In earlier studies we identified in a human genomic library a gene (human relaxin gene HI) coding for a relaxin-related peptide. We now have evidence that the human genome possesses an additional relaxin-related gene (designated human relaxin gene H2) which appears to be selectively expressed in the ovary during pregnancy. Nucleotide sequence analysis revealed striking differences in the predicted structures of relaxin encoded by these two genes. Chemical synthesis of biologically active relaxin based on the sequence obtained from ovarian cDNA clones confirmed that the expressed gene (H2) encodes an authentic human relaxin. The expressed gene appears to be transcribed into two different sized mRNAs and preliminary evidence suggests that the mRNA transcripts possess different 3'-untranslated regions. There was no evidence for the expression of human relaxin gene HI in the ovary and so far it is unclear whether gene Hl is expressed in another tissue or whether it represents a pseudogene. From the sequence data presented here it will now be possible to construct oligonucleotide probes and raise antibodies against synthetic peptides which could then be used to identify sites of relaxin biosynthesis and specifically quantitate the expression from either the Hl or H2 relaxin genes.

Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.

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%.

We have developed methods for the synthesis of peptide-oligodeoxyribonucleotide conjugate molecules in particular, and polyamide-oligonucleotide conjugates in general. Synthesis is carried out by a solid-phase procedure and involves the assembly of a polyamide on the solid support, conversion of the terminal amino group to a protected primary aliphatic hydroxy group by reaction with alpha, omega-hydroxycarboxylic acid derivatives, and finally oligonucleotide synthesis using phosphoramidite chemistry. The conjugate molecules can be used as DNA probes, with the polyamide component carrying one or more non-radioactive markers. These conjugates also have the potential to be used as anti-sense inhibitors of gene expression, with the peptide segment acting as a targeting moiety.