Applications for Bridged Nucleic Acids (BNAs)
BNA clamp oligonucleotides inhibit DNA primer extension on a single-stranded template and also arrest reverse transcription on a single-stranded RNA template. Clamp oligonucleotides are useful tools for sequence-specific control of gene expression as well as for a therapeutic agent. Clamping oligonucleotides are single-stranded nucleic acids recognized by a complementary (antisense) oligonucleotide to form a short double helix.
Oligopyrimidine-oligopurine helical formations are recognized by a third clamping oligonucleotide forming triple helices. Two oligonucleotides can be linked to each other such that they form a unique clamp with the target sequence on the single-stranded template (Giovannangeli et al. 1991).
Mutations on the epidermal growth factor receptor (EGFR) cause a variety of cancers including breast and lung cancers. The single mutation T790M on tyrosine kinase domain of EGFR signifies the response to the popular cancer drug gefitinib, which leads to the development of resistance to gefitinib. Detecting the mutation thus guide effective therapeutical options for patients who are in need of cancer drug treatments. BNANC clamping using real-time PCR enables effective detection of the EGFR single mutation T790M. Read the BSI Poster (Detection Method for the EGFR Single Mutation T790M).
Giovannangeli et al., Single-Stranded DNA as a Target for Triple-Helix Formation, J. Am. Chem. Soc., 113, 7775-7777 (1991).
BNANC Gapmers revert splicing and reduce RNA Foci in myotonic dystrophy type 1 (DM1) cells. DM1 is a multisystemic disease caused by an expanded CTG repeat in the 3’-untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. Gapmers targeting within the repetitive region of DMPK preferentially degrades the mutant allele.
Myotonic dystrophy is the most common type of muscular dystrophy in which the expanded CUG repeat RNA (CUGexp RNA) is retained in the nucleus where it forms RNA foci leading to defects in regulated alternative splicing events during development. However, if the development of CUG expanded repeat RNA foci could be prevented, the disorder will not materialize. Manning et al. used the antisense BNA gapmer strategy to degrade CUG expanded repeat (CUGexp) RNA in immortalized human TeloMyoD fibroblast cell lines expressing telomerase and containing a tetracycline-inducible MyoD to promote the myogenic program in response to growth. Visualization of RNA was achieved using a FISH probe targeting the repeat RNA of the RNA foci in the untreated and treated cells.
BNANC gapmers targeting within the repetitive region of DMPK preferentially degrade the mutant allele thereby decreasing RNA foci. Manning et al. were able to show that antisense BNANC gapmers could be used to potentially revert splicing defects in myotonic dystrophy type 1 (DM1) cells.
Daguenet E, Dujardin G, Valcárcel J. The pathogenicity of splicing defects: mechanistic insights into pre-mRNA processing inform novel therapeutic approaches. EMBO Rep. 2015;16(12):1640-55.
Manning KS, Rao AN, Castro M, Cooper TA. BNANC Gapmers Revert Splicing and Reduce RNA Foci with Low Toxicity in Myotonic Dystrophy Cells. ACS Chem Biol. 2017;12(10):2503-2509.
Antisense BNA-Peptide Conjugates
Antisense BNA/DNA-peptide-conjugates allow the treatment of drug-resistant infections.
The opportunistic patogen, Acinetobacter baumannii, primarily found in hospital-acquired infections has developed multi-resistance. The presence of the aac(6’ )-Ib gene give the pathogen resistance to amikacin and other aminoglycosides wich severely limits the effectiveness of these antibiotics.
Lopez et al. designed an antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6’)-Ib mRNA, overlapping the initiation codon, which efficiently inhibited translation in vitro. A nuclease-resistant hybrid oligomer composed of 2’, 4’ -bridge nucleic acid-NC(BNANC) residues and deoxynucleotides (BNA-NC/DNA) conjugated to the permeabilizing cell-penetrating peptide (RXR)4XB (CPPBD4) inhibited translation in vitro at the same levels observed when testing ODN4.
Lopez, Christina, Arivett, Brock A., Actis, Luis A., and Tolmasky, Marcelo E.; Inhibition of AAC(6’)-Ib-Mediated Resistance to Amikacin in Acinetobacter baumannii by an Antisense Peptide-Conjugated 2’ ,4’ -Bridge Nucleic Acid-NC (BNA-NC)/DNA Hybrid Oligomer. doi:10.1128/AAC.01304-15. Antimicrobial Agents and Chemotherapy.
CRISPR-Cas9 based Gene-Editing = CRISPR-BNAs
Adding BNAs (2′,4′-BNANC[N-Me]) to CRISPR-RNAs (crRNAs) dramatically improves the accuracy in CRISPR-based gene-editing. The replacement of natural crRNA molecules with synthetic crRNAs containing bridged nucleic acids, or BNAs, enhances the binding affinity to crRNAs.
Off-target effects or off-target cutting and the generation of additional mutations remain a significant barrier for using Cas9-based gene editing methods. BNA modified crRNAs improve the specificity of the CRISPR-Cas9 system and illustrate the power of recently developed synthetic nucleic acid technologies to solve problems in enzyme specificity as well.
Cromwell, Christopher R., Sung, Keewon, Park, Jinho, Krysler, Amanda R., Jovel, Juan, Kim, Seong Keun, and Hubbard, Basil P.; 2018. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity. Nature Communications 9, 1448.
BNAs as Molecular Tools
Molecular diagnostics is essential for drug development. Therefore, molecular tools are needed for identifying infections, screening of cancer or tumors, hepatitis, genetic disorders, and tissue screening to minimize the risks of tissue rejection.
Also, to allow utilization of available sequence information from genomic data efficiently, molecular tools are needed for the investigation of the molecular biology governing metabolic pathways in various species, including humans. Molecular diagnostic tools are particularly valuable for the detection of bacterial bloodstream infections. BNAs lend themselves as building blogs for the development of next-generation molecular tools.
BNAs as molecular tools.
Molecular diagnostic tools for bacterial bloodstream infections.
Tools for RNA targeting.
Genomic and Proteomic Tools.
Jean Pierre Rutanga and Therese Nyirahabimana, “Clinical Significance of Molecular Diagnostic Tools for Bacterial Bloodstream Infections: A Systematic Review,” Interdisciplinary Perspectives on Infectious Diseases, vol. 2016, Article ID 6412085, 10 pages, 2016. https://doi.org/10.1155/2016/6412085.
N6-methyladenosine (m6A) Analysis
Insertion of bridged nucleic acids (BNAs) into DNA probes increases the difference in melting temperature between N6-methyladenosine (m6A)-containing RNA and unmethylated RNA. This approach allows quantification of methylation efficiency at m6A in RNAs with high accuracy.
Inserting bridged nucleic acids (BNAs) into oligonucleotide probes increase the melting temperature (Tm) of the probes leading to an increase of the delta Tm (Δ Tm) between m6A-containing RNA and unmethylated RNA as compared with DNA probes.
Oshima T , Ishiguro K , Suzuki T , Kawahara Y .; Quantification of methylation efficiency at a specific N6-methyladenosine position in rRNA by using BNA probes. Chem Commun (Camb). 2018 Aug 23;54(69):9627-9630. doi: 10.1039/c8cc03713b.
Rapid Mutation Detection with BNAs
The enzyme DNA methyltransferase 3A (DNMT3A) methylates DNA by catalyzing the transfer of methyl groups to specific CpG regions in DNA. DNMT3A is point mutated in many myeloid malignancies, but the mutation frequency varies between different entities. For example, in adult acute myeloid leukemia (AML) DNMT3A mutations are found in 14–34% of cases from different series, 5–15% of MDS cases, 10% of chronic myelomonocytic leukemia (CMML) patients, 5.7% of primary myelofibrosis (PMF) patients, 12% of cases with systemic mastocytosis, and in approximately 18% of T cell acute lymphoblastic leukemia (T-ALL) cases.
Shivarov et al. in 2014 showed for the first time that BNA(NC)-modified probes can be used for the quantitative detection of DNMT3A R882 mutations using bead-based suspension assays. This assay can be successfully implemented in the diagnostics for patients with myeloid malignancies, as it is rapid, and reliable regarding specificity and sensitivity.
Shivarov V, Ivanova M, Naumova E (2014) Rapid Detection of DNMT3A R882 Mutations in Hematologic Malignancies Using a Novel Bead-Based Suspension Assay with BNA(NC) Probes. PLoS ONE 9(6): e99769. https://doi.org/10.1371/journal.pone.0099769.