Duplex-forming Ability Research Articles

Triplex- and Duplex-Forming Abilities of Oligonucleotides Containing 2'-Deoxy-5-trifluoromethyluridine and 2'-Deoxy-5-trifluoromethylcytidine.

A facile synthesis of 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine phosphoramidites from commercially available 2'-deoxyuridine and 2'-deoxycytidine was achieved, respectively. The obtained phosphoramidites were incorporated into oligonucleotides, and their binding affinity to double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) was evaluated by UV-melting experiments. The triplex-forming abilities of oligonucleotides including 5-trifluoromethylpyrimidine nucleobases with dsDNA were decreased. Especially, the stability of the triplex containing a trifluoromethylcytosine (CF3C)-GC base triplet was low, likely due to the low pKa of protonated CF3C by the electron-withdrawing trifluoromethyl group. A slight decrease in stability of the duplex formed with ssRNA by oligonucleotides including 5-trifluoromethylpyrimidine nucleobases was only observed, suggesting that they might be applicable to various ssRNA-targeted technologies using features of fluorine atoms.

Synthesis and Properties of 7-Deazapurine- and 8-Aza-7-deazapurine-Locked Nucleic Acid Analogues: Effect of the Glycosidic Torsion Angle.

Conformationally restricted nucleoside analogues 2',4'-BNA/LNA-7-deazaguanine (LNA-7cG) and 2',4'-BNA/LNA-8-aza-7-deazaguanine (LNA-8n7cG), which avoid extra hydrogen bond formation at the 7-position of the guanine nucleobase, were successfully synthesized and incorporated into oligonucleotides. While the LNA-7cG-containing oligonucleotides show high duplex-forming ability with complementary DNA and RNA similar to LNA-G, the LNA-8n7cG-containing oligonucleotide has lower binding affinity than that of natural 2'-deoxyguanosine. This disparity in thermostability is also observed in 7-deazaadenosine analogues (LNA-7cA, LNA-8n7cA). Thermodynamic parameters and computational chemistry revealed that an inappropriate glycosidic torsion angle χ of 2',4'-BNA/LNA-8-aza-7-deazapurine analogues destabilizes duplex formation in contrast to 2',4'-BNA/LNA-7-deazapurine analogues. This result indicates that the nucleobase rotation angle plays an important role in duplex binding affinity. In addition, LNA-7cG-modified oligonucleotide effectively suppresses aggregation even in a guanine-rich sequence.

Synthesis of scpBNA-mC, -A, and -G Monomers and Evaluation of the Binding Affinities of scpBNA-Modified Oligonucleotides toward Complementary ssRNA and ssDNA.

We previously reported the synthesis and evaluation of 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid (scpBNA) bearing a thymine (T) nucleobase. Oligonucleotides (ONs) modified with scpBNA-T exhibited strong binding affinity to complementary single-stranded RNA (ssRNA) and high enzymatic stability. These biophysical properties suggest that scpBNAs are well suited for use in antisense strategies. Herein, we describe the synthesis of scpBNA monomers bearing 5-methylcytosine (mC), adenine (A), and guanine (G) nucleobases for use in a variety of sequences. The prepared scpBNA monomers were incorporated into ONs at various positions. The scpBNA-modified ONs exhibited excellent duplex-forming ability with the complementary ssRNA comparable to ONs modified with 2'-O,4'-C-methylene-bridged nucleic acid (2',4'-BNA/LNA). Moreover, ON modified with scpBNA-mC, -A, and -G showed higher enzymatic stability than the corresponding 2',4'-BNA/LNA-modified ON. These results demonstrated a promising role for the incorporation of scpBNA monomers into therapeutic antisense ONs.

架橋部に複数のヘテロ原子を有する2’,4’-架橋型人工核酸の合成と機能

Nucleic acids bridged between 2’- and 4’-positions are attracting much attention because the oligonucleotides bearing these derivatives generally increase in the binding affinity with single-stranded RNA and have an increased resistance against nuclease. It is found that their properties are greatly affected by size and composition of the bridge. In particular, introduction of heteroatom into the bridge unit is considered to give an influence on network of surrounding water molecules in formation of nucleic acid complex. Therefore, it would be meaningful to investigate and understand the properties of various 2’,4’-bridged nucleic acids. Under such a background, this account focuses on 2’,4’-bridged nucleic acids containing multiple heteroatoms in the bridge structures, with five-, six- and seven-membered bridges, have recently been reported by us, and the synthesis, duplex-forming ability with single-stranded nucleic acid and nuclease resistance of their modified oligonucleotides are mainly described.

Effect of an N-substituent in sulfonamide-bridged nucleic acid (SuNA) on hybridization ability and duplex structure.

A sulfonamide-bridged nucleic acid without an N-substituent (SuNA[NH]) was successfully synthesized. A comparison of the SuNA[NMe]- and SuNA[NH]-modified oligonucleotides revealed that the duplex-forming abilities of the SuNA[NMe]-modified oligonucleotides with complementary DNA and RNA were higher than those of the SuNA[NH]-modified oligonucleotides. The crystal structures of DNA duplexes containing a SuNA[NR] revealed that the helical structures of the two duplexes and hydration patterns around the bridge moiety were different. These results provide insights into hydration patterns and rationale for the high RNA affinity of SuNA-modified oligonucleotides.

Synthesis and properties of 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (scpBNA), an analogue of 2',4'-BNA/LNA bearing a cyclopropane ring.

2'-O,4'-C-Spirocyclopropylene bridged nucleic acid (scpBNA), an analogue of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA/LNA) bearing a cyclopropane ring at the 6'-position, was synthesized and successfully incorporated into oligonucleotides. The scpBNA-modified oligonucleotides showed excellent duplex-forming ability with complementary single-stranded RNA and exhibited increased enzymatic stability as compared to the corresponding natural and 2',4'-BNA/LNA-modified oligonucleotides. Our results demonstrate the potential of scpBNA for gene therapeutics, such as antisense technology.

Synthesis and hybridization property of a boat-shaped pyranosyl nucleic acid containing an exocyclic methylene group in the sugar moiety

A boat-shaped pyranosyl nucleic acid (BsNA) having an exocyclic methylene group in the sugar moiety was synthesized to investigate the possibility that the axial H3′ of original BsNA is the cause of its duplex destabilization. The synthesized BsNA analog was chemically stable against various nucleophiles. From the thermal stability of duplex oligonucleotides including the BsNA analog, it was found that the duplex-forming ability can be sensitive to the size of functional groups at the 3′-position.

Hybridizing ability and nuclease resistance profile of backbone modified cationic phosphorothioate oligonucleotides

Various stereochemically pure cationic phosphorothioate oligonucleotides bearing aminoalkyl moieties were synthesized, and their duplex-forming ability against single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and triplex-forming ability against double-stranded DNA (dsDNA) were evaluated by UV melting experiments. The cationic Rp stereoisomers showed improved duplex-forming ability against ssDNA, triplex-forming ability against dsDNA and nuclease stability.

Synthesis and duplex-forming ability of oligonucleotides containing 4′-carboxythymidine analogs

Oligonucleotides containing 4'-carboxy-, 4'-methoxycarbonyl-, 4'-carbamoyl-, and 4'-methylcarbamoyl-thymidines, and their 2'-methoxy, 2'-amino or 2'-acetamido analogs were prepared. Their duplex-forming ability with DNA and RNA complements was evaluated by UV melting experiments. Interestingly, 4'-carboxythymidine existing in the S-type sugar conformation was found to lead to an increase in the stability of the duplex formed with RNA complements compared to natural thymidine.

Design, Synthesis, and Properties of Boat-Shaped Glucopyranosyl Nucleic Acid

A boat-shaped glucopyranosyl nucleic acid (BsNA) was synthesized to investigate the possibility that the lean of a nucleobase is a factor affecting duplex-forming ability of oligonucleotides. From the crystal structure of a BsNA nucleoside and the thermal stability of duplex oligonucleotides, it was found that not only the lean of the base but also the rotation angle of the glycosidic bond axis were important factors in a stable duplex formation.

Synthesis and properties of a novel artificial nucleoside that acts as a redox switch

Oligonucleotide that can reversibly alter the duplex-forming ability in response to an external factor has potential to be applied to various genome technologies, e.g. antisense and gene diagnosis technology. A novel bridged nucleoside which is capable of sensing an oxidant and reductant was designed and synthesized. The nucleoside formed a disulfide bridge between C2' and C4' positions, which would enhance its duplex-forming ability under oxidative conditions. Contrastingly, the disulfide bridge was cleaved under reductive conditions; it could cause decrease of duplex-forming ability.

Synthesis, Duplex-forming Ability and Fluorescent Property of OligoDNA Bearing Silylated Fluorophore

Novel pyrene derivative conjugated with dimethylsilyl moiety was prepared from bromopyrene. The compound was successfully converted to the corresponding phosphoramidite derivative and incorporated into oligoDNA. In uv-melting study, the resulted fluorescent oligoDNA can discriminate one-base mismatched complement only when the mismatched base is positioned around the middle of the duplex-forming region. On the other hand, the oligoDNA gives marked fluorescence upon binding with the full-matched complement. The intensity of the fluorescent signal of the oligoDNA in a duplex changes depending on the position of the mismatched base of the complement.

High-affinity RNA mimicking binding of 2′,4′-BNANC towards complementary strands: A comparative study with 2′,4′-BNA/LNA

2',4'-BNA(NC), a bridged nucleic acid analogue, which was designed and synthesized in our laboratory, showed very high binding affinity towards complementary RNA and DNA strands. Its duplex-forming ability towards a single-stranded RNA was similar to or slightly higher than that of 2',4'-BNA and the overall triplex-forming ability against a double-stranded DNA was also better than that of 2',4'-BNA. 2',4'-BNA(NC) exhibited higher RNA selectivity than 2',4'-BNA.

Watson–Crick Base‐Pairing Properties of Nucleic Acid Analogues with Stereocontrolled α and β Torsion Angles (α,β‐D‐CNAs)

Constrained nucleic acids (α,β-D-CNAs) have an ethylene bridge that locks the α and β backbone torsion angles of DNA in the canonical (gauche(−),trans) conformation, as in B-form DNA (see picture), or in the noncanonical (gauche(+),trans) conformation. Canonical α,β-D-CNAs enhance the duplex-forming ability of oligonucleotides with complementary DNA, while noncanonical α,β-D-CNAs stabilize distorted B-DNA structures. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2006/z504475_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Adjustment of the γ Dihedral Angle of an Oligonucleotide P3′→N5′ Phosphoramidate Enhances Its Binding Affinity towards Complementary Strands

Bridge to the right angle: A methylene bridge between the C3′ and N5′ atoms restricts the γ dihedral angle of an oligonucleotide P3′→N5′ phosphoramidate within the +sc orientation (see scheme), which improves duplex-forming ability and accelerates hydrolytic cleavage. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2005/z461942_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.