Triplex-Forming Oligonucleotides (TFOs) are synthetic nucleic acid strands designed to bind to double-stranded DNA (dsDNA) in a sequence-specific manner, forming a triple helix (triplex DNA). This binding occurs in the major groove of the DNA duplex via Hoogsteen or reverse Hoogsteen hydrogen bonds without denaturing the dsDNA. In this case, the TFO is used to bind to plasmid DNA.
Bridged Nucleic Acids (BNAs) are chemically modified nucleotides containing a sugar moiety bridge. This structural modification locks the sugar into a highly favorable conformation for hybridization with complementary nucleic acids. Adding BNAs to oligonucleotides increases their binding affinity by forming stable duplexes and triplexes with target DNA or RNA. Also, the modified backbone protects BNA-modified oligonucleotides from enzymatic degradation, resulting in enhanced nuclease resistance. Further, the rigid structure of BNAs can improve the ability to discriminate between perfect matches and single nucleotide mismatches, helping to overcome limitations of traditional TFOs, such as the requirement for long polypurine-polypyrimidine tracts and instability at neutral pH.
Modifying TFOs with BNAs and combining these with peptides results in conjugates with enhanced cellular delivery, increased triplex stability, improved nuclease resistance, broader target sequence recognition, and a higher sequence specificity, making them promising candidates for targeted cell delivery.
Girardin et al. reported a method for linking the peptide VVMVGEKPITITQHSVETEG to plasmid DNA (pDNA) via a triplex-forming oligonucleotide (TFO) targeting an oligopurine • oligopyrimidine site (TH). The TFO containing Bridged Nucleic Acids (BNAs) was conjugated to the peptide using click chemistry. The researchers evaluated the efficiency of the BNA TFO peptide conjugate using a five kbp pDNA encoding the luciferase gene and a 21 kbp pDNA encoding both the luciferase and a full-length dystrophin gene.
Girardin et al. linked a BNA TFO carrying a dibenzocyclooctyl (DBCO) group at the 3' end to the C-terminal of the azide-carrying peptide using click chemistry. The peptide-pDNA conjugate mediates the migration of the pDNA on microtubules via the triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH) were used, respectively, inserted outside the luciferase gene sequence.
Girardin et al. tested this conjugate in mice by in vivo transfection utilizing Hydrodynamic Limb Vein (HLV) injection. The conjugates formatted a 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. Non-invasive bioluminescence measurements determined the luciferase activity, showing a twofold increase in transfection efficiency in the hind limbs of mice. This conjugation method allows the design of pDNA targeting conjugates containing intracellular targeting molecules while preserving the full expression of encoded genes.
Reference
Gallardo J, Pérez-Illana M, Martín-González N, San Martín C. Adenovirus Structure: What Is New? Int J Mol Sci. 2021 May 15;22(10):5240. [PMC]
Girardin, C., Maze, D., Gonçalves, C. et al. Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement. Gene Ther 30, 271–277 (2023). [Gene Ther]
Horwitz MS. Function of adenovirus E3 proteins and their interactions with immunoregulatory cell proteins. J Gene Med. 2004 Feb;6 Suppl 1:S172-83. doi: 10.1002/jgm.495. PMID: 14978760. [J Gene Med]
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