Self-neutralizing oligonucleotides with enhanced cellular uptake

Abstract

There is tremendous potential for oligonucleotide (ON) therapeutics, but low cellular penetration due to their polyanionic nature is a major obstacle. We addressed this problem by developing a new approach for ON charge neutralization in which multiple branched charge-neutralizing sleeves (BCNSs) are attached to the internucleoside phosphates of ON by Aphosphotriester bonds. The BCNSs are terminated with positively charged amino groups, and are optimized to form ion pairs with the neighboring phosphate groups. The new modified ONs can be prepared by standard automated phosphoramidite chemistry in good yield and purity. They possess good solubility and hybridization properties, are not involved in non-standard intramolecular aggregation, have low cytotoxicity, adequate chemical stability, improved serum stability, and above all, display significantly enhanced cellular uptake. Thus, the new ON derivatives exhibit properties that make them promising candidates for the development of novel therapeutics or research tools for modulation of the expression of target genes.

DNA with zwitterionic and negatively charged phosphate modifications: formation of DNA triplexes, duplexes and cell permeability studies

“…65 In contrast, incorporation of BCNS (Fig. 3) groups into the DNA backbone led to self-neutralising ONs that did not induce a change of T m when binding complementary DNA sequences, 66 85 The Ts-modification stabilised duplex formation with RNA at 100 mM salt, but destabilised the RNA duplex at a low salt concentration.…”

“…65 Branched, chargeneutralising sleeves (BCNS, Fig. 3) incorporated on the ON backbone 66 formed selfneutralising ONs with good aqueous solubility, enhanced resistance to nucleases, low cytotoxicity and increased thermal stability in the context of 2ʼ-OMe-RNA duplexes. We hypothesised N+ONs should hybridise with complementary single-stranded DNA (ssDNA) or RNA with higher affinity than native ONs, due to both a reduced charge repulsion between negatively charged phosphates and their thermal stability being less dependent on the ionic strength of the solution.…”

DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies

“…The introduction of sulfonamide RNA (SaRNA monomers) to replace the phosphodiester backbone led to charge-neutral sulfonamide antisense oligonucleotides (SaASOs), which resulted in a lower destabilisation (a more stable) DNA–RNA duplex compared to a DNA–DNA duplex [ 55 ]. In contrast, the incorporation of branched, charge-neutralising sleeve (BCNS) groups onto the DNA backbone led to self-neutralising ONs that did not induce a change of T m when binding complementary DNA sequences [ 56 ], regardless of the number of BCNSs incorporated. This is in line with our results where increasing the number of N+ or Ts modifications showed no benefit in T m values of the antiparallel duplexes formed with complementary RNA or DNA.…”

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

“…These cationic internucleotide linkages were synthesized through conventional phosphoramidate chemistry with a slight variation. In contrast to the standard method, bis(diisopropylamino)chlorophosphine was first reacted with either of the three diaminoalcohol groups, before subsequent phosphitylation with the DMT-protected nucleosides [ 115 ]. After incorporation into ONs, conversion to monomers 78–81 was accomplished as shown in Table 8 .…”