Synthesis and Characterization of a Peptide Nucleic Acid Conjugated to a D-Peptide Analog of Insulin-like Growth Factor 1 for Increased Cellular Uptake Soumitra Basu † and Eric Wickstrom* Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 . Received January 27, 1997 X DNA therapeutics show great potential for gene-specific, nontoxic therapy of a wide variety of diseases. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to improve nuclease stability and cell membrane permeability. Recently, a new DNA derivative with an amide backbone instead ofa deoxyribose phosphate backbone, peptide nucleic acid (PNA), has shown tremendous potential as an antisense agent. Although PNAs hybridize very strongly and specifically to RNA and DNA, they are taken up by cells very poorly,limiting their potential as nucleic acid binding agents.To improve cellular uptake of a PNA sequence, it was conjugated to a D-amino acid analog of insulin-like growth factor 1 (IGF1), which binds selectively to the cell surface receptor for insulin-like growth factor 1 (IGF1R). The IGF1 D-peptide analog was assembled on (4-methylben- zhydryl)amine resin, and then the PNA was extended as a continuation of the peptide. The conjugate and control sequences were radiolabeled with 14 C or fluorescently labeled with fluorescein isothiocy- anate. Cellular uptake of the PNA-peptide conjugate,a control with two alanines in the peptide, and a control PNA without the peptide segment were studied in murine BALB/c 3T3 cells, which express low levels of murine IGF1R,in p6 cells, which are BALB/c 3T3 cells which overexpress a transfected human IGF1R gene, and in human Jurkat cells, which do not express IGF1R, as a negative control. The specific PNA-peptide conjugate displayed much higher uptake than the control PNA, but only in cells expressing IGF1R. This approach may allow cell-specific and tissue-specific application of PNAs as gene-regulating agents in vivo. INTRODUCTION Targeting oligonucleotides to a particular gene, or messenger RNA of the gene,to specifically inhibit the expression of that gene has developed into an attractive therapeutic strategy in recent years, especially for treat- ing cancers and viral diseases (1-3).Novel oligonucle- otide analogs have been synthesized to act as antisense/ antigene agents, to improve the biological stability, solubility, cellular uptake,and ease of synthesis.One of the recent additions to this group of modified oligo- nucleotide analogs is the peptide nucleic acid (PNA) 1 (Figure 1) (4). In these compounds, the entire deoxyri- bose phosphate backbone hasbeen replaced with a structurally homomorphous polyamide (peptide) back- bone composed of (2-aminoethyl)glycine units, leaving the oligomer uncharged. This synthetic DNA mimic exhibits enhanced affinity and specificity for its complementary nucleic acid target sequence. The PNAs have clear advantages over a variety of oligonucleotide analogs in severalproperties that are critical for antigene/antisense activity. Compared with other oligonucleotide derivatives, PNAs display the high- est T m values for duplexes formed with single-stranded DNA or RNA (5). PNAs are also resistant to both proteases and nucleases (6). Another major advantage is that PNAs can strand-invade duplex DNA, resulting in the formation of D-loops (7). This characteristic may make it possible to manipulate gene expression at the level of transcription. These complexes mediate the antigene/antisense effects of PNAs by steric hindrance * Address correspondence to this author at the Department of Microbiology and Immunology, Thomas Jefferson University, 1025 Walnut St., Suite 420, Philadelphia, PA 19107 [telephone (215) 955-4578; fax (215) 955-4580; e-mail ewick@lac.jci.tju.edu]. † Present address:Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520. X Abstract published in Advance ACS Abstracts, June 15, 1997. 1 Abbreviations: Boc, tert-butyloxycarbonyl;Bzl, benzyl; DMEM, Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; Fmoc, fluorenylmethoxycarbonyl; HPLC, high- performance liquid chromatography; IGF1, insulin-like growth factor; IGF1R, insulin-like growth factor 1 receptor; Mob, 4-methoxybenzyl;PNA, peptide nucleic acid; SDS, sodium dodecyl sulfate; SEM, standard error of the means; T m , melting temperature; Z, benzyloxycarbonyl. Figure 1.Structure of PNA and DNA. PNAs are peptide-based analogs of DNA in which the phosphate sugar backbone is replaced by (2-aminoethyl)glycine. 481 Bioconjugate Chem. 1997, 8, 481−488 S1043-1802(97)00065-7 CCC: $14.00 © 1997 American Chemical Society