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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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Structures of 2′,4′-BNA/LNA, ENA, 2′,4′-BNANC, PrNA and 2′,4′-BNACOC monomers.
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Figure 2: Structures of 2′,4′-BNA/LNA, ENA, 2′,4′-BNANC, PrNA and 2′,4′-BNACOC monomers.

Mentions: Antisense oligonucleotides are now attracting interest for their potential to be developed as a new class of drugs for treatment of inveterate diseases such as cancer and viral diseases. For practical application of antisense methodology, it is essential to develop modified oligonucleotides, which strongly interact with single-stranded RNA (ssRNA) in a sequence-specific manner (1–3). The sugar moiety of natural nucleosides and single-stranded oligonucleotides exists in an individual equilibrium mixture between S-type and N-type conformations. However, it is well known that the B-form DNA duplex possesses the S-type sugar conformation, and that a range of N-type sugar conformation (pseudorotation phase angle, 0 ≤ P ≤ 36°) are adapted to the A-form RNA duplex structure (Figure 1) (4–6). Therefore, modified oligonucleotides, which have sugar moiety restricted to the S-type or N-type conformations in advance, are expected to have high binding affinity with complementary ssDNA or ssRNA respectively. We have so far developed various kinds of bridged nucleic acids (BNAs) (7–19), the sugar conformation of which is restricted or locked by introduction of an additional bridged structure to the furanose skeleton. It has been observed that 2′,4′-BNA (9,10)/LNA [The 2′,4′-BNA was independently synthesized by the group of Wengel et al. immediately after our first report (9), and it is called a locked nucleic acid (LNA). See refs (3,20–22)] (Figure 2), which is a BNAs with its sugar moiety fixed to an N-type conformation by five-membered ring, prominently hybridizes to ssRNA targets. An X-ray crystallographic analysis of 2′,4′-BNA showed that the maximum out-of-plane (νmax) value was 57°, and that this value is larger than an adapted value (νmax = 38.6° ± 3°) to natural A-form RNA duplex (4–6), so the N-type nature of 2′,4′-BNA was emphasized due to the restriction of the sugar moiety by the small five-membered ring. Other nucleic-acid analogues with a different type of bridged structure between the 2′- and 4′-positions, which have six- and seven-membered ring, have been reported (16–19,23–29). In one of these studies, we described the synthesis and properties of 2′-O,4′-C-aminomethylene BNA (2′,4′-BNANC) (Figure 2), and showed that oligonucleotides containing 2′,4′-BNANC have high hybridizing affinity with RNA compliments (16–19). X-ray crystallographic analysis revealed that the P and νmax values of 2′,4′-BNANC[NMe] were 23° and 49°, respectively (19). These values indicated that the conformation of 2′,4′-BNANC[NMe] is restricted to the N-type conformation as seen in natural A-form RNA duplex. This suggested that the sugar conformation restricted by the bridged structure between the 2′- and 4′-positions approximated to the adapted sugar conformation of the natural A-form RNA duplex because of increased ring size. Recently, we designed 2′,4′-BNACOC, bearing a seven-membered bridged structure, and successfully synthesized the 2′,4′-BNACOC monomers having pyrimidine nucleobases (Figure 2) (30). The oligonucleotides containing these monomers show high affinity with complementary ssRNA. An X-ray crystallographic analysis of 2′,4′-BNACOC bearing thymine showed that the P and νmax values were 17° and 38°, respectively. This revealed that the sugar conformation of 2′,4′-BNACOC, which is restricted by a large seven-membered ring, is identical with the N-type sugar puckering fit to canonical A-form RNA duplex, and that 2′,4′-BNACOC has the closest sugar conformation among nucleic-acid analogues with a different type of bridged structure between the 2′- and 4′-positions.Figure 1.


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)

Structures of 2′,4′-BNA/LNA, ENA, 2′,4′-BNANC, PrNA and 2′,4′-BNACOC monomers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651773&req=5

Figure 2: Structures of 2′,4′-BNA/LNA, ENA, 2′,4′-BNANC, PrNA and 2′,4′-BNACOC monomers.
Mentions: Antisense oligonucleotides are now attracting interest for their potential to be developed as a new class of drugs for treatment of inveterate diseases such as cancer and viral diseases. For practical application of antisense methodology, it is essential to develop modified oligonucleotides, which strongly interact with single-stranded RNA (ssRNA) in a sequence-specific manner (1–3). The sugar moiety of natural nucleosides and single-stranded oligonucleotides exists in an individual equilibrium mixture between S-type and N-type conformations. However, it is well known that the B-form DNA duplex possesses the S-type sugar conformation, and that a range of N-type sugar conformation (pseudorotation phase angle, 0 ≤ P ≤ 36°) are adapted to the A-form RNA duplex structure (Figure 1) (4–6). Therefore, modified oligonucleotides, which have sugar moiety restricted to the S-type or N-type conformations in advance, are expected to have high binding affinity with complementary ssDNA or ssRNA respectively. We have so far developed various kinds of bridged nucleic acids (BNAs) (7–19), the sugar conformation of which is restricted or locked by introduction of an additional bridged structure to the furanose skeleton. It has been observed that 2′,4′-BNA (9,10)/LNA [The 2′,4′-BNA was independently synthesized by the group of Wengel et al. immediately after our first report (9), and it is called a locked nucleic acid (LNA). See refs (3,20–22)] (Figure 2), which is a BNAs with its sugar moiety fixed to an N-type conformation by five-membered ring, prominently hybridizes to ssRNA targets. An X-ray crystallographic analysis of 2′,4′-BNA showed that the maximum out-of-plane (νmax) value was 57°, and that this value is larger than an adapted value (νmax = 38.6° ± 3°) to natural A-form RNA duplex (4–6), so the N-type nature of 2′,4′-BNA was emphasized due to the restriction of the sugar moiety by the small five-membered ring. Other nucleic-acid analogues with a different type of bridged structure between the 2′- and 4′-positions, which have six- and seven-membered ring, have been reported (16–19,23–29). In one of these studies, we described the synthesis and properties of 2′-O,4′-C-aminomethylene BNA (2′,4′-BNANC) (Figure 2), and showed that oligonucleotides containing 2′,4′-BNANC have high hybridizing affinity with RNA compliments (16–19). X-ray crystallographic analysis revealed that the P and νmax values of 2′,4′-BNANC[NMe] were 23° and 49°, respectively (19). These values indicated that the conformation of 2′,4′-BNANC[NMe] is restricted to the N-type conformation as seen in natural A-form RNA duplex. This suggested that the sugar conformation restricted by the bridged structure between the 2′- and 4′-positions approximated to the adapted sugar conformation of the natural A-form RNA duplex because of increased ring size. Recently, we designed 2′,4′-BNACOC, bearing a seven-membered bridged structure, and successfully synthesized the 2′,4′-BNACOC monomers having pyrimidine nucleobases (Figure 2) (30). The oligonucleotides containing these monomers show high affinity with complementary ssRNA. An X-ray crystallographic analysis of 2′,4′-BNACOC bearing thymine showed that the P and νmax values were 17° and 38°, respectively. This revealed that the sugar conformation of 2′,4′-BNACOC, which is restricted by a large seven-membered ring, is identical with the N-type sugar puckering fit to canonical A-form RNA duplex, and that 2′,4′-BNACOC has the closest sugar conformation among nucleic-acid analogues with a different type of bridged structure between the 2′- and 4′-positions.Figure 1.

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.

Show MeSH
Related in: MedlinePlus