International Journal of Advanced Computer Science, Vol. 2, No. 6, Pp. 237-241, Jun., 2012.

Chemical RNA Editing as a Possibility Novel Therapy for Genetic Disorders

Vu Thi Luyen, Yu Ooka, Shafiul Alam, Hitoshi Suzuki, Kenzo Fujimoto & Toshifumi Tsukahara
Manuscript
Received: 7,Oct., 2011 Revised: 5,Feb., 2011 Accepted: 21,May, 2012 Published: 15,Jul., 2012

Keywords
RNA editing, Photochemical reaction, Therapeutic approach, Deamination, Leigh syndrome

Abstract We are trying to alter the coded message of RNA transcripts by photochemical RNA editing to treat genetic diseases. A mutation of mitochondrial DNA (mtDNA) T8993C in Leigh syndrome as a model, we subjected site-directed deamination of Cytosine (C) to Uridine (U) by reversible photoligation using hairpin-type oligonucleotides with carboxyvinyldeoxy-uridine at the 5-terminal. We observed significant objective base substituted fragment with ODN2 as a 72mer ODN as a target. Then, we tried to genetic restoration experiments as patient derived samples as targets. ODN2 could restore 10% of the mutated C to U, when in-vitro-synthesizedfull-length RNA was used as a target. Moreover, the site-directed deamination was performed even toward total RNA from the patients cells as a target.

1. Introduction
RNA editing is a biological step to modify gene-encoded information and to generate functionally distinct proteins from a single gene in eukaryotes. In mammalian cells, site-directed genetic modifications are achieved by deamination from cytidine or adenosine into uracil or inosine which serve as guanine [1, 2]. If we can control RNA editing, genetic diseases that result in T>C or G>A point mutations could be treated. Non-enzymatic, template-directed ligation of oligodeoxyribonucleotides and oligoribonucleotides in aqueous solution holds considerable promise for the control of RNA editing. Various template-directed chemical ligation reactions join oligonucleotides by either a native phosphodiester bond. [3-6] or non-native linkages [7-9] have been reported. In addition, there are some reports of photochemical methods, the first of which is based on the photochemistry of native DNA [10], and two others of which are based on the photochemistry of DNA containing appended coumarins [11] or stilbenes [9]. In the first photochemical ligation

method category, Fujimoto et al. succeeded a site-specific transition of cytidine (C) to uridine (U) by using 5-carboxyvinyldeoxyuridine-(CVU)-containing oligodeoxy -nucleotides [12]. In this protocol, they performed a template-directed DNA photoligation mediated by artificial oligodeoxynucleotides, then caused the heat-induced transition of C to U with high efficiency without any side reaction. The base substitution from the C to U has been performed at almost 100% efficiency in the sequence-specific manner. Consequently, it is possible that we treat genetic disorders caused by T>C point mutation by using this technology. Leigh syndrome (also known as Leighs disease, subacute necrotizing encephalomyelopathy, SNEMs) is an inherited disorder usually begins in early childhood until 2 years old. The disease symptom includes psychomotor regression and brain-stem dysfunction, and the first signs are often poor sucking ability, loss of head control, and loss of acquired motor skills or movement [13]. As the disorder progresses, symptoms may also include generalized weakness, lack of muscle tone, and episodes of lactic acidosis. The disease is not a monogenic, however mutations in mitochondrial DNA (mtDNA) at nucleotide (nt) 8993 or 9176 were found in more than 20 % of Leigh syndrome patients. These mutations are located in the ATPase 6 gene; therefore, it is important to establish a treatment of ATPase 6 gene in Leigh syndrome. Here, we studied a completely new genetic therapy for inherited diseases, as a Leigh syndrome patient with the mtDNA T8993C mutation as a model. This method is based on chemical modification of nucleotides. In chemical structure, when the amino group of the base of cytidine at the fourth pyrimidine ring is reversed by deamination, the base converts into the base of uridine. Naturally, uridine base in DNA is recognized as thymidine. Therefore, if nt 8993 cytidine converts into uridine in a Leigh syndrome patient having T8993C mutation, the modification changes the mutant sequence to wild-type sequence. Since ATPase 6 is coded in mtDNA and transcribed into mRNA, there are a couple of target nucleotide species in vivo. Thus, in order to establish a novel genetic therapy, it is important to find out the optimal condition in this study.

This work was supported by JAIST. Vu Thi Luyen, Yu Ooka, Shafiul Alam, Hitoshi Suzuki, Kenzo Fujimoto & Toshifumi Tsukahara are with School of Materials Science and at the same time Hitoshi Suzuki & Toshifumi Tsukahara are with Center for Nano Materials and Technology, Japan Advanced Institute of Science and Technology. Asahidai, Nomi, Ishikawa 923-1292, JAPAN Email: tuka hara@jaist.ac.jp
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D. Preparation of Mutated ATPase 6 DNAs and RNA as Targets A full-length ATPase 6 cDNA was amplified by high-fidelity PCR as the purified whole DNA from the patients cells as a template, then cloned into a pGEM-T Easy Vector (Promega). The sequence was confirmed by dideoxy sequencing. A single-stranded ATPase DNA of 731nt was prepared by asymmetric PCR. A double-stranded 681nt-full-length ATPase DNA was amplified by using ExTaq enzyme. An 813nt-ATPase 6 RNA was prepared by in vitro transcription by using SP6 RNA polymerase. E. Photochemical Deamination The site-directed substitution by deamination has been done as described by Fujimoto and his colleague. At first, the responsive oligodeoxynucleotide (ODN) to conversion anneals to the target site of the mutated gene. The mutated cytidine was cross-linked with the responsive ODN by ultra-violet (366 nm UV), then received heat-treatment. Finally, the cross-linked nucleotide is cleaved by the photospliting operation (UV radiation; 312 nm UV). In the trial experiment, a synthetic 72nt oligonucleotide of mutated ATPase 6 sequence was used as a target. Then, DNAs or RNAs derived from the Leigh syndrome patient were used as targets. F. Confirmation of Base Substitution Base substitution from C to U was confirmed by PCR followed by restriction fragment length polymorphism (RFLP). Briefly, the same sequence as the trail 72nt synthetic target was amplified by PCR after photodeamination protocol. Amplified samples were subjected to RFLP analysis by using Mva I enzyme. Mva I could cut CCTGG but not CCCGG. This means wild type sequence could be cut but mutated sequence could not by the restriction enzyme (Fig. 2A).

Fig. 1 Schematic representation of artificial transition. A. The steps of artificial translation. A responsive ODN should be annealed with the test sequence which includes a targeted C.Photo- ligation of 5-terminal CVU with the targeted C was performed by photoirradiation. An amino residue of the targeted C was deaminated by heat-treatment. After photosplitting of an ODN, site-directed transition was completed. Please refer to Fujimoto et al. [12] for details. B. Deamination of Cytidine to Uridine

2. Materials and Methods

A. Oligonucleotides All oligonucleotides were synthesized by an automated DNA synthesizer according to the conventional amidite chemistry. An oligonucleotide from mtDNA 8957 to 9028, 72nt, with T8993C mutation, was used for a synthetic target in trial experiments. 5- CVU-containing oligonucleotides were synthesized as reported previously [14]. B. Leigh Syndrome Patients Cells A patient derived primary cultured fibroblast was obtained from a sample of muscle biopsy from a patient with Leigh syndrome and provided by Dr. Y. Goto, National Institute of Neuroscience, National Center of Neurology and Psychiatry under approval by the local research ethics committee [15]. The cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 20% fetal bovine serum. C. Preparation of Whole DNA and RNA from Leigh Syndrome Patients Cells Whole genomic DNA and RNA were purified from Leigh syndrome patients cells by using Flexi Gene DNA Kit (Qiagen) or TRIzol Reagent (Invitrogen) according to suppliers protocols, respectively.

3. Results
A. Confirmation of the T8993C Mutation in the Leigh Syndrome Patient First, we confirmed abundance of mutated mtDNA in the Leigh syndrome patient, because cells have multi copies of mitochondria. We performed RFLP analysis by using whole DNA from patients cells. Restriction enzyme, Mva I, could cut wild type sequence but not mutated sequence. As shown in Fig. 2A, we could not find any digested band from patients DNA. The result suggests this patient seems to have homoplasmic mitochondrial DNA. These results were confirmed by sequencing (Fig. 2B). B. Site-Directed Deamination toward a 72nt-Target As described before, the efficiency of the base substitution was almost 100% when the responsive ODN and a 20-mer target were used in vitro [12]. The target must be much longer than 20-mer in vivo. Therefore, we made a
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synthetic target elongate to 72-mer ODN (single strand 72nt). The sequence was based on ATPase 6 gene, including a part of sequence of T8993C mutated ATPase 6 which was found in the Leigh syndrome patient.

Fig. 2 Confirmation of a mutation of the patient. A mutation of the patient was confirmed by RFLP analysis, A, and sequencing analysis, B. WT; wild type, PT; patient.

5C for annealing. To subject photoligation, the samples were irradiated by 366 nm UV on ice using a UV-LED irradiation device that can irradiate narrow peak UV. Then, the deamination reaction was performed at 90C for 2 hours. Then, the samples were irradiated by 312 nm UV using a UV transilluminator for photosplitting. Finally, we confirmed a possibility of photochemical base substitution by using RFLP following PCR. Restored mutation (C>U) must be cut by Mva I, therefore, we can observe efficient photochemical deamination. To determine the photoligation efficiency of 5 ODNs with a single-stranded-72nt-target, the 72-nt target was subjected to photoligation reactions with ODN 1, 2, 3, 4, and 5, independently, and the photoligation products were subjected to electrophoresis followed by SYBR green-staining. As shown in Fig. 3A, all five ODNs resulted in shifted bands (lanes 2, 4, 6, 8 and 10) but not the negative controls without photoirradiation (lanes 1, 3, 5, 7 and 9).

For effecters of deamination, we designed and synthesized five different ODNs (ODN1 to 5) as shown in Table 1 for the trial experiment of site-directed photochemical deamination. They have different length of complementary sequences to the boundary sequence of the mutated C in ATPase 6 gene, hairpin structures and a photoresponsive nucleobase (CVU) in 5-terminus, respectively.
TABLE 1 SEQUENCES OF EFFECTOR ODNS. EACH EFFECTOR ODN HAS COMPLEMENTARY SEQUENCES OF BOUNDARY OF THE TARGET C, HAIRPIN STRUCTURES AND PHOTORESPONSIVE CVU IN 5-TERMINUS

Name
CV

Sequences
UGCGTGTTTTC ACGCAGGGGCTA TTGGTTG (30 nt)
CV

Complement -ary Sequence

GGGGCTATTG GTTG (14 nt) GGGGCTATTG GTTGAATGA GTA (22 nt)

Hairpin Structure
UGCGTG ACGCAC

ODN1

ODN2

UGCGTGTTTTC ACGCAGGGGCTA TTGGTTGAATGAG TA (38 nt)

CV

UGCGTG ACGCAC

Fig. 3 Efficiency of photoligation between 5 ODNs and a single-stranded 72nt oligonucleotide after 5 minutes of photoirradation at 366nm. A, a single-stranded 72nt-target was subjected to-photoligation-and the products were then analyzed by denaturating gel electrophoresis followed by SYBR green-staining. B. Densitometric analysis of the shifted bands in A.

ODN3

UGCGTGACACT TTTGTGTCACGCA GGGGCTATTGGTT G (38 nt)

CV

GGGGCTATTG GTTG (14 nt)

UGCGTGACAC ACGCACTGTG

ODN4

UGCGTGACACT TTTGTGTCACGCA GGGGCTATTGGTT GAATGAGTA (46 nt)

CV

GGGGCTATTG GTTGAATGA GTA (22 nt)

UGCGTGACAC ACGCACTGTG

ODN5

UGCGTGACTTT TGTCACGCAGGG GCTATTGGTTGAA TGAGTA (42 nt)

GGGGCTATTG GTTGAATGA GTA (22 nt)

UGCGTGACAC ACGCACTGTG

For photochemical base substitutions, mixtures including 10nM 72nt-target, 100nM effecter ODN and 2mM MgCl2, in PBS were heated at 90C, then chilled at
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The efficiency of treatment depends on both the concentration of the effecter ODN and the times of photoligation and photosplitting. Accordingly, to determine the optimum conditions for ligation, we investigated the photochemical reaction with different concentration of ODN1 and at different photoligation and photosplitting times with a constant concentration of the 72nt-target (10nM). When the 72nt-target was irradiated at 366nm in the absence of ODN1, no photoligation products were observed (Fig.4; lanes 1, 2, 3 and 4); however, in the presence of 1, 15 and 30 M ODN1 independently, the expected photoligation products were produced (Fig. 4; lanes 8, 11 and 14). When the ODN1 concentration was 0.01 M, no photoligation products were apparent (Fig.4; lanes 5, 6 and 7). When the 72nt-target was irradiated at 312nm, photosplitting be occurred and the photoligation products were not present (Fig. 4; lanes 9, 10, 12, 13, 15 and 16). Thirty minutes is sufficient for photosplitting (Fig.

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4; lanes 9, 12, and 15). From these results, we concluded that the optimum conditions for the ligation of the single-stranded 72nt-target were 1 M ODN1, 5 minutes of irradiation at 366nm and 30 minutes of irradiation at 312nm.

1 2 3

4 5 6 7 8 9 10 11 12 13 14 15 16 Fig. 6 Efficiency of site-directed deamination toward various targets. Various targets including purified samples from the patients cells were subjected to photochemical deamination, then analyzed by RFLP. ODN2 was used as an effecter. A: Result of RFLP, target was described on the top of each lane. A red box is corresponding bands with digested samples. B: Densitometric results of base substitutions from Fig.6A.

Fig. 4 Conditions for ligation of the 72nt-target. The 72nt-target was subjected to photochemical ligation, and then analyzed by native gel followed by SYBR green-staining. The ODN concentrations were 0, 0.01, 1, 15 and 30 M, and irradiation was performed at 366nm for 0 and 5 minutes and at 312nm for 0, 30 and 60 minutes.

The results of these experiments showed that the most effective ODN was ODN2 (Fig. 5).

D. Site-Directed Deamination at 37C In previous experiments, we used 90C in the heat treatment for deamination. However, we cannot use unphysiological condition for disease treatment. It is possible that deamination process progresses in low temperature. Therefore, we subjected longer incubation (48 hours) at 37C instead of 90C for 2 hours.

Fig. 5 Efficiency of site-directed deamination by 5 ODNs as a synthetic 72nt as a target. A 72nt-target were subjected to photochemical deamination, then analyzed by RFLP. A: Result of RFLP, WT; wild type, MT; mutant, ODN*; ODN1 to 5 and no ODN. A red box is corresponding bands with digested samples. B: Densitometric results of base substitutions from Fig. 5A.

C. Site-Directed Deamination toward Various Targets Physiological targets of the effecter ODN are the mtDNA and mRNA in vivo. Therefore, we prepared series of targets, including ss 731nt (single strand 731-mer DNA), ds 681nt (double strand 681nt DNA), whole DNA (purified genome and mitochondrion DNA from the patients cells), full-length ATPase 6-RNA (813nt) and total RNA (purified from the patients cells). Then, each 10nM of ss 72nt, ss 731nt, ds 681nt and full-length RNA or each 5 ng/l of whole DNA and total RNA was added to the reaction mixture with 100nM ODN2 and was subjected to photochemical deamination, essentially same as described above. As shown in Fig. 6, photochemical base substitution was succeeded to series of the target nucleotide even whole DNA and total RNA as a target.

Fig.7 Efficiency of site-directed deamination toward various targets at 37C. Various targets were subjected to photochemical deamination, then analyzed by RFLP as described in Fig. 6 except for heat treatment. A: Result of RFLP, target was described on the top of each lane. A red box is corresponding bands with digested samples. B: Densitometric results of base substitutions from Fig. 7A.

Surprisingly, the efficiency of the base substitution toward full-length ATPase 6-RNA and total RNA from the patients cells increased when the deamination reaction was performed at 37C instead of 90C for 2 hours (Fig. 7). By repeating experiments, almost 10% of site-directed deamination was observed. This condition is acceptable for the novel genetic therapy in vivo.

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4. Discussion
Genetic restoration is one of major tactics for genetic disorders. However, there are many difficulties in expression controls of exogenous genes, even though mutated genes in patients express mutated mRNA. Therefore, if we can repair a part of mutated mRNA, phenotype of patients should be improved. In this research, we tried to restore genetic code of mutated RNA into healthy RNA. In this paper, we tried site -directed photochemical deamination technology to repair a T>C mutation. Among examined 5 ODNs, ODN2 could convert C to U most effectively (Fig. 5). As shown in table 1, ODNs having longer complementary sequences of the target seems to work well. This result is reasonable that longer sequences should bind stronger to the target. In contrast, longer hairpin structure did not show efficient deamination. It is possible that longer exogenous sequences may result of nonspecific sticking of ODNs. Better results were observed when the heat- treatment was performed at 37C instead of 90C by using RNA as a target (please compare Fig.7 and Fig.6). It is known that RNA is destroyed quickly in higher temperature. The result might be caused by stabilization of RNA in lower temperature. We succeeded a sequence-specific photochemical base substitution toward whole DNA and total RNA from patients cells used as a target (Fig. 6 and Fig.7). This result suggests the possibility of restoration of mutated mRNA. Because DNA is present as double-stranded form in cells in usual, RNA should be target for the site-directed restoration of genetic code. We found that almost 10% of RNA was targeted deamination in vitro. It is not efficient enough to treat a genetic disorder. However, we believe that the sitedirected photochemical deamination technology could be improved to develop more efficiently. In this paper, we demonstrated that the site-directed deamination for genetic restoration in vitro. In vivo study includes cultured cells and model animals must be done in the near future.

References
[1] S. Farajollahi & S. Maas, Molecular diversity through RNA editing: a balancing act, (2010) Trends in Genet., vol. 26, pp.221-230. [2] J. Shrestha, Regulation of ADAR2 activity by RNA editing, (2002) MURJ Rep., vol. 6, pp.45-48. [3] E. Kanaya & H. Yanagawa, Template-directed polymerization of oligoadenylates using cyanogen bromide, (1986) Biochemistry, vol. 25, pp.7423-7430. [4] D. Sievers & G. von Kiedrowski, Self-replication of complementary nucleotide-based oligomers, (1994) Nature, vol.369, pp.221224. [5] R. Rohatgi, D. P. Bartel & J. W. Szostak, Kinetic and Mechanistic Analysis of Nonenzymatic, Template-Directed Oligoribonucleotide Ligation, (1996) J. Am. Chem. Soc., vol. 118, pp.33323344. [6] T. H. Li, D. S. Weinstein & K. C. Nicolaou, The chemical end-ligation of homopyrimidine oligodeoxyribonucleotides within a DNA triple helix, (1997) Chem. Biol., vol. 4(3), pp.209-214. [7] A. E. Ferentz & G. L. Verdine, Disulfide-Crosslinked Oligonucleotides, (1991) J. Am. Chem. Soc., vol. 113, pp.4000-4002. [8] S. M. Gryaznov & R. L. Letsinger, Chemical ligation of oligonucleotides in the presence and absence of a template, (1993) J. Am. Chem. Soc., vol.115, pp.38083809. [9] R. L. Letsinger, T. Wu & R. Elghanian, Chemical and photochemical ligation of oligonucleotide blocks, (1997) Nucleosides Nucleotides, vol.16, pp.643652. [10] R. J. Lewis & P. S. Hanawalt, Ligation of oligonucleotides by pyrimidine dimers-a missing 'link' in the origin of life?, (1982) Nature, vol.298, pp.393396. [11] G. P. Royer, K. A. Cruickshank & L. E. Morrison Template-directed photoligation, (1989) European patent 0324626A2. [12] K. Fujimoto, S. Matsuda, Y. Yoshimura, T. Matsumura, M. Hayashi, & I. Saito, Site-specific transition of cytosine to uracil via reversible DNA photoligation, (2006) Chem. Comm., pp. 3223-3225. [13] J. Finsterer, Leigh and Leigh-Like Syndrome in Children and Adults, (2008) Pediatr. Neurol. vol. 39, pp. 223-235. [14] K. Fujimoto, N. Ogawa, M. Hayashi, S. Matsuda & I. Saito, Template directed photochemical synthesis of branched oligodeoxynucleotides via 5-carboxyvinyl- deoxyuridine, (2000) Tetrahedron Lett., vol. 41, pp.9437-9440. [15] M. Makino, S. Horai, Y. Goto & I. Nonaka, Mitochondrial DNA mutations in Leigh syndrome and their phylogenetic implications, (2000) J. Hum. Genet, vol. 45, pp.69-75.

5. Conclusion
In this research, we observed an efficient site-directed base-substitution via photochemical deamination in vitro. The C>U substitution means restoration of genetic code in diseases caused T>C point mutations. Therefore, we conclude that photochemical site-directed deamination technology could be useful for novel genetic restoration therapy towards genetic disorders caused T>C point mutations.

Acknowledgment
This research was supported in part by a JAIST's Research Grant and a Grant-in-Aid from the Japan Society for the Promotion of Science.
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