Polyamine‐Functionalized 2′‐Amino‐LNA in Oligonucleotides: Facile Synthesis of New Monomers and High‐Affinity Binding towards ssDNA and dsDNA

Abstract

Attachmento fc ationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charger epulsion between the two negatively charged strandso faduplex, but also to augment their in vivo stability against nucleases.I nt his study, polyamine functionality was introduced into ONs by means of 2'-amino-LNAs caffolds. The resulting ONs exhibited efficient binding towardsssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying at riaminated linker showed them ostp ronouncedd uplex-and triplex-stabilizing effect. Molecular modelling revealed that favourable conformationala nd electrostatic effects led to salt-bridge formation between positivelyc harged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showedi ncreased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.

Cationic oligonucleotide derivatives and conjugates: A favorable approach for enhanced DNA and RNA targeting oligonucleotides

“…This was demonstrated to be a successful design as both monomers showed very high duplex stabilizing properties towards RNA (T m +10.0 °C) and DNA (T m +8.5 °C). Additionally, nuclease resistance was shown to be high [95] which was in agreement with other 2'-aminoalkylated-LNA monomers [94]. The triplex stability of the monomers was determined, and the spermidine variant carrying three cationic charges (57) had the highest triplex stabilizing effect [95].…”

“…The triplex stability of the monomers was determined, and the spermidine variant carrying three cationic charges ( 57 ) had the highest triplex stabilizing effect [ 95 ]. Interestingly, at biologically relevant pH (7.0), two incorporations of the nor-spermidine variant ( 53 ) [ 94 ], and the triazole-linker variant carrying three cationic charges ( 57 ) [ 95 ] stabilized the formed triplex by 28.0 °C and 30.5 °C, respectively [ 94 – 95 ]. These cationic 2’-amine-functionalized LNA modifications ( 51 – 57 ) all gave high binding affinity towards RNA with excellent nuclease resistance, making them ideal ASO modifications as only a limited number of modifications is needed for a substantial effect.…”

“…In a follow-up study, after being introduced into bisLNAs, the piperazinomodified 2'-amino-LNA-T nucleotide was compatible with invasion into dsDNA targets in vitro [56]. In further studies utilizing the same amide coupling, nor-spermidine with different group lengths (52 and 53), a glycol-amine-functionalized group (54), and a bis-C6-amine-functionalized group (55) were attached to 2'-amino-LNA-T. All these modifications demonstrated high duplex stabilizing capabilities combined with high nuclease resistance [94,95]. Additionally, the nor-spermidine and amino-glycol modified 2'-amino-LNA-T when incorporated into TFOs all induced excellent triplex stability at pH 7.0 [94] (Table 6).…”

“…Additionally, nuclease resistance was shown to be high [95] which was in agreement with other 2'-aminoalkylated-LNA monomers [94]. The triplex stability of the monomers was determined, and the spermidine variant carrying three cationic charges (57) had the highest triplex stabilizing effect [95]. Interestingly, at biologically relevant pH (7.0), two incorporations of the nor-spermidine variant (53) [94], and the triazole-linker variant carrying three cationic charges (57) [95] stabilized the formed triplex by 28.0 °C and 30.5 °C, respectively [94,95].…”

Convertible and Constrained Nucleotides: The 2’-Deoxyribose 5’-C-Functionalization Approach, a French Touch

“…Following the same synthetic strategy, they also introduced a positively charged piperazino group [ 143 ] or diamino groups [ 144 ] to increase binding affinity and resistance to 3’-exonucleotlytic degradation. Finally, they also relied on a convertible approach with the 2’-alkynyl-2-amino-LNA phosphoramidites, allowing up to two post synthetic modifications by different polyamines [ 145 ].…”