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3rd Generation Bridged Nucleic Acids (BNA) - A superior alternative to LNA & PNA
 
Super-functional Nucleic Acid for Gene Targeting

Multi Functional Bridged Nucleic Acid (BNA), An Alternative to LNA and PNA

Development of artificial nucleic acids is essential for the design of new efficient tools for molecular biology as diagnostic probes or the design of potentially new drugs for oligonucleotide-based therapies. Of particular interest are artificial nucleic acids with the following characteristics:

  • The ability to increase base-discrimination
  • Increase stability of duplex or triplex formation

As a pioneer in oligonucleotide synthesis, Bio-Synthesis Inc. has been pursuing the development of new oligonucleotide based technologies that can provide compounds that have superior binding affinity and chemical/biological stability. As such, Bio-Synthesis Inc. is introducing its third generation Bridged Nucleic Acids (BNA) . This new technology is based on multi-functional synthetic RNA analogues that can be used in place of the first generation bridged nucleic acids known as Locked Nucleic Acids (LNA). These RNA analogues can be synthesized and spiked with DNA or RNA in order to modify the formation of nucleic acid helices. Also, when compared to Peptide Nucleic Acids (PNA), BNA allows for better base-pair stacking and a high stability of the resulting oligonucleotide complexes, making BNA based oligonucleotides an ideal solution for the detection of small or highly similar DNA or RNA targets.

Bio-Synthesis Inc. is now providing synthetic oligonucleotides containing BNA which are deprotected, desalted or HPLC purified. All oligonucleotides are quality checked by MALDI-TOF Mass Spectrometry.

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BNA oligonucleotides exhibit significantly higher affinity to their complementary strands when compared to previous generations of constrained nucleic acids (PNA & LNA). Their extraordinary level of sensitivity, specificity for nuclease-resistant activity, makes these multi-functional BNA nucleic acids a superior tool for developing high value detection systems and therapeutic products

Benefit of the BNA Technology

  • Improved hybridization selectivity and specificity over PNA & LNA
  • Ideal for the detection of short RNA and DNA targets
  • Facilitate Tm normalization
  • Increased thermal duplex stability of duplexes
  • Increased thermal stability of triplexes
  • Capable of single nucleotide discrimination
  • Resistance to exo- and endonucleases resulting in high biological stability for in vivo and in vitro application
  • Superior antisense inhibition and potency
  • Strand invasion enable detection of "hard to access" sample
  • Flexible probe designs regardless of GC content
  • Easily adaptable to many DNA or RNA detection system

BNA Applications

  • Antigene inhibition
  • Gapmer antisense research
  • In vivo, in vitro delivery
  • RNAi research
  • Inhibition of RNA function
  • Real-time/ qPCR
  • SNP detection /allele specific PCR
  • In situ hybridization
  • DNAzymes and Ribozymes
  • Biosensor and more..

Bio-Synthesis, home of BNATM oligonucleotides

Bio-Synthesis under a licensing agreement with BNA Inc, is the exclusive provider of synthetic BNAs; purchase of research use of BNA’s only, carries a research use only license; clinical/therapeutic and/or commercial applications of BNA’s required a separate commercial license; we will be glad to discuss licensing terms with interested parties.

Bridged Nucleic Acids (BNA) Technology

Natural nucleic acids have a higher degree of freedom in their chemical structure. This feature is thermodynamically unfavorable for DNA-DNA and RNA-RNA double strand formation (hybridization) and is often subject to degradation by both endo and exonucleases. Improving binding affinity (hybridizing capability) is yet unresolved for highly sensitive gene-targeting applications.

Bridged nucleic acid 2',4'-BNANC (2'-O,4'-aminoethylene bridged nucleic acid) RNA analogue, containing a six-member bridged structure with an N-O linkage, was developed by Professor Emeritus Takeshi Imanishi of Osaka University. These novel nucleic acid analogues can be synthesized and incorporated into oligonucleotide. When compare to the earlier generation of 2', 4' BNA (LNA)- modified oligonucleotides, 2', 4'-BNANC [N-Me] analogues were found to possess:

  • Higher binding affinity against an RNA complement with excellent single-mismatch discriminating power,
  • Enhanced RNA selective binding,
  • Stronger and more sequence selective triplex-forming characters, and
  • Stronger nuclease resistance to endo and exo-nucleases, even higher than the S(p)-phosphorothioate analogue.

2',4'-BNA(NC)-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.

These nucleic acid analogs can be easily incorporated into natural oligonucleotide strands. They provide flexibility in designing BNA/DNA and BNA/RNA hybrid oligos to satisfy the need for very high and sequence-specific hybridization with natural nucleic acids. Additionally, they possess a strong nuclease-resistant property. While first generation BNA (also known as LNA) is still used in various applications, Bio-Synthesis Inc. now offers third generation, six member bridged 2', 4' BNANC which has shown to possess superior properties to the earlier generation of locked nucleic acids.

Synthesis of BNA Oligonucleotides:

BNA oligos allow greater flexibility in the design of primers and probes. They can be mixed with DNA, RNA and other nucleic acid analogs using standard phosphoramidite synthesis chemistry. BNA oligonucleotides are also easily labeled or modified with standard oligonucleotide tags such as DIG, fluorescent dyes, biotin, amino-linkers, etc.

For additional information please contact us

Structure of basic BNA, LNA and Natural RNA Base

Type of BNA Bases

References:

  • 1.Zon, G.; Geiser, T. G., “Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions”, Anticancer Drug Des. 1991, 6, 539-568.
  • 2.Prakash, T. P.; Bhat, B., “2'-Modified oligonucleotides for antisense therapeutics”, Curr. Top. Med. Chem. 2007, 7, 641-649.
  • 3.Manoharan, M., “2'-carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation”, Biochim. Biophys. Acta 1999, 1489, 117-130.
  • 4.Dellinger, D. J.; Sheehan, D. M.; Christensen, N. K.; Lindberg, J. G.; and. Caruthers, M. H., “Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides”J. Am. Chem. Soc., 2003, 125, 940950.
  • 5.Summerton, J., Weller, D., “Morpholino antisense oligomers: design, preparation, and properties” Antisense Nucleic Acid Drug Dev., 1997, 7:187-195.
  • 6.Hyrup, B.; Nielsen, P. E., “Peptide nucleic acids (PNA): synthesis, properties and potential applications”, Bioorg. Med. Chem. 1996, 4, 5-23.
  • 7.Stein, C.A; Cohen, J. S., “Oligodeoxynucleotides as inhibitors of gene expression: a review”, Cancer Res., 1988, 48, 2659-2668.
  • 8.Sanghvi, Y. S., “A status update of modified oligonucleotides for chemotherapeutics applications”, Curr. Protoc. Nucleic Acid Chem., 2011, Chapter 4, Unit 4.1.1-22.
  • 9.Prakash, T. P.,“An overview of sugar-modified oligonucleotides for antisense therapeutics”, Chem. Bioliers., 2011, 8, 1616-1641.
  • 10.Yamamoto, T.; Nakatani, M.; Narukawa, K.; Obika, S., “Antisense drug discovery and development”, Future Med. Chem. 2011, 3, 339-365.
  • 11.Veedu, R. N.; Wengel, J., “Locked nucleic acids: promising nucleic acid analogs for therapeutic applications”, Chem. Bioliers., 2010, 7, 536-542.
  • 12.Rahman, S.M.; Seki, S.; Utsuki, K.; Obika, S.; Miyashita, K.; Imanishi, T., “2',4'-BNA(NC): a novel bridged nucleic acid analogue with excellent hybridizing and nuclease resistance profiles”, Nucleosides Nucleotides Nucleic Acids., 2007, 26, 1625-1628.
  • 13.Rahman, S. M.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita, K.; Imanishi, T., “Design, synthesis, and properties of 2',4'-BNA(NC): a bridged nucleic acid analogue”, J. Am. Chem. Soc., 2008, 130, 4886-4896.
  • Yamamoto, T.; Harada-Shiba, M.; Nakatani, M.; Wada, S.; Yasuhara, H.; Narukawa, K.; Sasaki, K.; Shibata, M.-A.; Torigoe, H.; Yamaoka, T.; Imanishi, T.; Obika, S., “Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice”, Mol. Ther. Nucleic Acids, 2012, 1, e22.

Enhance your applications with Bridged Nucleic Acid (BNA)

Better Tm modulation which offers the most effective solution to investigate short RNA and DNA targets with extremely high sensitivity, specificity and discriminatory power1

BNA can be mixed with DNA, RNA and other nucleic acid analogs within the oligonucleotide to facilitate changes of Tm (melting temperature) without losing specificity. They allow for shorter probe design while maintaining the same Tm. The Tm of a nucleotide duplex can be modulated by varying the BNA content. This feature can be used to normalize the Tm across a population of short sequences with varying GC-content. For AT-rich nucleotides with low melting temperatures,

The addition of BNA can be used to raise the duplex Tm. This enables the design of BNA oligonucleotides with a narrow Tm range, which is beneficial in microRNA research, PCR, microarray and applications where hybridization sensitivity and specific binding to many different targets must occur under the same condition.

Stronger biostability due to Increased thermal duplex stability and nuclease resistance, an ideal solution for in vivo antisense, siRNA application 2,3

Older generations of 2',4'-LNA do not posses sufficient resistant to nuclease, nor the flexibility required for efficient triplex formation. The increased duplex and triplex-forming ability of BNA conforms with the Watson-Crick binding and gives BNATM oligonucleotides a high binding affinity to ssRNA and dsDNA. This strand invading property makes BNATM an excellent tool for in vivo applications. Incorporating BNA into oligonucleotides, further enhanced resistance against nuclease degradation and leads to high in vitro and in vivo stability, making BNANC a promising therapeutic agent.

Multi-functional nucleic acid analogs provide for flexibility and ease-of-use

The physiological properties of BNA® such as water solubility are very similar to those of DNA and RNA. BNA can therefore be easily adapted to conventional experimental protocols. It can be incorporated with most oligonucleotide synthesis chemistries and analysis methods.

Reference:

  • 1.Miyashita, K.; Abdur Rahman, S. M.; Seki, S.; Obika, S.; Imanishi, T., "N-Methyl substituted 2',4'-BNANC: a highly nuclease-resistant nucleic acid analogue with high-affnity RNA selective hybridization
  • 2.Yamamoto, T.; Harada-Shiba, M.; Nakatani, M.; Wada, S.; Yasuhara, H.; Narukawa, K.; Sasaki, K.; Shibata, M.-A.; Torigoe, H.; Yamaoka, T.; Imanishi, T.; Obika, S., “Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice”, Mol. Ther. Nucleic Acids, 2012, 1, e22.
  • 3.Abdur Rahman, S. M.; Sato, H.; Tsuda, N.; Haitani, S.; Narukawa, K.; Imanishi, T.; Obika, S., " RNA interference with 2',4'-bridged nucleic acid analogues", Bioorganic & Mdicinal Chemistry 18 (010) 3474-3480

BNA is a super-functional tool for nucleic acids research.

RNAi and antisense oligonucleotides

Although siRNA elicit RNAi activity in cell culture, their in vivo usage as a drug remains questionable because of low biostability and undesirable toxicity (off-target effects). The effort to use nucleotide analogues to overcome these problems and to improve pharmacokinetics and delivery of siRNA are increasing daily. Example of using Locked Nucleic Acids (LNA), 1st generation of Bridged Nucleic Acids have been reported to have good RNA specific binding affinity. Nevertheless, the enzymatic resistance is significantly lower than that obtained by the PS oligonucleotides and the consecutive LNA bases, and fully modified LNA analogue are very rigid, resulting in inefficient triplex formation. Peptide nucleic acids (PNA) also possessed promising RNA selective binding affinity. However, their mode of RNA targeting was achieved via triplex formation, which requires the use of two folds of antisense nucleic acids. In addition to that, PNA has limited aqueous solubility, poor cellular uptake and ambiguity in binding complementary DNA/RNA in both parallel and antiparallel orientations. Several reports have shown that nucleotide analogue 2', 4'-BNA NC is substantially compatible with the in vivo siRNA or antisense technology. siBNANC offers:

  • Higher binding affinity against an RNA complement with excellent single-mismatch discriminating power,
  • Much better RNA selective binding
  • Stronger and more sequence selective triplex-forming characters
  • Immensely higher nuclease resistance, even higher than phosphorothioate analogue

BNA oligonucleotide in DNA Research

  • BNA PCR Clamp
  • Real-time/quantitative PCR
  • SNP detection/allele specific PCR
  • In situ hybridization
  • PCR clamping
  • Methylation analysis
  • Bead-base applications
  • Chromsomal FISH
  • Comparative genome hybridization
  • Antigene inhibition
  • Mutagenesis
  • Proteomic of isolated chromatin segments (PICh)

BNA oligonucleotide in mRNA Research

  • Real-time/quantitative PCR
  • In situ hybridization
  • Northern blotting
  • Bead-base applications
  • Fluorescence activated cell sorting isolation
  • Inhibition of RNA function
  • Microarray analysis
  • RNA modification
  • DNAzymes

BNA oligonucleotide in miRNA Research

  • Real-time/quantitative PCR
  • In situ hybridization
  • Microarray analysis
  • Northern blotting
  • Bead-base applications
  • Inhibition of RNA function
  • RNA modification
  • Functional analysis

BNA oligonucleotide in ncRNA Research

  • Real-time/quantitative PCR
  • In situ hybridization
  • Fluorescence activated cell sorting
  • Microarray analysis
  • Northern blotting
  • Inhibition of RNA function
  • RNA modification

BNA oligonucleotide can also be use in

  • PCR based approached
  • Hybridization base approaches
  • In vivo based approaches

 

 

 

Links and Resources

Reference/Citing:

  1. Tsuyoshi Yamamoto, et al.: Superior Silencing by 2',4'-BNA NC-based Short Antisense Oligonucleotides Compared to 2',4'-BNA/LNA-based Apolipoprotein B Antisense Inhibitors; Journal of Nucleic Acid, Vol. 2012
  2. T. Yamamoto et al.: Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholersterolemic Mic; Molecular TherapyNucleic Acid Researech. 2012.
  3. M. Nishida. et al.: Synthesis, RNA selective hybridization and high nuclease resistance of an oligonucleotide containing novel bridged nucleic acid with cyclic urea structure; ChemComm 2012.
  4. S.M. Abdur Rahman et al.: Highly Stable Pyrimidine-Motif Triplex Formation at Physiological pH Values by a Bridged Nculeci acid Analogues; Angeu. Chem. Int. Ed. 2007.
  5. S.M. Abdur Rahman et al.: Design, Synthesis, and Properties of 2',4'-BNANC: A Bridged Nculeic acid Analogue; JACS, 2008.

Visit our literature vaults for more references and citings.