2′‐O‐Lysylaminohexyl Oligonucleotides: Modifications for Antisense and siRNA

Abstract

A novel type of oligonucleotide has been developed, characterized by the attachment of a lysyl moiety to a 2'-O-aminohexyl linker. A protected lysine building block was tethered to 2'-O-aminohexyluridine, and the product was converted into the corresponding phosphoramidite. Up to six modified nucleosides were incorporated in dodecamer DNA and RNA oligonucleotides using standard phosphoramidite chemistry. Each of the building blocks contributes one positive charge to the oligonucleotide instead of the negative charge of a wild-type nucleotide. Thermal denaturation profiles indicated a stabilizing effect of 2'-O-lysylaminohexyl chains that was more pronounced in RNA duplexes. Incubation of the oligonucleotides with 5'-exonuclease revealed an exceptionally high stability against enzymatic degradation. Incorporation of up to three modifications into functional antisense and siRNA oligonucleotides targeted at ICAM-1 showed that the gene-silencing activity was higher with an increasing number of lysylaminohexyl nucleotides. Compared with wild-type antisense or siRNA, compounds with three modifications led to equal or higher ICAM-1 downregulation.

Polyamine–Oligonucleotide Conjugates: A Promising Direction for Nucleic Acid Tools and Therapeutics

“…Lysine-containing groups have been incorporated into ODNs and ORNs at positions close to the 5´-terminal positions ( Table 2; structure 11). 2´-O-Lysylaminohexyl residues were found to decrease the thermal stability of ODN-DNA duplexes, whereas for the same ORN-RNA sequences, a moderate stabilizing effect on the duplex was observed (ΔT M ≈ +1°C per modification) [57,58]. The modified ODNs benefited from an increased nuclease resistance and some enhanced silencing activity (200 nM treatment for 24 h) was also noted for ODNs containing 5´-terminal and interdispersed 2´-O-lysylaminohexyl residues, respectively.…”

“…They are also finding use as PCR primers and hybridization probes with increased sensitivity of detection [39][40][41][42]. • The effect of polyamine conjugation to the 2´-O-position of ribose on hybridization affinity is somewhat variable [34][35][50][51][52][53][55][56][57][58][59][60]. All of the modifications, however, showed increased nuclease resistance, and as a likely consequence, when tested, an increased silencing activity.…”

“…The modified ODNs benefited from an increased nuclease resistance and some enhanced silencing activity (200 nM treatment for 24 h) was also noted for ODNs containing 5´-terminal and interdispersed 2´-O-lysylaminohexyl residues, respectively. In contrast, little benefit was gained by incorporation of lysylaminohexylnucleotides substitutions into siRNAs (50 nM treatment for 24 h) [58].…”

“…However, in contrast to the terminal positions of oligonucleotides, the stability of ODN-DNA and ODN-RNA duplexes is very sensitive to modifications of the ribose structure and conformation. Although most investigations have focused on the conjugation of amines and polyamines to the 2´-O positions of riboses of ODNs and oligoribonucleotides (ORNs) with various linkers ( Table 2; structures 6-18) [34,[48][49][50][51][52][53][54][55][56][57][58][59][60][61], in one study the C-4´ position of a deoxyribose was used to conjugate aminoalkyl groups ( Table 2; structures 19-20) [60]. Incorporation of 4´-aminomethyl, -ethyl-and -propyl-groups into ODNs at various positions slightly increased melting temperatures of duplexes formed with a complementary DNA target by 0.5-1°C per modification.…”

EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins

“…5) and HT-29 (Supplementary Materials). The siRNA was designed as a 2'-O-methyl gapmer sequence (see Materials and Methods), with the flanking region consisting of 2'-O-methyl ribonucleotides and the central part of unmodified ribonucleotides, which provides higher stability against degrading enzymes and reduces off-target effects, especially immune cell activation by interferon-release (37)(38)(39)(40).…”