Peptide BNA/LNA oligonucleotide conjugates are hybrid molecules combining a peptide with an oligonucleotide modified with nucleic acid analogs known as Bridged Nucleic Acids (BNAs) or Locked Nucleic Acids (LNAs). The conjugation of peptides to oligonucleotides is possible through the use of chemical conjugation methods. The connection of peptides to oligonucleotides is possible at internal or terminal positions. The resulting conjugates are valuable research tools in molecular biology and medicine due to their potential applications in diagnostics and therapeutics.
Locked Nucleic Acids (LNAs) and Bridged Nucleic Acids (BNAs)
LNAs, considered as first-generation BNAs, are bicyclic nucleotide analogs where the furanose sugar ring is constrained or "locked" by a methylene bridge between the 2'-oxygen and the 4'-carbon. This structural constraint locks the sugar in a C3'-endo (N-type) conformation, characteristic of the A-form RNA. The molecular structure of bridged or locked nucleic acids enhances binding affinities, resulting in a higher binding affinity to complementary DNA and RNA strands compared to unmodified nucleic acids. The increased affinity of modified oligonucleotides leads to a higher melting temperature (Tm) for duplexes.
The rigid structure improves mismatch discrimination, making BNA/LNA-containing oligonucleotides excellent for detecting single-nucleotide polymorphisms (SNPs) or other subtle sequence variations. Additionally, the modified backbone offers resistance to enzymatic degradation by nucleases, thereby increasing its stability in biological environments.
LNAs are the earliest and most well-known type of BNAs. Other BNA derivatives have been developed with different bridge structures, such as 2'-O, 4'-aminoethylene-bridged nucleic acid (BNANC). BNAs offer further improvements in efficiency, bioavailability, and reduced toxicity.
Peptides are short chains of amino acids linked by peptide bonds. Cell-penetrating peptides (CPPs) facilitate the uptake of conjugated molecules into cells, potentially overcoming the poor cellular delivery associated with naked nucleic acids.
Peptides can be designed to bind to specific receptors on cell surfaces or within tissues, enabling the targeted delivery of BNA/LNA conjugates to desired locations. Further, peptides can also contribute to the overall biological activity of the conjugate, for example, by interacting with proteins or modulating cellular pathways.
Peptide BNA/LNA conjugates are created by covalently linking a peptide and a BNA/LNA-modified oligonucleotide together. Several chemical strategies, such as post-synthetic conjugation, stepwise solid-phase synthesis, or internal incorporation, allow the synthesis of peptide BNA/LNA conjugates.
In post-synthetic conjugation, the peptide and modified oligonucleotide are synthesized separately and then joined together using "click chemistry" or other ligation methods. In stepwise solid-phase synthesis, peptides and oligonucleotides are produced sequentially on a solid support. During internal incorporation, peptides are incorporated directly into the oligonucleotide sequence via modified BNA/LNA scaffolds.
The unique properties of BNA/LNA and specific functions of peptides make these conjugates promising research tools for the development of antisense therapeutics that target specific mRNA sequences. Well-designed conjugates can inhibit gene expression, offering potential treatments for genetic disorders, viral infections, and certain types of cancer.
BNA/LNA conjugates may allow modulating splicing, activating or inhibiting gene expression, or even participating in gene repair strategies. The high binding affinity and specificity of these conjugates make them excellent probes for detecting DNA and RNA targets in various diagnostic assays, including qPCR, SNP detection, and in situ hybridization. Correctly selected peptides can enhance the delivery of the BNA/LNA component to target cells and tissues, improving the bioavailability and efficacy of oligonucleotide-based drugs.
Rosenbohm et al. (2003) reported a revised and improved synthesis of the 2’-amino-LNA monomer, and Jørgensen et al. (2013) reported the synthesis of an alkyne-LNA, a valuable monomer for the synthesis of clickable fluorescent LNA/DNA probes. Also in 2013, Astakhova et al. reported the synthesis of peptide-LNA oligonucleotide conjugates (POCs) utilizing a 2’-alkyne-2’-amino-LNA scaffold.
To create the POC, the researchers first synthesized 21-mer oligonucleotides containing single and double internal insertions of the 2′-alkyne-LNA monomer M1. The monomer M1 combines the bicyclic LNA with a 2′-alkyne group. The 2′-alkyne group enables post-synthetic click chemistry and precise positioning of attached modifications within nucleic acid sequences or complexes. Using this approach, Astakhova et al. modified oligonucleotides with enkephalin peptides via CuAAC conjugation with azide-functionalized peptide derivatives.
| 2’-Amino-LNA | 2’-Amino-LNA phosphoramidite | 2’-Amino-alkyne-LNA phosphoramidite |
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Since the number of nucleotides between two peptide modifications can affect the distance, orientation, and interactions between the peptides and the oligonucleotide scaffolds, a varied number of nucleotides can be inserted between the peptide residues by double insertion of monomers. This approach also allows the preparation and study of fluorescent LNA/DNA probes.
Reference
Astakhova, I.A., Hansen, L.H., Vester, B., and Wengel, J.; Peptide–LNA oligonucleotide conjugates. Org. Biomol. Chem., 2013, 11, 4240-4249. [pdf]
Bioconjugation
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