoro-2’-O-methyl-4-thiouridine (FSU)^[12] and 5-chloro-2’-deoxy-4-thiouridine (ClSdU).^[13] Similarly to the native 4SU, 5-fluoro-4-thiouridine and 5-chloro-4-thiouridine show a high photoreactivity towards thymidine. However, unlike 4SU which mostly forms 6-4 and 5-4 pyrimidine-pyrimidone adducts with thymidine,^[14] 5-fluoro-4-thiouridine and 5-chloro-4-thiouridine undergo photocycloaddition with thymidine producing a unique pair of diastereomeric, highly fluorescent and thermally stable tricyclic adducts.^[15] It should be noted that FSU exhibits photoreactivity much higher than that of the 5-chloro derivative. As we have already demonstrated the photoreaction occurs both under selective excitation monomeric 5-fluoro-4-thiouridine in the presence of thymidine,^[15] as well as in double stranded oligonucleotides leading to fluorescent interstrand crosslink formation with nearly quantitative yield.^[12-13]

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. For application purposes, a DNA fluorescent probe sequence must bind specifically to the target DNA. In the case of interstrand DNA photocrosslinking, Watson-Crick base pairing and strand complementarity meet this requirement. However, in the absence of the target sequence, there is a risk of competitive intrastrand photocrosslinking reactions. Therefore, it is necessary to study the possibility of intrastrand crosslinks formation, as it can lead to false detection signals (Figure 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. The probe DNA sequence labeled with FSU also will be tested for detection of pseudotarget from DNA of HPV-16 virus.

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.^[16]

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.

Table 1. Sequences of studied oligodeoxynucleotides.

Oligonucleotide	Sequence (5’-3’)
ODN 1	CGATACGA(FSU)A
ODN 2	A(FSU)AGCATAGC
ODN 3	ATAGCA(FSU)AGC
ODN 4	TGCTATGCTATT

We have previously tested photochemical reactivity of ODN 1 and ODN 2 in the presence of complementary oligonucleotide and we have observed almost quantitative formation of interstrand photocrosslinks between FSU and T.^[12] We decided to performed analogous experiment in the case of ODN 3. We irradiated ODN 3 with λ = 355 nm in the presence of 1.1 molar equivalent of ODN 4. While total conversion of ODN 3 was noticed, 66% of ODN 4 (based on HPLC) remained unreacted (Figure 2A). Different than before, apart from the formation of fluorescent interstrand photocrosslinks, we also observed fluorescent photoproducts with shorter retention times (Figure 2A). The shorter retention times of these photoproducts and their absorption and emission properties (higher ratio of the intensity of the absorption bands at 370 nm to the band at 260 nm than in the case of interstrand photocrosslinks) prompted us to conclude that these are intrastrand photocrosslinks. Moreover, we also observed its formation in the experiment of irradiation of ODN 3 without ODN 4 (Figure 2B). Although duplex ODN3 : ODN 4 was irradiated at 10℃, well below the melting temperature of the duplex to ensure complete hybridization of the strands and with different excess of ODN 4 (1-2 molar equivalents), intrastrand photocrosslinking reaction was always observed. The above encouraged further studies on the intrastrand photocrosslinking for oligonucleotides ODN 1-3.

Aqueous solutions of ODN 1-3 in 0.1M phosphate buffer, pH 7.0, were irradiated at 20℃ and the progress of reaction was monitored by HPLC (Figure 2C-E). Under these conditions (λ = 355 nm) selective excitation of FSU is achieved. For all oligonucleotides, after irradiation for 150 s 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 (Figure 2C, 2D, 2E). Since ODNs 1-3 have the same nucleoside composition, photoproducts (a-d) obtained from ODNs 1-3, respectively, have similar UV absorption spectra (Figure S1). For ODN 1, fluorescent photoproducts 1a and 1b were formed as the main photoproducts (Figure 2C), while for ODN 2, only trace amounts of photoproducts 2a and 2b were formed (Figure 2D). For fluorescence spectra of 1a and 1b see Supplementary Fig. S2. Non-fluorescent photoproduct 2c was observed in the mixture after irradiation of ODN 2 as the dominant one (Figure 2D). 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 irradiation of ODN 3 (Figure 2E). In the case of ODN 2 and ODN 3, photoproducts 2d and 3d were observed respectively (Figure 2C, 2D), while no analogous photoproduct was formed in the case of ODN 1.

In the case of ODN 3 the formation of photoproducts with retention times between 15-17 minutes was observed (Figure 2E, marked with *). Although they are found in relatively high yield after irradiation for 150 s, we noticed their total conversion to photoproducts 3a, 3b and 3d after irradiation of ODN 3 for 10 minutes (Figure 2B). The absorption spectra of these intermediate photoproducts are presented in Figure S3. The starting ODN 3 has absorption maximum at 340 nm (Figure S3, A) while in case of photoproducts this band is definitely less intense (Figures S3, C and D) or shifted to the shorter wavelengths (Figures S3, B). 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.^{[14, 17]} We proposed that this process in the case of reaction of 5-fluoro or 5-chloro-4-thiouridine with T is followed by thietane ring opening leaving the thiol on C6 of the pyrimidine part.^[15] We were able to observe the thietane experimentally for duplexes labeled with 5-chloro-2’-deoxy-4-thiouridine.^[13] Taking into account the photochemical instability of the observed photoproducts after short irradiation of ODN 3 and it’s absorption properties we strongly believe that these are isomers of postulated intermediates.

The main photoproducts after irradiation of ODN 1-3 were isolated and separated by HPLC and their UV absorption (Figure 3), fluorescence (Figure S2) and MALDI TOF MS spectra (Table S1, Figure S4-10) 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,^[15] and were identified as two diastereomeric (a,b) intrastrand photocrosslinks of FSU with T (Figure 3).

The photoproducts 2d and 3d exhibit UV absorption maximum at 260 nm, and a lack of the band with a maximum at 340 nm, characteristic for a thiocarbonyl group, is observed (Figure S1). In the MALDI TOF MS spectrum of product 3d (Figure S9 and S10), the signal corresponding to mass reduced by 16 compared to ODN 3 is present. This prompts us to claim that 3d and 2d are the products of photooxidation of FSU to 5-fluorouridine (Figure 3). To identify photoproduct 2c, an additional experiment was performed by irradiating FSU in the presence of cytidine in 0.1M phosphate buffer, pH 7.0. The triple molar equivalent of cytidine was used to minimize the formation of photoadducts of FSU itself.^[18] Comparing the absorption spectra of the products obtained after irradiation of the mixture of 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 (Figure S12). This product was isolated from the reaction mixture and based on the result obtained from its ESI MS spectrum (Figure S13) and MALDI TOF MS spectrum of photoproduct 2c (Figure S6 and S7) we suggest that in the case of ODN 2 the intrastrand photocrosslink with C is formed (Figure 3). The photoadduct formation of 4SU with C observed in E. coli tRNA is well described.^{[14, 17]} We propose analogous pathway for the photoaddition of FSU to C with elimination of H₂S and formation of non-fluorescent 4-5 photoadduct c (Figure 4).

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 (Figure 5) and most likely they react with FSU to form non-fluorescent adduct c.

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 Figure 2. Different conversion of the starting ODN was observed (Figure S14) 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 the 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.

Considering the reactivity of ODN 1-3 in intra and interstrand photocrosslinking^[12] we concluded that the location of FSU near the 3’ end of the modified strand has an advantage in the context of the application for fluorescence based detection of specific DNA sequences. Consequently, we designed and synthesized the FSU probe oligonucleotide (Table 2, Figure S15), with the sequence complementary to the fragment of the E6 gene of Human papillomavirus (Alphapapillomavirus 9) (HPV-16)^[^19] (Table 2, target 1). All thymidines in the FSU probe (Table 2) were replaced with deoxyuridine (dU) to ensure fluorescence signal is only generated during interstrand photocrosslinking of FSU probe with T from target DNA.

Table 2. Sequences of FSU probe and tested pseudotarget oligonucleotides.

Oligonucleotide	Sequence (5’-3’)
FSU probe	GCdUCdUGdUGCA(FSU)A
target 1	TTATGCACAGAGC
target 2	TTAGTATAGTGAG
target 3	TTATGCGTGAGAT

FSU probe (10 mM in 0.1 M phosphate buffer, pH 7.0) was exposed to irradiation with 355 nm in the presence of specific targets (1.2 molar eq.) for 5 minutes at room temperature. The fluorescence signal at 450 nm was measured before and after irradiating of the FSU probe in the presence of different targets.

Figure 6 shows that the intense fluorescence signal was only generated when the FSU probe was irradiated with target 1. This indicates that although all targets have T in close proximity to FSU, the FSU probe is highly selective, and efficiently forms photocrosslinking product with T only in the presence of fully complementary target 1.

The results showed that the intrastrand photocrosslinking reaction can occur in short DNA fragments between non-adjacent bases. 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. When FSU is located near 3’ end (ODN 1) the reaction of FSU with T is observed, while for the reversed sequence (ODN 2) the photocrosslink with C is formed. However, only in the case of the reaction of FSU with T photocrosslinking product is fluorescent. The location of FSU closer to the middle of the strand, as in the case of ODN 3, results in limitation of conformational flexibility what makes FSU difficult to arrange properly for cycloaddition to the thymidine from the complementary strand (ODN 3 : ODN 4 duplex). Moreover, taking into account the photochemical reactivity of ODN 3, this limitation is also reflected in the appearance of photooxidation as a competitive reaction to intrastrand photocrosslinking.

Although intrastrand reactions were observed for FSU labeled oligonucleotides, the interstrand photocrosslinking of FSU with T is almost quantitative when FSU is located near the 3’ end. The FSU-labeled probe was synthesized and tested in photocrosslinking reaction with oligonucleotide targets 1-3, showing high selectivity toward pseudo-target from HPV-16 DNA sequence (target 1).

The obtained results have great significance for designing the FSU-labeled probes for fluorescence-based detection of specific DNA sequences. Since the formation of fluorescent photocrosslink is only possible with T, substituting T with dU can be considered when designing a probe with FSU to avoid a false detection signal observed 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) and at fluorescence measurement plates. Absorption spectra were measured on a JASCO V750. Fluorescence spectra were recorded on plate reader TECAN Infinite M200. 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

Synthesis of ODN 1-4 and FSU probe was performed on DNA/RNA Synthesizer H-6 (K&A Laborgeraete) applying 0.2 μmol protocol and ultramild CPG supports. Oligonucleotides labeled with FSU were synthesized using previously published method.^[16] Oligodeoxynucleotides were purified by reversed phase HPLC (Waters XBridge OST C18 Column, 2.5 μm, 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% to 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.

Oligonucleotides targets 1-3 were purchased from Genomed (Warsaw, Poland) and were used without further purification.

Irradiation experiments and isolation of photocrosslinks

Duplex ODN 3: ODN 4 (1 : 1.1 eq) was dissolved in 0.1 M phosphate buffer (pH = 7) to obtain solution with A₂₆₀ = 2.3. The irradiation was performed at 10°C with Coherent Genesis CX-355-100 cw laser with 80 mW optical laser power for 10 minutes. 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 80 mW optical laser power.

The progress of the reaction was monitored by HPLC (analyses were performed on a Waters XBridge OST C18 Column, 2.5 μm, 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 (Figure 1A-B) and on a Agilent Poroshell 120 EC-C18 Column, 2.7 μm, 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 (Figure 1C-E)). The reaction mixture was concentrated to small volume and photocrosslinks were separated by reversed phase HPLC (Agilent Poroshell 120 EC-C18 Column, 2.7 μm, 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% to 15%B in 25 minutes, flow rate: 0.5 ml/min. The residue was evaporated and passed through Waters XBridge OST C18 Column, 2.5 μm, 10 x 50 mm, 40℃, with mobile phases: A = 0.01M phosphate buffer (pH 7.0), B = acetonitrile/water, 80/20, v/v with a diode array detector monitoring at 260 nm using the following solvent gradient: 0% to 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-C18 Column, 2.7 μm, 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).

Irradiation experiments of FSU probe with targets 1-3

The solution of FSU probe (10mM) with target (1-3) (12mM) in 0.1M phosphate buffer (pH 7.0) (100 μl), was irradiated with Coherent Genesis CX-355-100 cw laser with 80mW optical laser power for 5 minutes at room temperature at fluorescence measurement plate. The experiment was repeated 3 times for each duplex. The fluorescence signal at 450 nm (λ_ex 370 nm) was measured before and after irradiation using plate reader TECAN Infinite M200.

MD simutions

The molecular dynamics simulations were run in explicit solvent using ParmBsc1^[20] force field with OL15 modifications^[21] implemented in Amber20 software. The single stranded ODN1 and ODN2 were generated using Nucgen software.^[22] The modified nucleotide was generated using ICM MolSoft program.^[23] 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.^[24] The post-simulation analysis was carried out using VMD software.^[25]

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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.

The authors declare no competing interests.

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Intrastrand Photocrosslinking of 5-Fluoro-2’-O-Methyl-4-Thiouridine Modified Oligonucleotides and its Implication for Fluorescence-Based Detection of DNA Sequences

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Abstract

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Introduction

Results And Discussion

Conclusion

Experimental Section

References

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Additional Declarations

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Status:

Journal Publication

Version 2