2'-O-AMINOPROPYL RIBONUCLEOTIDES - A ZWITTERIONIC MODIFICATION THAT ENHANCES THE EXONUCLEASE RESISTANCE AND BIOLOGICAL-ACTIVITY OF ANTISENSE OLIGONUCLEOTIDES

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl )cytidine (APC), 2'-O-(aminopropyl)guanosine (APG), and 2'-O-(aminopro pyl)uridine (APU) have been prepared in high yield from the ribonucleo side, protected, and incorporated into an oligonucleotide using conven tional phosphoramidite chemistry. Molecular dynamics studies of a dinu cleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleosid e but does not form a tight ion pair with the proximate phosphate. A 5 -mer with the sequence ACTUC has been characterized using NMR. As pred icted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine ro tates freely and is not bound electrostatically to any phosphate group , as evidenced by the different sign of the NOE between sugar proton r esonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modi fied oligomers does not decrease the affinity for complementary RNA co mpared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold in crease in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophys ical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioa te oligonucleotide capped with two phosphodiester aminopropyl nucleoti des targeted against C-raf mRNA has been transfected into cells via el ectroporation. This oligonucleotide has 5-10-fold greater activity tha n the control phosphorothioate for reducing the abundance of C-raf mRN A and protein.