A brief history of Bridged Nucleic Acids (BNAs)


By Klaus D. Linse   01/03/2013

 A quest for better oligonucleotide mimics.

The quest for oligonucleotide mimics with improved characteristics and stabilities useful for molecular diagnostics and therapeutics that also show minimal side effects has led to the design and synthesis of novel bridged nucleic acid monomers and oligonucleotides. These synthetic oligonucleotide mimics may be used as tools for gene validation, as antisense (targeting mRNA) and antigene (targeting DNA) agents, for selective regulation of gene expression and as a potentially new class of drugs for the treatment of diseases such as cancer, inflammation, viral diseases and other pathological diseases. The 3D structures for A-RNA and B-DNA were used as a template for the design of the BNA monomers. The goal for the design is to find derivatives that posses high binding affinities with complementary RNA and/or DNA strands.

Chemical structures for A-RNA and B-DNA are shown below.

MovieStructures 1

Movie: A-RNA:

Movie of B-DNA:

RNA contains ribose rather than 2’-deoxyribose in its backbone. The ribose has a hydroxyl group at the 2’-position. Furthermore, RNA contains the nucleic acid uracil in place of thymine and is usually found as a single polynucleotide chain. While RNA is typically single stranded, RNA chains can frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences. The presence of 2’-hydroxyls in the RNA backbone favors a structure that resembles the A-form structure of DNA. The flexible five-membered furanose ring in nucleotides exists in equilibrium of two preferred conformations of the N- (C3’-endo, A-form) and the S-type (C2’-endo, B-form) as depicted in the insert. A closer look at the two forms is shown below. An increased conformational inflexibility of the sugar moiety in nucleosides (oligonucleotides) should result in a gain of high binding affinity with complementary single-stranded RNA and/or double-stranded DNA.

Puckerings

closer look

comparison

Synthetic oligonucleotides are now important, established tools for life scientists and have many applications in molecular biology and genetic diagnostics, and are poised to become important tools in the emerging field of molecular medicine as well. While unmodified oligodeoxynucleotides can form DNA:DNA and DNA:RNA duplexes they are sometimes unstable and labile to nucleases. Therefore a variety of nucleic acid analogs have been developed to enhance high-affinity recognition of DNA and RNA targets, enhancing duplex stability and assist with cellular uptake.

Bridged nucleic acids (BNAs) are molecules that contain a five-membered or six-membered bridged structure with a “fixed” C3’-endo sugar puckering (Saenger 1984). The bridge is synthetically incorporated at the 2’, 4’-position of the ribose to afford a 2’, 4’-BNA monomer. The monomers can be incorporated into oligonucleotide polymeric structures using standard phosphoamidite chemistry. BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability.

The incorporation of BNAs into oligonucleotides allows the production of modified synthetic oligonucleotides with:

(i)            Equal or higher binding affinity against an DNA or RNA complement with excellent single-mismatch discriminating power,

(ii)           Better RNA selective binding,

(iii)          Stronger and more sequence selective triplex-forming characters, and

(iv)          Pronounced higher nuclease resistance, even higher than  Sp-phosphorthioate analogues,

(v)           Good aqueous solubility of the resulting oligonucleotides when compared to regular DNA or RNA oligonucleotides.

BNAs can be synthesized using standard phosphoramidite chemistry.

The first synthesis of bridged 2’-O, 4’-C-methyleneuridine and –cytidine monomers were described by Obika et al. in 1997 (in Imanishi’s group). The same group showed in 1998 that these monomers allowed the formation of stable oligonucleotide duplexes in both DNA and RNA based synthetic 12 meric oligonucleotides. Chemical structures for nucleosides and a bridged nucleoside are shown below.

Structures 2

Koshkin et al. in 1998 demonstrated that these monomers can be used to synthesize oligonucleotides that can form stable complexes with DNA and RNA oligonucleotides. Furthermore, the group gave these monomers a new name and called them “Locked Nucleic Acids” (LNAs). The synthesis of these bridged nucleic acids could be achieved by standard phosphoramidite chemistry.

Jesper Wengel in 1999 describes the synthesis of 3’-C- and 4’-C-branched oligonucleotides and the development of locked nucleic acids as well as their use as DNA/RNA mimics.

Christensen et al. in 2001 used stopped-flow kinetic measurements to study the thermodynamics of LNA oligonucleotide based complexes.

Obika at al. in 2001 report that 2′-O, 4′-C-methylene bridged nucleic acids (2′,4′-BNAs = LNA) have unprecedented binding affinities towards their complementary RNA. The researchers showed that 2′,4′-BNA oligonucleotides can be used as antisense molecules and demonstrated their potent inhibitory effect on gene expression of Intercellular Adhesion Molecule-1 (ICAM-1) in living cells. Furthermore, the contribution of RNase H to this antisense effect and adequate stability of 2′,4′-BNA oligonucleotides to enzymatic degradation were also demonstrated. These results showed that BNAs can be used to find natural RNA sequences and target them for destruction.

Torigoe et al. also in 2001 report that 2′-O, 4′-C-methylene bridged nucleic acids (LNAs) can be used to synthesize modified oligonucleotides that can form triplexes with DNA at physiological pH. LNAs are the best studied and characterized bridged nucleic acids so far.

Also in 2001 Obika et al. introduced a 3’-amino-2’,4’-BNA monomer and a 2’,4’-BNA that contained a 2-pyridone group as the base that showed duplex and triplex forming abilities when used in oligonucleotides.

Structures 3

Another bridged nucleic acid monomer was synthesized and introduced in 2001 by Morita et al. called 2’-O, 4’-C-ethylene-bridged nucleic acid (ENA).  The 2′-O,4′-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation. The ethylene-bridged nucleic acids showed a high binding affinity for the complementary RNA strand (ΔTm = +5°C per modification) and were approximately 400 and 80 times more nuclease-resistant than natural DNA and BNA/LNA, respectively. These results indicate that ENA have better antisense activities than BNA/LNA.

Hari et al. in 2003 developed a novel nucleoside analogue that allowed for the effective recognition of CG interruption in a homopurine–homopyrimidine tract of double-stranded DNA (dsDNA). The scientists succeeded in the synthesis of a triplex-forming oligonucleotide (TFO) containing the novel 2’,4’-BNA (QB) bearing 1-isoquinolone as a nucleobase. The triplex-forming ability and sequence-selectivity of the TFO (TFO-QB) were examined. Melting temperature (Tm) measurements found that the TFO-QB formed a stable triplex DNA in a highly sequence-selective manner under near physiological conditions.

Tolstop et al. in 2003 published a paper that described a software tool called “OligoDesign” that allowed for the ‘in-silico” design of LNA based oligonucleotides. The software provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence are included. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. The OligoDesign program is freely accessible at http://lnatools.com/.

The bioinformatics tools was designed to optimize the design of modified oligonucleotides used for the following applications:

  1. Microarray probes,
  2. Probes for in situ hybridization,
  3. Oligonucleotides for antisense inhibition,
  4. FISH probes,
  5. SNP detection as well as others.

Antisense oligonucleotides that contain LNAs show improved silencing potency but cause significant hepatoxicity in animals. This was noticed in 2006 by Swayze at el. when designing antisense oligonucleotides for the silencing of TRADD and ApoB genes in cell cultures.  These results indicated that LNAs may need to be used with caution for antisense purposes. These characteristics led to design newer generations of BNAs.

Miyashita et al. in 2007 (in Imanishi’s group) report the design and synthesis of a new type of BNA, a N-methyl substituted 2’,4’-BNANC. This is a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization. The monomer was designed to fine tune the BNA structure.

Structures 4

The research group synthesized a novel bridged nucleic acid 2’,4’-BNANC[N–Me] and showed that it has high-affinity hybridization similar to that of 2’,4’-BNA (LNA) against an RNA complement. Furthermore, the scientists report that, the nucleic acid analogue displayed RNA selectivity superior to that of 2’,4’-BNA (LNA) and other structural analogues of 2’,4’-BNA (LNA). Nuclease resistance of this nucleic acid analogue is abundantly higher than that of 2’,4’-BNA (LNA) and also slightly higher than that of a phosphorthioate. The hydrophobic methyl substituent on the backbone might present an additional advantage resulting in cellular uptake of the oligonucleotides. All of these reported characteristics of the BNA are essential for antisense applications.  In the same year Rahman et al. report that 2’,4’-BNANC form highly stable pyrimidine-motif DNA triplexes at physiological pH. These triplexes are involved in the regulation of gene expression, site-specific cleavage of DNA, gene mapping and isolation, maintenance of folded chromosome confromations, and gene-targeted mutagenesis. In a pyrimidine-motif triplex DNA the triplex forming oligonucleotide binds with the homopurine tract of the target duplex DNA in a sequence specific manner through Hoogsteen hydrogen bonds to form T●A:T and C+●G:C triads.  In the same year Obika et al report that 5’-amino-BNAs can be used to digest oligonucleotides triggered by triplex formation.

In 2008 Imanishi’s group (Rahman et al. 2008) introduced  three new bridged nucleic acid analogues called 2’,4’-BNANC[NH], 2’,4’-BNANC[NMe], and 2’,4’-BNANC[NBn]. Structures of these analogs are shown below. The new analogs were designed by taking the length of the bridged moiety into account. A six-membered bridged structure with a unique structural feature (N-O bond) in the sugar moiety was designed to have a nitrogen atom. This atom can act as a conjugation site and improve the formation of duplexes and triplexes by lowering the repulsion between the negatively charged backbone phosphates. Furthermore, the nitrogen atom on the bridge can be functionalized by hydrophobic and hydrophilic groups, by adding groups that introduce steric bulk or any desired functional moiety. These modifications allow to control affinity towards complementary strands, regulate resistance against nuclease degradation and the synthesis of functional molecules designed for specific applications in genomics. The properties of these analogs were investigated and compared to those of previous 2’,4’-BNA (LNA) modified oligonucleotides.

Structures 5

Compared to 2’,4’-BNA (LNA)-modified oligonucleotides, 2’,4’-BNANC congeners were found to possess:

(i)        Equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power,

(ii)       Much better RNA selective binding,

(iii)      Stronger and more sequence selective triplex-forming characters, and

(iv)     Immensely higher nuclease resistance, even higher than the Sp-phosphorthioate analogue.

The researchers state that “2’,4’-BNANC-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.”

More recently Yamamoto et al. in 2012 demonstrated successfully that BNA-based antisense therapeutics inhibited hepatic PCSK9 expression, resulting in a strong reduction of the serum LDL-C levels of mice. The findings support the hypothesis that PCSK9 is a potential therapeutic target for hypercholesterolemia. This appears to be the first time that researchers were able to show that BNA-based antisense oligonucleotides (AONs) induced cholesterol-lowering action in hypercholesterolemic mice.  A moderate increase of aspartate aminotransferase, ALT, and blood urea nitrogen levels was observed whereas the histopathological analysis revealed no severe hepatic toxicities. The same group, also in 2012, report that the 2’,4’-BNANC[NMe] analog when used in antisense oligonucleotides showed significantly stronger inhibitory activities which is more pronounced in shorter (13- to 16mer) oligonucleotides. Their data led the researchers to conclude that the 2’,4’-BNANC[NMe] analog may be a better alternative to conventional LNAs.

Wada et al. in 2012 aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2′,4′-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia.  The researchers designed a potentially therapeutic siRNA, siL2PT-1M, and modified it with phosphorothioate (PS) and 2′,4′-BNA/LNA in its sense strand and with 2′-methoxy (2′-OMe) nucleotides in its immunostimulatory motif. The administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2′,4′-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect. The researchers results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5′-UG-3′ in the sense strand would ameliorate the negative immune response.

Action mechanism of antisense oligonucleotides

 The proposed action mechanism for antisense oligonucleotides may involve translation arrest, mRNA degradation mediated by RNase H and splicing arrest. This is illustrated in the following figure.

Antisense Actions

References

Ulla CHRISTENSEN, Nana JACOBSEN., Vivek K. RAJWANSHI, Jesper WENGEL and Troels KOCH. Stopped-flow kinetics of locked nucleic acid (LNA)–oligonucleotide duplex formation : studies of LNA–DNA and DNA–DNA interactions Biochem. J. (2001) 354, 481-484.

Yoshiyuki Hari, Satoshi Obika, Mitsuaki Sekiguchi and Takeshi Imanishi; Selective recognition of CG interruption by 2’,4’-BNA having

1-isoquinolone as a nucleobase in a pyrimidine motif triplex formationqTetrahedron 59 (2003) 5123–5128.

Makoto KOIZUMI; 2’-O,4’-C-Ethylene-Bridged Nucleic Acids (ENATM) as Next-Generation Antisense and Antigene Agents. Biol. Pharm. Bull. 27(4) 453-456 (2004).

Koizumi M.; ENA oligonucleotides as therapeutics. Curr Opin Mol Ther. 2006 Apr;8(2):144-9.

Koshkin AA, SK Singh, P Nielsen, VK Rajwanshi, R Kumar, M Meldgaard, CE Olsen, and J Wengel 1998 LNA (Locked Nucelic Acid): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54: 3607-3630.

Koshkin, A.A., Nielsen, P., Meldgaard, M., Rajwanshi, V. K., Singh S. K. and Wengel, J. (1998). LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes. J. Am. Chem. Soc. 120, 13252 – 13253.

Kazuyuki Miyashita, S. M. Abdur Rahman, Sayori Seki, Satoshi Obikaab and Takeshi Imanishi;   N-Methyl substituted 2’,4’-BNANC: a highly nuclease-resistant nucleic acid analogue with high-affinity RNA selective hybridization.  Chem. Commun., 2007, 3765–3767.

Koji Morita, Chikako Hasegawa, Masakatsu Kaneko, Shinya Tsutsum P, Junko Sone, Tomio Ishikawa, Takeshi Imanishi and Makoto Koizumi; 2′-O,4′-C-Ethylene-bridged nucleic acids (ENA) with nuclease resistance and high affinity for RNA.  2001. Nucleic Acids Research Supplement No. 1 241-242.

Nomenclature for polynucleotide chains including for the sugar puckering can be found at:  http://www.chem.qmul.ac.uk/iupac/misc/pnuc2.html

Satoshi Obika, Daishu Nanbu, Yoshiyuki Hari, Ken.ichiro Morio, Yasuko In, Toshimasa Ishida, and Takeshi Imanishi;  Synthesis of 2′-O,4′-C-Methyleneuridine and -cytidine. Novel Bicyclic Nucleosides Having a Fixed C3 ,-endo Sugar Puckering. Tetrahedron Letters, Vol. 38, No. 50, pp. 8735-8738, 1997.

Obika S, D Nanbu, Y Hari, J-i Andoh, K-i Morio, T Doi, and T Imanishi 1998. Stability and structural features of the duplexes containing nulcoeside analogs with a fixed N-type conformation. 2’-O, 4’-C methylene ribonucleosides. Tetrahedron Lett 39: 5401-5404.

Satoshi Obika, Mayumi Onoda, Koji Morita, Jun-ichi Andoh, Makoto Koizumi and Takeshi Imanishi; 3’-Amino-2’,4’-BNA: novel bridged nucleic acids having an N3’->P5’ phosphoramidate linkage. Chem. Commun., 2001, 1992–1993. Note: BNA/DNA; BNA/dsDNA.

Satoshi Obika, Yoshiyuki Hari, Mitsuaki Sekiguchi, and Takeshi Imanishi; A 2′,4′-Bridged Nucleic Acid Containing 2-Pyridone as a Nucleobase: Efficient Recognition of a C●G Interruption by Triplex Formation with a Pyrimidine Motif.  Angew. Chem. Int. Ed. 2001, 40, No. 11, 2079-2081.

Obika S, Hemamayi R, Masuda T, Sugimoto T, Nakagawa S, Mayumi T, Imanishi T.;  Inhibition of ICAM-1 gene expression by antisense 2′,4′-BNA oligonucleotides. Nucleic Acids Res Suppl. 2001;(1):145-6.

Satoshi Obika, Masaharu Tomizu, Yoshinori Negoro, Ayako Orita, Osamu Nakagawa, and Takeshi Imanishi;  Double-Stranded DNA-Templated Oligonucleotide Digestion Triggered by Triplex Formation. ChemBioChem 2007, 8, 1924 – 1928. Note: Triplex triggered cleavage of oligonucleotides.

S. M. Abdur Rahman, Sayori Seki, Satoshi Obika, Sunao Haitani, Kazuyuki Miyashita, and Takeshi Imanishi; Highly Stable Pyrimidine-Motif Triplex Formation at Physiological pH Values by a Bridged Nucleic Acid Analogue. Angew. Chem. Int. Ed. 2007, 46, 4306 –4309.

Saenger, W.; Principles of Nucleic Acid Structure, Springer-Verlag, new York, 1984.

Eric E. Swayze,* Andrew M. Siwkowski, Edward V. Wancewicz, Michael T. Migawa, Tadeusz K. Wyrzykiewicz, Gene Hung, Brett P. Monia, and and C. Frank Bennett;  Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals Nucleic Acids Res. 2007 January; 35(2): 687–700.

Torigoe H, Y Hari, M Sekiguchi, S Obika, and T Imanishi 2001; 2’-O, 4’-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiologic pH. J Biol Chem 276: 2354-2360. Note: TFO for therapeutics.

Tsuyoshi Yamamoto, Mariko Harada-Shiba, Moeka Nakatani, Shunsuke Wada, Hidenori Yasuhara, Keisuke Narukawa, Kiyomi Sasaki, Masa-Aki Shibata, Hidetaka Torigoe, Tetsuji Yamaoka, Takeshi Imanishi and Satoshi Obika;  Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice Molecular Therapy–Nucleic Acids (2012) 1, e22; oi:10.1038/mtna.2012.16.

Tsuyoshi Yamamoto, Hidenori Yasuhara, FumitoWada, Mariko Harada-Shiba, Takeshi Imanishi, and Satoshi Obika; Superior Silencing by 2’,4’-BNANC-Based Short Antisense Oligonucleotides Compared to 2’,4’-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors. Hindawi Publishing Corporation, Journal of Nucleic Acids Volume 2012, Article ID 707323, 7 pages

Yong You, Bernardo G. Moreira, Mark A. Behlke, and Richard Owczarzy; Design of LNA probes that improve mismatch discrimination. Nucleic Acids Research, 2006, Vol. 34, No. 8 e60.



Categories: Artificial Nucleic Acids, BNA RNA, BNAs, Bridged Nucleic Acid, Bridged Nucleic Acids, Centromere, conjugation, DNA, DNA Analysis, DNA Editing, Epigenetics, Gene Expression, Long noncoding RNA, miRNA, non-coding RNAs, Oligonucleotide Synthesis, RNA Editing, RNA silencing, RNA World, RNAi, Synthesis, Uncategorized, Vaccines

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