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A bridged nucleic acid, 2',4'-BNA COC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2',4'-BNA COC monomers and RNA-selective nucleic-acid recognition.

Mitsuoka Y, Kodama T, Ohnishi R, Hari Y, Imanishi T, Obika S - Nucleic Acids Res. (2009)

Bottom Line: Oligonucleotides (BNA(COC)) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation.Furthermore, mismatched sequence studies showed that BNA(COC) generally improved the sequence selectivity with Watson-Crick base-pairing compared to the corresponding natural DNA and RNA.A CD spectroscopic analysis indicated that the BNA(COC) formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.

ABSTRACT
Recently, we synthesized pyrimidine derivatives of the 2'-O,4'-C-methylenoxymethylene-bridged nucleic-acid (2',4'-BNA(COC)) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNA(COC)) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNA(COC) consisting of 2',4'-BNA(COC) monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2',4'-BNA(COC) monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNA(COC)/BNA(COC) duplex possessed excellent thermal stability and that the BNA(COC) increased thermal stability with a complementary RNA strand. On the other hand, BNA(COC)/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNA(COC) generally improved the sequence selectivity with Watson-Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNA(COC) formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.

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(A) CD spectra of BNACOC duplex 27/30 (red line), BNACOC/RNA heteroduplexes 27/29 (orange line) and 26/30 (green line), DNA duplex 25/28 (black line), RNA duplex 26/29 (blue line); (B) CD spectra of duplexes the BNACOC/DNA heteroduplexes 27/28 (red line) and 25/30 (green line) and DNA/RNA duplexes 25/29 (black line) and 26/28 (blue line). The spectra were recorded in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl at 12°C. The concentration of each strand was 4 μM.
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Figure 4: (A) CD spectra of BNACOC duplex 27/30 (red line), BNACOC/RNA heteroduplexes 27/29 (orange line) and 26/30 (green line), DNA duplex 25/28 (black line), RNA duplex 26/29 (blue line); (B) CD spectra of duplexes the BNACOC/DNA heteroduplexes 27/28 (red line) and 25/30 (green line) and DNA/RNA duplexes 25/29 (black line) and 26/28 (blue line). The spectra were recorded in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl at 12°C. The concentration of each strand was 4 μM.

Mentions: Circular dichroism spectroscopic analyses of BNACOC-hybridizing duplexes were performed to investigate the structural preferences. CD spectra were recorded at 10°C for the BNACOC duplex 27/30, the BNACOC/DNA heteroduplexes 27/28 and 30/25, the BNACOC/RNA heteroduplexes 27/29 and 30/26, as well as for the control DNA duplex 25/28, the RNA duplex 26/29 and the DNA/RNA duplexes 25/29 and 28/26 (Figure 4). The CD spectra of BNACOC duplex 27/30 and the BNACOC/RNA heteroduplexes 27/29 and 30/26 were similar to that of the natural RNA duplex. These spectra showed a large positive Cotton band around 265 nm and a large negative Cotton band at 210 nm, indicating a typical A-form helix formation (Figure 4A). In the case of the BNACOC/DNA and the RNA/DNA heteroduplexes, all spectra were very similar to each other and the shoulder band around 280 nm suggested an intermediate between A- and B-form geometry. This revealed that BNACOC formed duplexes with complementary DNA in the same manner the natural RNA did. Additionally, the CD profile of the natural DNA duplex belonged to no other types of duplexes in this study.Figure 4.


A bridged nucleic acid, 2',4'-BNA COC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2',4'-BNA COC monomers and RNA-selective nucleic-acid recognition.

Mitsuoka Y, Kodama T, Ohnishi R, Hari Y, Imanishi T, Obika S - Nucleic Acids Res. (2009)

(A) CD spectra of BNACOC duplex 27/30 (red line), BNACOC/RNA heteroduplexes 27/29 (orange line) and 26/30 (green line), DNA duplex 25/28 (black line), RNA duplex 26/29 (blue line); (B) CD spectra of duplexes the BNACOC/DNA heteroduplexes 27/28 (red line) and 25/30 (green line) and DNA/RNA duplexes 25/29 (black line) and 26/28 (blue line). The spectra were recorded in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl at 12°C. The concentration of each strand was 4 μM.
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Related In: Results  -  Collection

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Figure 4: (A) CD spectra of BNACOC duplex 27/30 (red line), BNACOC/RNA heteroduplexes 27/29 (orange line) and 26/30 (green line), DNA duplex 25/28 (black line), RNA duplex 26/29 (blue line); (B) CD spectra of duplexes the BNACOC/DNA heteroduplexes 27/28 (red line) and 25/30 (green line) and DNA/RNA duplexes 25/29 (black line) and 26/28 (blue line). The spectra were recorded in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl at 12°C. The concentration of each strand was 4 μM.
Mentions: Circular dichroism spectroscopic analyses of BNACOC-hybridizing duplexes were performed to investigate the structural preferences. CD spectra were recorded at 10°C for the BNACOC duplex 27/30, the BNACOC/DNA heteroduplexes 27/28 and 30/25, the BNACOC/RNA heteroduplexes 27/29 and 30/26, as well as for the control DNA duplex 25/28, the RNA duplex 26/29 and the DNA/RNA duplexes 25/29 and 28/26 (Figure 4). The CD spectra of BNACOC duplex 27/30 and the BNACOC/RNA heteroduplexes 27/29 and 30/26 were similar to that of the natural RNA duplex. These spectra showed a large positive Cotton band around 265 nm and a large negative Cotton band at 210 nm, indicating a typical A-form helix formation (Figure 4A). In the case of the BNACOC/DNA and the RNA/DNA heteroduplexes, all spectra were very similar to each other and the shoulder band around 280 nm suggested an intermediate between A- and B-form geometry. This revealed that BNACOC formed duplexes with complementary DNA in the same manner the natural RNA did. Additionally, the CD profile of the natural DNA duplex belonged to no other types of duplexes in this study.Figure 4.

Bottom Line: Oligonucleotides (BNA(COC)) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation.Furthermore, mismatched sequence studies showed that BNA(COC) generally improved the sequence selectivity with Watson-Crick base-pairing compared to the corresponding natural DNA and RNA.A CD spectroscopic analysis indicated that the BNA(COC) formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.

ABSTRACT
Recently, we synthesized pyrimidine derivatives of the 2'-O,4'-C-methylenoxymethylene-bridged nucleic-acid (2',4'-BNA(COC)) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNA(COC)) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNA(COC) consisting of 2',4'-BNA(COC) monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2',4'-BNA(COC) monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNA(COC)/BNA(COC) duplex possessed excellent thermal stability and that the BNA(COC) increased thermal stability with a complementary RNA strand. On the other hand, BNA(COC)/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNA(COC) generally improved the sequence selectivity with Watson-Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNA(COC) formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.

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Related in: MedlinePlus