Preparation and Characterization of Antisense Oligonucleotide-Peptide Hybrids Containing Viral Fusion Peptides

Abstract

We have developed a strategy for the synthesis of novel oligodeoxynucleotide (ODN)-peptide conjugates on a scale suitable for the investigation of their potential as antisense inhibitors of gene expression. These conjugates have the 3'-terminus of the antisense oligodeoxynucleotide linked covalently to the N-terminus of a peptide. This strategy allows the preparation of conjugates containing a peptide segment designed to facilitate intracellular delivery of the antisense oligodeoxynucleotide as well as providing protection against 3'-exonuclease digestion. To illustrate the synthetic approach we describe the preparation of a series of conjugates comprising antisense oligonucleotides to human immunodeficiency virus type 1 (HIV) linked to fusion peptides derived from the HIV transmembrane glycoprotein gp41. The conjugates were prepared by the total synthesis method, in which the peptide is assembled first by the N-(fluorenylmethoxycarbonyl) (Fmoc) solid-phase methodology. This is followed by derivatization of the amino terminus by reaction with an alpha,omega-hydroxycarboxylic acid derivative which converts the terminus to a protected aliphatic hydroxy group on which standard solid phase DNA synthesis by the phosphoramidite method is performed. The purified conjugates were characterized extensively by several analytical techniques including ion spray mass spectrometry. Thermal denaturation studies showed that the interaction of the ODN-peptide conjugate with its complementary strand was similar to that of unmodified oligonucleotides. Preparation by the total synthesis method gave the purified conjugate with overall yields in the range of 6-14%.

Peptide antisense nanoparticles

“…However, molecular methods of preparing these materials are relatively inefficient, limit sequence diversity because of incompatible chemistries, require orthogonal protecting groups on nucleosides and amino acids (44), and can generate undesired side products (45). Thus, the synthesis and purification of heterofunctional molecules is a challenge.…”

MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users

“…Examples include conjugation of oligonucleotides to viral fusogenic peptides (37,38), staphylococcal nuclease and other enzymes (39,40), neoglycopeptides (41), phospholipids (42), thiolated polysaccharides (43), vitamin E (44), lipophilic groups such as cholesterol (45)(46)(47), and nonradioactive detection labels (48)(49)(50).…”

Synthesis of Oligonucleotide–Peptide Conjugates for Biomedical and Technological Applications

“…In the post-synthetic conjugation approach, the two moieties are prepared independently using solid-phase synthesis, and then thiols and maleimido groups are specifically introduced to link the two molecules together (1). In the stepwise synthesis approach, oligonucleotide-peptide conjugates are prepared by the addition of protected amino acids and nucleotides during solid-phase synthesis on the same solid support (2)(3)(4). In this case, the main challenge is to develop an effective protection strategy for peptide synthesis that is compatible with oligonucleotide synthesis.…”

“…For example, during the final steps of solidphase peptide synthesis a treatment with acid is usually required, which can lead to partial depurination of DNA oligonucleotides. In the case of the synthesis of oligonucleotide 3'-peptide conjugates, the solution to this issue is the use of t-butoxycarbonyl (Boc)-protected amino acids carrying fluorenylmethyl (Fm) or fluorenylmethoxycarbonyl (Fmoc) groups for the protection of the side chains (2)(3)(4). This unusual Boc/Fmoc strategy, described in several recent reviews (5)(6)(7)(8)(9), is compatible with oligonucleotide synthesis, and most of the required amino acid derivatives can be obtained from commercial sources.…”