Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA-Gapmer Approach.

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a "gapmer" architecture comprised of one zwitterionic central segment ("gap") containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA-gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON-RNA duplexes with base-pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA-gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents.

Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes.

Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, ON1-ON4, incorporating the DMTr phosphorodiamidite monomer of dUHBI, 2, and the corresponding dUDFHBI, 5b, monomer. ON1-ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 with complementary 2'-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔTm 6 °C). Furthermore, addition of ON1-ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/µL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, ON3 and ON4 are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.

Synthesis of 2'-Deoxyuridine Modified with a 3,5-Difluoro-4-Methoxybenzylidene Imidazolinone Derivative for Incorporation into Oligonucleotide Probes for Detection of HER2 Breast Cancer Marker.

Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn-on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non-fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid-phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5-(6-p-Methoxybenzylidene imidazolinone-1-hexene)-2'-deoxyuridine (dUMBI ) is a synthetic monomer to which 4-methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single-strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5-Difluoro-4-methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dUDFMBI as a monomer of fluorescent turn-on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 5-[(6)-3,5-difluoro-4-methoxybenzylidene imidazolinone-1-hexene]-2'-deoxyuridine.