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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

To clarify the geometric effect of the ultra-fast photocrosslinking reaction of photoreactive oligodeoxyribonucleotide containing 3-cyanovinylcarbazole nucleoside (CNVK) on uridine in complementary RNA strands, pseudouridine (Ψ), which is an isomer of uridine with a C5–C1′ glycosidic bond, was introduced to the photocrosslink site of CNVK in complementary RNA instead of U. The photoreactivity of CNVK toward Ψ was two-fold lower than that of U, suggesting that the geometry between the vinyl moiety on CNVK and the reactive double bond in the pyrimidine base has a large affect on the photoreactivity of CNVK. Contrary to the case of U, the reactivity of the CNVK toward Ψ was decreased by the decrease of reaction temperature below the Tm of heteroduplex, suggesting that the flexible structure of the duplex is advantageous for the photocrosslinking reaction with Ψ, whose reactive double bond possesses unfavorable geometry for the photocrosslinking reaction with CNVK. These basic findings might contribute to the development of a geometry selective photocrosslinking reaction. This is the first example of a sequence specific photocrosslinking reaction toward Ψ, which is the most abundant posttranscriptionally modified nucleoside.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The sequence specific photocrosslinking reaction between nucleic acid strands is a promising tool for specific photoregulation of gene expression. For example, psoralen-modified oligonucleotide had been used as photodynamic antisense [1-3] and antigene [4] oligonucleotide, and these oligonucleotides successfully regulate the gene expression in a photoresponsive manner. The photocrosslinking reaction is also useful for the construction and/or the stabilization of nanostructured DNA [5].

As an effective photocrosslinker, in a previous study, we successfully developed oligodeoxyribonucleotides (ODNs) containing 3-cyanovinylcarbazole nucleoside (CNVK), which can photocrosslink to a pyrimidine base in complementary DNA or RNA within a few seconds of photoirradiation [6]. This quick photocrosslinking reaction is applicable for antisense strategy [7], selection of miRNAs [8], and construction and/or stabilization of nanostructured DNA [9-11]. Thus, we believe that CNVK has tremendous potential for biological and nanotechnological applications. From the computational structural prediction of the duplexes containing CNVK, it is assumed that the quick photocrosslinking properties are obtained by the favorable geometry between the reactive vinyl group on CNVK and the target double bond in the pyrimidine base, however, there is no experimental evidence to support this assumption.

In this study, to clarify the geometric effect on the photocrosslinking reaction, we adopted pseudouridine (Ψ), which is an isomer of uridine (U) with a C5–C1' glycosidic bond as the target nucleoside having a different geometry of a reactive double bond compared with U (Fig. 1 and Figures S1 and S2, see Supplementary Materials). The photoreactivity between CNVK and U or Ψ under various conditions was investigated.

image

Figure 1. Denaturing PAGE analysis of the heteroduplexes of CNVK–ODN/ORN. [CNVK–ODN] = [ORN] = 20 μM in 50 mM Na-Cacodylate buffer (pH 7.4) containing 100 mM NaCl. Photoirradiation was performed at 20°C. Square means cyclobutane ring formation caused by [2 + 2] photocycloaddition.

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Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Preparation of oligonucleotide

Oligodeoxyribonucleotides sequences were synthesized by the conventional phosphoramidite method by using an automatic DNA synthesizer (3400 DNA synthesizer, Applied Biosystems, CA, USA). The coupling efficiency was monitored with a trityl monitor. The coupling efficiency of crude mixture of CNVK was 97% yield. The coupling time for CNVK was 999 s. They were deprotected by incubation with 28% ammonia for 4 h at 65°C and were purified on a InertSustain C18 column (GL Science, 10 × 150 mm) by reverse phase HPLC; elution was with 0.05 M ammonium formate containing 3–20% CH3CN, linear gradient (30 min) at a flow rate of 3.0 mL/min. Preparation of oligonucleotides was confirmed by MALDI-TOF-MS analysis: Cy3–CNVK–ODN [(M + H)+], calcd. 3288.77, found 3288.99, CNVK-ODN [(M + H)+], calcd. 2781.53, found 2781.17. Other oligoribonucleotide (ORNs) and ODNs were purchased from Nihon Gene Research Laboratories (Miyagi, Japan) or Greiner Bio-One (Tokyo, Japan) and used without further purification.

Photoirradiation

Photoirradiation was performed with an LED lamp (ZUV, 366 nm, 1600 mW/cm2, Omron Corporation, Kyoto, Japan) and a transilluminator (312 nm, Funakoshi) on an aluminum block incubator or water bath.

Polyacrylamide gel analysis

Polyacrylamide gel electrophoresis (PAGE) was performed with 18% polyacrylamide containing 7 M urea and 25% formamide. After the electrophoresis (160 V, 80 min), fluorescent image was taken by a luminescent image analyzer (LAS3000, Fuji, Tokyo, Japan).

Ultrahigh performance liquid chromatography analysis

Ultrahigh performance liquid chromatography (UPLC) was performed with a UPLC system (Aquity, Waters, Milford, MA, USA) equipped with BEH Shield RP18 column (1.7 μm, 2.1 × 50 mm, elution was with 0.05 M ammonium formate containing 1–11.8% CH3CN, linear gradient (7 min) at a flow rate of 0.5 mL/min, 60°C). Chromatograms were monitored at 260 nm.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Photoreactivity of CNVK–ODN and ORN containing Ψ

To evaluate the photoreactivity of CNVK to Ψ, an equimolar mixture of Cy3-labeled ODN containing CNVK (Cy3–CNVK–ODN) and ORN containing Ψ (ORN [Ψ]) was irradiated at 366 nm, and then analyzed by denaturing PAGE (Fig. 1). A band having low mobility compared with Cy3–CNVK–ODN appeared and such band did not appear in the absence of ORN and also in the presence of mismatch ORNs, such as ORN(A), ORN(C) or ORN(G), suggesting that Cy3–CNVK–ODN clearly crosslinked to ORN(Ψ) by photoirradiation in a sequence specific manner. In the case of ORN(U), the same band was also observed, indicating that CNVK in DNA/RNA heteroduplex has photoreactivity not only to U, as previously reported [6], but also to Ψ.

To confirm the reversibility of the photocrosslinking reaction, photodimers consisting of Cy3–CNVK–ODN and ORN(Ψ) were irradiated with 312 nm light and then analyzed with denaturing PAGE (Fig. 2). The band identical to the photodimer was clearly decreased and the band identical to the Cy3–CNVK–ODN appeared, indicating that the photosplitting reaction occurred and the photodimers reverted to the original ODN and ORN. UPLC analysis after the photosplitting reaction, the peak having the same retention time and the same molecular mass of ORN(Ψ) was appeared (Figure S3). Thus, we concluded that the photocrosslinking reaction to the ORN(Ψ) is photoreversible, the same as the case of U and other unmodified pyrimidine bases in our previous report [12-14]. This is the first report about the site-specific chemical reaction toward Ψ in RNA strands.

image

Figure 2. Denaturing PAGE analysis of the photosplitting reaction of heteroduplexes consisting of CNVK–ODN and ORN. [CNVK–ODN] = [ORN] = 20 μM in 50 mM Na-Cacodylate buffer (pH 7.4) containing 100 mM NaCl. Photoirradiation was performed at 37°C. Square means cyclobutane ring formation caused by [2 + 2] photocycloaddition.

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As shown in Fig. 3 and Table 1, the photocrosslinking rate constant of the heteroduplex of CNVK–ODN/ORN(Ψ) was two-fold lower than that of CNVK–ODN/ORN(U), suggesting that the difference in the geometry between the vinyl group on CNVK and the reactive double bond on Ψ or U affects the photoreactivity of CNVK in DNA/RNA heteroduplex. In the case of the thymine dimerization in DNA strand, the importance of the geometry of thymine bases for the photocycloaddition was also pointed out [15], suggesting that our finding was compelling one, although the properties of the excited state was not same as in the case of thymine dimerization. Contrary to the photocrosslinking reaction, the rate constants of the photosplitting reactions were the same. On the other hand, the reactivity of the photocrosslinking reaction to Ψ in DNA double strand was 6.9- and 28-fold lower than that in the case of U and T, respectively (Table 1, Figure S4). The results suggest that the photocrosslinking reaction between CNVK and the pyrimidine base in DNA and RNA strand was largely affected by the geometry between the vinyl moiety on the CNVK and the reactive double bond on the pyrimidine base. Moreover, two-fold selectivity of our photocrosslinking reaction toward U and Ψ has the potential for the selective detection of the posttranscriptional modification in U to Ψ in RNA strands.

Table 1. Photocrosslinking and photosplitting rate constant between CNVK–ODN and Ψ, U or T in RNA or DNA strand
Complementary ORN or ODN strandaRate constant/s−1
PhotocrosslinkingPhotosplitting
  1. a

    Calculated by exponential fitting with the assumption of first-order kinetics (Fig. 3 and Figure S5).

  2. ORN = oligoribonucleotide

ORN(Ψ)0.750.06
ORN(U)1.50.05
ODN(Ψ)0.33
ODN(U)2.3
ODN(T)9.5
image

Figure 3. Time course of the photocrosslinking (a) and photosplitting (b) reaction of the CNVK–ODN/ORN heteroduplexes containing Ψ or U.

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Identification of photoproduct

To discuss the details of the photocrosslinking reaction between CNVK–ODN and ORN(Ψ), the photocrosslinking reaction was monitored with UPLC using unlabeled CNVK–ODN and ORN(Ψ) (Fig. 4 and S1; see supplementary materials). As the peaks of ORN(Ψ) and CNVK–ODN were clearly decreased by 366 nm photoirradiation, the crosslinking reaction clearly occurred between CNVK-ODN and ORN(Ψ). Contrary to the case of ORN(U), two different peaks having the same molecular mass, which is identical to the photodimer consisting of CNVK–ODN and ORN(Ψ) ([(M + H)+], calcd. 5673.97, found 5674.13 and 5672.79), appeared, suggesting that these photodimers might have a different chemical structure. Photoadducts consisting of CNVK and Ψ were detected in the HPLC analysis and MALDI-TOF-MS analysis of the enzymatic-digested product of the photodimer (Figure S6a), and also the nucleosides Ψ and U clearly generated after the photosplitting of photoadducts from the photodimers of CNVK-ODN/ORN(Ψ) and CNVK-ODN/ORN(U), respectively (Figure S6b), suggesting that the photocrosslinking reaction occurred with the covalent bond formation between CNVK and Ψ or U. As the photoadducts from two different photodimers consisting of CNVK–ODN and ORN(Ψ) observed in UPLC analysis have a different retention time (Fig. 4a), there are two different coupling schemes of photoadducts in the case of the photoadduct consisting of CNVK and Ψ.

image

Figure 4. Ultrahigh performance liquid chromatography analysis of the photocrosslinking reaction between CNVK–ODN and ORN(Ψ) or ORN(U). Chromatograms were monitored at 260 nm.

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Temperature dependence of the photocrosslinking reaction between CNVK–ODN and ORN(Ψ)

To investigate the temperature dependence of the photocrosslinking reaction between CNVK–ODN and ORN(Ψ), the conversion after 1 s irradiation of 366 nm at different temperatures was compared with the case of ORN(U) (Fig. 5a). The conversion of the photocrosslinking reaction between CNVK–ODN and ORN(U) was decreased at the higher temperature compared with Tm (Fig. 5b), although the conversion was constant at the lower temperature. This phenomenon can be explained by the dissociation of heteroduplex at a higher temperature than Tm. On the other hand, in the case of ORN(Ψ), the conversion was both decreased at lower and higher temperature ranges, suggesting that the flexibility of the reaction point in the heteroduplex is favorable for the photocrosslinking reaction between CNVK and Ψ, whose reactive double bond possesses unfavorable geometry for the photocrosslinking reaction with CNVK compared with U in the predicted static structure (Figure S2). The ratio of the minor peak among the two peaks that appeared with the photocrosslinking between CNVK–ODN and ORN(Ψ) was increased with the increase in the reaction temperature (Figure S7), suggesting that the minor product was mainly provided from the flexible structure of the heteroduplex.

image

Figure 5. Temperature dependence of the conversion of the photocrosslinking reaction between CNVK–ODN and ORN(Ψ) or ORN(U) (a) and the UV melting profiles of the heteroduplexes of CNVK–ODND/ORN(Ψ) and CNVK–ODND/ORN(U).

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In this study, we found that Ψ in RNA strand is photocrosslinked with CNVK within a few seconds of 366 nm irradiation and that the reaction rate was different from U. As posttranscriptional modification in U to Ψ plays an important role in the translation [16, 17] and processing of small nuclear RNA [18, 19], specific detection and a regulation method of Ψ are now important issues for the understanding and regulation of biological events including Ψ. The basic findings described here may contribute to the development of chemical tools for the detection or regulation of biological events including Ψ.

In conclusion, we successfully demonstrated that the geometry of the vinyl group on CNVK and the reactive double bond in the pyrimidine base affected the reactivity of the photocrosslinking reaction between CNVK–ODN and ORN or ODN, and that the favorable geometry of U in DNA/RNA heteroduplex enabled a highly efficient photocrosslinking reaction. Ψ in DNA/RNA heteroduplex was also reactive with CNVK, although the reactivity was lower than that of U. This is the first example of the site-specific chemical modification in Ψ in RNA strands. Further development based on the basic findings described here might contribute to the selective and sequence specific detection and regulation of Ψ in RNA strands.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study was partly supported by the Grant-in-Aid for Scientific Research (B), Scientific Research (S) and Scientific Research on Innovative Areas (Molecular Robotics) (90293894, K.F.) of The Ministry of Education, Science, Sports and Culture of Japan.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Higuchi, M., A. Kobori, A. Yamayoshi and A. Murakami (2009) Synthesis of antisense oligonucleotides containing 2′-O-psoralenylmethoxyalkyl adenosine for photodynamic regulation of point mutations in RNA. Bioorg. Med. Chem. 17, 475483.
  • 2
    Higuchi, M., A. Yamayoshi, T. Yamaguchi, R. Iwase, T. Yamaoka, A. Kobori and A. Murakami (2007) Selective photo-cross-linking of 2′-O-psoralen- conjugated oligonucleotide with RNAs having point mutations. Nucleos. Nucleot. Nucl. 26, 277290.
  • 3
    Murakamia, A., A. Yamayoshia, R. Iwasea, J.-I. Nishidab, T. Yamaokaa and N. Wake (2001) Photodynamic antisense regulation of human cervical carcinoma cell growth using psoralen-conjugated oligo (nucleoside phosphorothioate). Eur. J. Pharm. Sci. 13, 2534.
  • 4
    Musso, M., J. C. Wang and M. W. Van Dyke (1996) In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides. Nucleic Acids Res. 24, 49244932.
  • 5
    Liu, J., Y. Geng, E. Pound, S. Gyawali, J. R. Ashton, J. Hickey, A. T. Woolley and J. N. Harb (2011) Metallization of branched DNA origami for nanoelectronic circuit fabrication. ACS Nano 5, 22402247.
  • 6
    Yoshimura, Y. and K. Fujimoto (2008) Ultrafast reversible photo-cross-linking reaction: Toward in situ DNA manipulation. Org. Lett. 10, 32273230.
  • 7
    Shigeno, A., T. Sakamoto, Y. Yoshimura and K. Fujimoto (2012) Quick regulation of mRNA functions by a few seconds of photoirradiation. Org. Biomol. Chem. 10, 78207825.
  • 8
    Yoshimura, Y., T. Ohtake, H. Okada and K. Fujimoto (2009) A new approach for reversible RNA photocrosslinking reaction: Application to sequence-specific RNA selection. Chem. Bio. Chem. 10, 14731476.
  • 9
    Fujimoto, K., K. Hiratsuka-Konishi, T. Sakamoto, T. Ohtake, K.-I. Shinohara and Y. Yoshimura (2012) Specific and reversible photochemical labeling of plasmid DNA using photoresponsive oligonucleotides containing 3-cyanovinylcarbazole. Mol. BioSyst. 8, 491494.
  • 10
    Gerrard, S. R., C. Hardiman, M. Shelbourne, I. Nandhakumar, B. Nordén and T. Brown (2012) A new modular approach to nanoassembly: Stable and addressable DNA nanoconstructs via orthogonal click chemistries. ACS Nano 6, 92219228.
  • 11
    Tagawa, M., K.-I. Shohda, K. Fujimoto and A. Suyama (2011) Stabilization of DNA nanostructures by photo-cross-linking. Soft Matter 7, 1093110934.
  • 12
    Fujimoto, K., K. Konishi-Hiratsuka, T. Sakamoto and Y. Yoshimura (2010) Site-specific photochemical RNA editing. Chem. Commun. 46, 75457547.
  • 13
    Fujimoto, K., K. Konishi-Hiratsuka, T. Sakamoto and Y. Yoshimura (2010) Site-specific cytosine to uracil transition by using reversible DNA photo-crosslinking. Chem. Bio. Chem. 11, 16611664.
  • 14
    Fujimo, K., K. Konishi-Hiratsuka and T. Sakamoto (2013) Quick, selective and reversible photocrosslinking reaction between 5-methylcytosine and 3-cyanovinylcarbazole in DNA double strand. Int. J. Mol. Sci. 14, 57655774.
  • 15
    Schreier, W. J., T. E. Schrader, F. O. Koller, P. Gilch, C. E. Crespo-Hernández, V. N. Swaminathan, T. Carell and W. ZinthB. Kohler(2007) Thymine dimerization in DNA is an ultrafast photoreaction. Science 315, 625629.
  • 16
    Ofengand, J. (2002) Ribosomal RNA pseudouridines and pseudouridine synthases. FEBS Lett. 514, 1725.
  • 17
    Charette, M. and M. W. Gray (2000) Pseudouridine in RNA: What, where, how, and why. IUBMB Life 49, 341351.
    Direct Link:
  • 18
    Reddy, R., T. S. Ro-Choi, D. Henning, H. Shibata, Y. C. Choi and H. Busch (1972) Modified nucleosides of nuclear and nucleolar low molecular weight ribonucleic acid. J. Biol. Chem. 247, 72457250.
  • 19
    Yu, A. T., J. Ge and Yu Y. T. (2011) Pseudouridines in spliceosomal snRNAs. Protein Cell 2, 712725.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
php12118-sup-0001_FigS1-S7.pdfapplication/PDF1341K

Figure S1. Reversible photocrosslinking reaction between CNVK and Ψ or U in DNA/RNA heteroduplex.

Figure S2. Predicted structure of DNA/RNA heteroduplexes containing CNVK and Ψ or U.

Figure S3. Ultrahigh performance liquid chromatography analysis of the equimolar mixture of CNVK–ODN and complementary oligoribonucleotide (ORN) containing Ψ or U after the photocrosslinking and photosplitting.

Figure S4. Ultrahigh performance liquid chromatography analysis of the equimolar mixture of CNVK–ODN and complementary ODN containing Ψ, U or T before and after 4 s of photoirradiation.

Figure S5. Time course of the photocrosslinking reaction between CNVK–ODN and complementary ODN containing Ψ, U or T.

Figure S6. HPLC analysis of the degraded product of the two photodimers consisting CNVK–ODN and oligoribonucleotide (ORN) (Ψ).

Figure S7. Temperature dependence of the yield of minor product consisting of CNVK–ODND and ORN(Ψ).

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