Oligonucleotides with Cationic Backbone and Their Hybridization with DNA: Interplay of Base Pairing and Electrostatic Attraction

Abstract

Non‐natural oligonucleotides represent important (bio)chemical tools and potential therapeutic agents. Backbone modifications altering hybridization properties and biostability can provide useful analogues. Here, we employ an artificial nucleosyl amino acid (NAA) motif for the synthesis of oligonucleotides containing a backbone decorated with primary amines. An oligo‐T sequence of this cationic DNA analogue shows significantly increased affinity for complementary DNA. Notably, hybridization with DNA is still governed by Watson–Crick base pairing. However, single base pair mismatches are tolerated and some degree of sequence‐independent interactions between the cationic NAA backbone and fully mismatched DNA are observed. These findings demonstrate that a high density of positive charges directly connected to the oligonucleotide backbone can affect Watson–Crick base pairing. This provides a paradigm for the design of therapeutic oligonucleotides with altered backbone charge patterns.

Oligonucleotide analogues with cationic backbone linkages

“…These considerations have stimulated our design of a new artificial internucleotide linkage named 'nucleosyl amino acid (NAA)-modification' ( Fig. 5 ) [ 72 – 74 ]. In principle, the NAA-modification is inspired by 'high-carbon' nucleoside structures (i.e., nucleosides having more than five carbon atoms in the sugar unit) found in naturally occurring nucleoside antibiotics [ 75 – 77 ].…”

“…The phosphoramidite-based synthetic strategy depicted in Scheme 5 was not suitable to reach this goal as it furnishes phosphate diester linkages at least at every second position within a given sequence. Therefore, a different synthetic route was developed ( Scheme 6 ) [ 74 ]. The envisioned fully cationic thymidine-derived oligomers 55a ( all -( S )-configured at the 6'-positions) and 55b ( all -( R )-configured at the 6'-positions) were assembled by manual Fmoc-based solid phase-supported peptide synthesis using the monomeric 3'-amino-nucleosyl amino acids ( S )- 56 and ( R )- 56 , respectively, as building blocks.…”

“…The envisioned fully cationic thymidine-derived oligomers 55a ( all -( S )-configured at the 6'-positions) and 55b ( all -( R )-configured at the 6'-positions) were assembled by manual Fmoc-based solid phase-supported peptide synthesis using the monomeric 3'-amino-nucleosyl amino acids ( S )- 56 and ( R )- 56 , respectively, as building blocks. The synthesis of thymidinyl amino acids 56 was again started from a corresponding 5'-aldehyde 57 using Wittig–Horner olefination and catalytic asymmetric hydrogenation as key steps (reactions not shown) [ 74 ].…”

“…The properties of fully cationic oligonucleotide analogues 55a and 55b were studied in detail [ 74 ]. Thermal denaturation experiments demonstrated a strong hybridization of both thymidinyl oligomers with native complementary A 14 DNA, with T m values being 9 and 17 °C higher, respectively, than the T m value of an unmodified T 14 –A 14 DNA reference duplex.…”

“…The hampered base-pairing fidelity of 55a and 55b raised the question if the hybridization of these oligocations with oligoanionic DNA was dependent on Watson–Crick base-pairing at all or if it was mainly mediated by electrostatic attraction. Thermal denaturation studies of mixtures of 55a or 55b , respectively, with a fully mismatched DNA counterstrand (G 6 TTG 6 ) showed a pronounced hyperchromicity upon heating in both cases, but also indicated that no transition between two defined states occurred [ 74 ]. It was derived from these results that 55a and 55b probably formed less defined, unspecific aggregates with the fully mismatched counterstrand, which then disassembled at elevated temperatures.…”