Preparation and characterization of antisense oligonucleotide-peptide hybrids containing viral fusion peptides.
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%.
Soukchareun
S
S
Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, Australia.
Tregear
G W
GW
Haralambidis
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015700:HIV Envelope Protein gp41 / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry*; D011489:Protein Denaturation; D013696:Temperature; D014760:Viral Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry*
D015700:HIV Envelope Protein gp41; D016376:Oligonucleotides, Antisense; D014760:Viral Fusion Proteins