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Brief history of Bridged Nucleic Acids (BNAs)

A brief history of Bridged Nucleic Acids (BNAs) - 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 potential new class of drugs for the treatment of diseases such as cancer, inflammation, viral infections, and other pathological disorders. Researchers used the 3D structures for A-RNA and B-DNA as templates to enable the design of the bridged nucleic acid (BNA) monomers. The design goal is to find derivatives that possess high binding affinities with complementary RNA and DNA strands.


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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, 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 an equilibrium of two preferred conformations of the N-type (C3’-endo, A-form) and the S-type (C2’-endo, B-form) as depicted in the next figure.


N- and S-type sugar puckering. The N-type exists predominantly in A-RNA and the S-type in duplexes with B-DNA helical structure.

Illustrated below is a closer look at the two forms. A conformationally constrained sugar moiety in nucleosides or oligonucleotides will gain high binding affinity with complementary single-stranded RNA and double-stranded DNA.

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Synthetic oligonucleotides are now essential, established tools for life scientists and have many applications in molecular biology and genetic diagnostics, and are poised to become crucial tools in the emerging field of molecular medicine. While unmodified oligodeoxynucleotides can form DNA:DNA, and DNA:RNA duplexes, they are sometimes unstable and labile to nucleases. Therefore, various nucleic acid analogs have been developed to enhance high-affinity recognition of DNA and RNA targets, enhancing duplex stability, and assisting 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). Synthetic incorporation of the bridge 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 phosphoramidite chemistry. BNAs are structurally rigid oligonucleotides 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 a 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 analogs, 

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

Standard phosphoramidite chemistry allows the synthesis of BNAs. 

[1997] Obika et al. (in Imanishi’s group) described the first synthesis of conformationally restricted bridged 2’-O, 4’-C-methylene-uridine, and bridged 2’-O, 4’-C-methylene-cytosine monomers. The same group showed in 1998 that these monomers allowed the formation of stable oligonucleotide duplexes in both DNA- and RNA-based synthetic 12mer oligonucleotides. The next figure shows the chemical structures for uridine,  cytosine and the 2’-O, 4’-C-methyleneuridine monomer.

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[1998] Koshkin et al. demonstrated that bridged nucleic acid monomers allowed the synthesis of oligonucleotides with the ability to form stable complexes with DNA and RNA oligonucleotides and studied the thermal stability of formed duplexes. The research group compared the chemical structure of these bridged nucleic acids with the structure of RNA, the analogs peptide nucleic acids (PNAs), 2’-Fluoro-N3’-P5’-phosphoramidates, and 1’,5’-anhydrohexitol nucleic acids (HNAs). Furthermore, the group gave these monomers a new name and called them “Locked Nucleic Acids” (LNAs). The use of standard phosphoramidite chemistry enabled the synthesis of these bridged nucleic acids.

[1999] Jesper Wengel described the synthesis of 3’-C- and 4’-C-branched oligonucleotides, the development of locked nucleic acids and their use as DNA/RNA mimics. C-branched monomers allow attachment of ligands to predefined positions in oligonucleotides.


[2001] Christensen et al. used stopped-flow kinetic measurements to study the thermodynamics of LNA oligonucleotide-based complexes. The reactions of the DNA octamer 5’-CAGGAGCA-3’ with its complementary DNA octamer 5’-TGCTCCTG-3’, and the LNA octamers 5’-TLGCTCCTG-3’ (LNA-1), 5’-TLGCTLCCTG-3’ (LNA-2) and 5’-TLGCTLCCTLG-3’ (LNA-3), containing respectively one, two or three thymidine 2’-O,4’-C-methylene-(D-ribofuranosyl) nucleotide monomers, designated TL, were studied to confirm the enhanced duplex stability of DNA-LNA duplexes.


Obika’s group reported 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 scientist also explained the contribution of RNase H to this antisense effect and the adequate stability of 2',4'-BNA oligonucleotides to enzymatic degradation. These results showed that BNAs could be used to find natural RNA sequences and target them for destruction.


Also, 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.

I
n addition, 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 exhibits duplex and triplex-forming abilities when used in oligonucleotides.

Chemical structures of various BNA analogs are shown next.

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Another bridged nucleic acid monomer was synthesized and introduced 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), approximately 400 and 80 times more nuclease-resistant than natural DNA and BNA/LNA, respectively. These results indicated that ENA has better antisense activities than BNA/LNA.

[2002]
Imanishi and Obika described the general properties of bridged nucleic acids 2’,4’-BNA/LNA and their interactions when forming duplexes and triplexes.

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

Tolstrup et al. published a paper that described a software tool called “OligoDesign” that allowed for the ‘in-silico” design of LNA-based oligonucleotides. The software provides the 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 to minimize cross-hybridization with non-target sequences. The software included routines for predicting melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, and secondary structure prediction of the target nucleotide sequence. However, this tool is no longer freely available.


Optimal designed modified oligonucleotides enhance the following probes: 

1. microarray probes, 

2. probes for in situ hybridization, 

3. oligonucleotides for antisense inhibition, 

4. FISH probes, 

5. SNP detection probe, and many others.

[2006] Antisense oligonucleotides that contain LNAs show improved silencing potency but caused significant hepatoxicity in animals. Swayze et al. noticed this when designing antisense oligonucleotides to silence TRADD and ApoB genes in cell cultures. Their results indicated that LNAs might need to be used with caution for antisense purposes. These characteristics led to the design of newer generations of BNAs.

[2007] Miyashita et al. (in Imanishi’s group), reported the design and synthesis of a new type of BNA, an N-methyl substituted 2’,4’-BNANC. The result was a highly nuclease-resistant nucleic acid analog with high-affinity RNA selective hybridization. The new design fine-tuned the BNA monomer structure. 

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The research group synthesized a novel bridged nucleic acid 2’,4’-BNANC[N–Me] exhibiting high-affinity hybridization similar to that of 2’,4’-BNA (LNA) against an RNA complement. Furthermore, the scientists reported that the nucleic acid analog displayed RNA selectivity superior to 2’,4’-BNA (LNA) and other structural analogs of 2’,4’-BNA (LNA). Nuclease resistance of this nucleic acid analog is abundantly higher than that of 2’,4’-BNA (LNA) and 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 regulate gene expression, site-specific cleavage of DNA, gene mapping and isolation, maintenance of folded chromosome conformations, 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.

[2008] Imanishi’s group (Rahman et al. 2008) introduced three new bridged nucleic acid analogs called 2’,4’-BNANC[NH], 2’,4’-BNANC[NMe], and 2’,4’-BNANC[NBn]. The structures of these analogs are illustrated below.  Rahman et al. considered the length of the bridged moiety during design.  The research group designed a six-membered bridged structure with a unique structural feature (N-O bond) in the sugar moiety with 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 allows functionalization with hydrophobic and hydrophilic groups by adding bulky steric groups or any desired functional moiety. These modifications allow to control affinity towards complementary strands, regulate resistance against nuclease degradation, and synthesize active 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. The chemical structures of the three 2’,4’-BNANC analogs are shown below. 

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Compared to 2’,4’-BNA (LNA)-modified oligonucleotides, 2’,4’-BNANC congeners 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 analog.

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

[2012] More recently, Yamamoto et al. demonstrated successfully that BNA-based antisense therapeutics inhibited hepatic PCSK9 expression, resulting in a substantial reduction of the serum LDL-C levels of mice. The findings support the hypothesis that PCSK9 is a potential therapeutic target for hypercholesterolemia. Apparently, this was the first time that researchers were able to show that BNA-based antisense oligonucleotides (AONs) induced cholesterol-lowering action in hypercholesterolemic mice.  The study observed a moderate increase of aspartate aminotransferase, ALT, and blood urea nitrogen levels.  However, 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.


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. The following figure illustrates the proposed action mechanism. 

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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.


Niels Tolstrup, Peter S. Nielsen, Jens G. Kolberg, Annett M. Frankel, Henrik Vissing, Sakari Kauppinen, OligoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling, Nucleic Acids Research, Volume 31, Issue 13, 1 July 2003, Pages 3758–3762, 
https://doi.org/10.1093/nar/gkg580 , https://academic.oup.com/nar/article/31/13/3758/2904202?login=true

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 
doi:10.1155/2012/707323.

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.

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