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Synthesis and characterization of 2'-modified-4'-thioRNA: a comprehensive comparison of nuclease stability.

Takahashi M, Minakawa N, Matsuda A - Nucleic Acids Res. (2009)

Bottom Line: The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2'-fluoroRNA (FRNA), 2'-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t(1/2) values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3'-exonuclease).Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t(1/2) = 1631 min) compared with FRNA (t(1/2) = 53.2 min) and MeRNA (t(1/2) = 187 min), whose modifications are currently used as components of therapeutic aptamers.The results presented in this article will, it is hoped, contribute to the development of 2'-modified-4'-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

ABSTRACT
We report herein the synthesis and physical and physiological characterization of fully modified 2'-modified-4'-thioRNAs, i.e. 2'-fluoro-4'-thioRNA (F-SRNA) and 2'-O-Me-4'-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2'-modified oligonucleotides (ONs) and 4'-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T(m) value (+16 degrees C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2'-fluoroRNA (FRNA), 2'-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t(1/2) values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3'-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t(1/2) = 1631 min) compared with FRNA (t(1/2) = 53.2 min) and MeRNA (t(1/2) = 187 min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2'-modified-4'-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.

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Sequences of modified ONs.
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Figure 2: Sequences of modified ONs.

Mentions: As reported in our previous article (18), we have synthesized 2′-deoxy-2′-fluoro-4′-thiouridine, -thiocytidine and -thioadenosine, the components of F-SRNA. However, despite further attempts to prepare 2′-deoxy-2′-fluoro-4′-thioguanosine, we did not obtain the desired compound in sufficient quantity to carry out ON synthesis. For the synthesis of a series of 2′-O-Me-4′-thioribonucleosides, the components of Me-SRNA, uridine, cytidine, adenosine and guanosine derivatives were all prepared according to methods similar to those used for their 4′-O-congener (19,20). Thus, treatment of 2,2′-O-anhydro-4′-thiouridine (1) (9) with a mixture of trimethyl borate and trimethyl orthoformate in the presence of a catalytic amount of sodium bicarbonate afforded 2′-O-Me-4′-thiouridine (2) in 86% yield (Scheme 1). The resulting 2 was converted into its cytosine derivative, and then the corresponding phosphoramidite units, respectively, using the standard procedure (details of the synthetic scheme and experimental procedures are given in the Supplementary Data; Scheme S2). In the case of the purine derivatives, a direct methylation of the 2′-hydroxy group was employed. In Scheme 2, the synthesis of the 2′-O-Me-4′-thioguanosine units is shown as an example, since the chemical conversion of the nucleobase differs slightly from that of our previous method (10). Thus, immediately after the Pummerer reaction (8), compound 3 was heated in NH3/EtOH, followed by MeNH2/MeOH at room temperature to give the diamino derivative 4. When the resulting 4 was treated with MeI in DMF in the presence of NaH at –40°C, the 2′-O-Me derivative 5 was obtained in 85% yield. Selective protection of the 2-amino group of 5 by isobutyryl chloride afforded 6. The remaining 6-amino group of 6 was then hydrolyzed via diazotization to give the guanine derivative. Since a partial deprotection of the TIPDS group on the sugar portion took place during the reaction, the resulting mixture was subsequently treated with Et3N•3HF to give 7 in 73% yield in two steps. Protection of the 5′-hydroxy group with a dimethoxytrityl (DMTr) group, followed by reaction with N,N-diisopropylchlorophosphoramidite in the presence of Hünig′s base afforded the 2′-O-Me-4′-thioguanosine phosphoramidite unit 9. The corresponding adenosine unit was also prepared in a similar manner (details of the synthetic scheme and experimental procedures are given in the Supplementary Data; Scheme S3). Using these phosphoramidite units, we synthesized 2′-modified-4′-thioRNAs along with other ONs in a DNA/RNA synthesizer following the standard procedure. The sequences used in this study are shown in Figure 2. The series of ON1s includes Me-SRNA (Me-SRNA1), which is the same as that used in our previous report (10), while the sequence of ON2s includes F-SRNA (F-SRNA2), since the 4′-thioguanosine phosphoramidite unit of this modified ON was not available. For a comprehensive comparison of their properties, MeRNAs (MeRNA1 and MeRNA2), FRNAs (FRNA1 and FRNA2), and SRNAs [SRNA1 (10) and SRNA2] along with natural RNAs (RNA1 and RNA2) and DNAs (DNA1 and DNA2) were also prepared.Figure 2.


Synthesis and characterization of 2'-modified-4'-thioRNA: a comprehensive comparison of nuclease stability.

Takahashi M, Minakawa N, Matsuda A - Nucleic Acids Res. (2009)

Sequences of modified ONs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651785&req=5

Figure 2: Sequences of modified ONs.
Mentions: As reported in our previous article (18), we have synthesized 2′-deoxy-2′-fluoro-4′-thiouridine, -thiocytidine and -thioadenosine, the components of F-SRNA. However, despite further attempts to prepare 2′-deoxy-2′-fluoro-4′-thioguanosine, we did not obtain the desired compound in sufficient quantity to carry out ON synthesis. For the synthesis of a series of 2′-O-Me-4′-thioribonucleosides, the components of Me-SRNA, uridine, cytidine, adenosine and guanosine derivatives were all prepared according to methods similar to those used for their 4′-O-congener (19,20). Thus, treatment of 2,2′-O-anhydro-4′-thiouridine (1) (9) with a mixture of trimethyl borate and trimethyl orthoformate in the presence of a catalytic amount of sodium bicarbonate afforded 2′-O-Me-4′-thiouridine (2) in 86% yield (Scheme 1). The resulting 2 was converted into its cytosine derivative, and then the corresponding phosphoramidite units, respectively, using the standard procedure (details of the synthetic scheme and experimental procedures are given in the Supplementary Data; Scheme S2). In the case of the purine derivatives, a direct methylation of the 2′-hydroxy group was employed. In Scheme 2, the synthesis of the 2′-O-Me-4′-thioguanosine units is shown as an example, since the chemical conversion of the nucleobase differs slightly from that of our previous method (10). Thus, immediately after the Pummerer reaction (8), compound 3 was heated in NH3/EtOH, followed by MeNH2/MeOH at room temperature to give the diamino derivative 4. When the resulting 4 was treated with MeI in DMF in the presence of NaH at –40°C, the 2′-O-Me derivative 5 was obtained in 85% yield. Selective protection of the 2-amino group of 5 by isobutyryl chloride afforded 6. The remaining 6-amino group of 6 was then hydrolyzed via diazotization to give the guanine derivative. Since a partial deprotection of the TIPDS group on the sugar portion took place during the reaction, the resulting mixture was subsequently treated with Et3N•3HF to give 7 in 73% yield in two steps. Protection of the 5′-hydroxy group with a dimethoxytrityl (DMTr) group, followed by reaction with N,N-diisopropylchlorophosphoramidite in the presence of Hünig′s base afforded the 2′-O-Me-4′-thioguanosine phosphoramidite unit 9. The corresponding adenosine unit was also prepared in a similar manner (details of the synthetic scheme and experimental procedures are given in the Supplementary Data; Scheme S3). Using these phosphoramidite units, we synthesized 2′-modified-4′-thioRNAs along with other ONs in a DNA/RNA synthesizer following the standard procedure. The sequences used in this study are shown in Figure 2. The series of ON1s includes Me-SRNA (Me-SRNA1), which is the same as that used in our previous report (10), while the sequence of ON2s includes F-SRNA (F-SRNA2), since the 4′-thioguanosine phosphoramidite unit of this modified ON was not available. For a comprehensive comparison of their properties, MeRNAs (MeRNA1 and MeRNA2), FRNAs (FRNA1 and FRNA2), and SRNAs [SRNA1 (10) and SRNA2] along with natural RNAs (RNA1 and RNA2) and DNAs (DNA1 and DNA2) were also prepared.Figure 2.

Bottom Line: The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2'-fluoroRNA (FRNA), 2'-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t(1/2) values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3'-exonuclease).Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t(1/2) = 1631 min) compared with FRNA (t(1/2) = 53.2 min) and MeRNA (t(1/2) = 187 min), whose modifications are currently used as components of therapeutic aptamers.The results presented in this article will, it is hoped, contribute to the development of 2'-modified-4'-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.

ABSTRACT
We report herein the synthesis and physical and physiological characterization of fully modified 2'-modified-4'-thioRNAs, i.e. 2'-fluoro-4'-thioRNA (F-SRNA) and 2'-O-Me-4'-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2'-modified oligonucleotides (ONs) and 4'-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T(m) value (+16 degrees C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2'-fluoroRNA (FRNA), 2'-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t(1/2) values of > 24 h against S1 nuclease (an endonuclease) and 79.2 min against SVPD (a 3'-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t(1/2) = 1631 min) compared with FRNA (t(1/2) = 53.2 min) and MeRNA (t(1/2) = 187 min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2'-modified-4'-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.

Show MeSH