Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides.

Huntington's disease is a neurodegenerative, trinucleotide repeat (TNR) disorder affecting both males and females. It is caused by an abnormal increase in the length of CAG•CTG TNR in exon 1 of the Huntingtin gene (HTT). The resultant, mutant HTT mRNA and protein cause neuronal toxicity, suggesting that reduction of their levels would constitute a promising therapeutic approach. We previously reported a novel strategy in which chemically modified oligonucleotides (ONs) directly target chromosomal DNA. These anti-gene ONs were able to downregulate both HTT mRNA and protein. In this study, various locked nucleic acid (LNA)/DNA mixmer anti-gene ONs were tested to investigate the effects of varying ON length, LNA content, and fatty acid modification on HTT expression. Altering the length did not significantly influence the ON potency, while LNA content was critical for activity. Utilization of palmitoyl-modified LNA monomers enhanced the ON activity relatively to the corresponding nonmodified LNA under serum starvation conditions. Furthermore, the number of palmitoylated LNA monomers and their positioning greatly affected ON potency. In addition, we performed RNA sequencing analysis, which showed that the anti-gene ONs affect the "immune system process, mRNA processing, and neurogenesis." Furthermore, we observed that for repeat containing genes, there is a higher tendency for antisense off-targeting. Taken together, our findings provide an optimized design of anti-gene ONs that could potentially be developed as DNA-targeting therapeutics for this class of TNR-related diseases.

Carbazole modified oligonucleotides: synthesis, hybridization studies and fluorescence properties.

Synthesis of the novel thiophenyl carbazole phosphoramidite DNA building block 5 was accomplished in four steps using a Suzuki-Miyaura cross-coupling reaction from the core carbazole and it was seamlessly accommodated into a 9-mer DNA-based oligonucleotide by incorporation at the flanking 5'-end in combination with a central insertion of an LNA-T nucleotide. The carbazole-containing oligonucleotide was combined in different duplex hybrids, which were characterized by thermal denaturation, circular dichroism and fluorescence studies. The carbazole monomer modulates the duplex stability in various ways. Thus, monomer Z increased the thermal stability of the 9-mer towards the complementary 9-mer/15-mer DNA duplex by 4.2 °C. Furthermore, indications of its intercalation into the duplex were obtained by modeling studies and robust decreases in fluorescence emission intensities upon duplex formation. In contrast, no clear intercalating tendency was corroborated for monomer Z within the DNA/RNA hybrid duplex as indicated by moderate quenching of the fluorescence and similar duplex thermal stabilities relative to the corresponding control duplex. The recognition efficiencies of the carbazole modified oligonucleotide toward single nucleotide mismatches were studied with two 15-mer model targets (DNA and RNA). For both systems, mismatches positioned at the juxtaposition of the carbazole monomer showed pronounced deceases in thermal denaturation temperature. Steady-state fluorescence emission studies of all mismatched duplexes with incorporation of Z monomer typically displayed efficient fluorescence quenching.

Novel assemblies based on oligonucleotides containing intercalating nucleic acid monomers.

This is the first report exploring the capability of twisted intercalating nucleic acid (TINA) and naphthalene-functionalized non-nucleosidic linkers to stabilize and engage in double-helical structures. Four designs were studied with respect to the formation of duplexes and/or other types of self-assemblies. One of the constructs involving TINA provides a thermostable duplex. The biophysical properties of the individual constructs were investigated by UV thermal melting experiments, circular dichroism, and fluorescence emission spectroscopy. Molecular modeling studies were performed in attempts of explaining the biophysical measurements for the duplex based on the TINA-containing oligonucleotide strands.

Improved i-motif thermal stability by insertion of anthraquinone monomers.

In order to gain insight into how to improve thermal stability of i-motifs when used in the context of biomedical and nanotechnological applications, novel anthraquinone-modified i-motifs were synthesized by insertion of 1,8-, 1,4-, 1,5- and 2,6-disubstituted anthraquinone monomers into the TAA loops of a 22mer cytosine-rich human telomeric DNA sequence. The influence of the four anthraquinone linkers on the i-motif thermal stability was investigated at 295 nm and pH 5.5. Anthraquinone monomers modulate the i-motif stability in a position-depending manner and the modulation also depends on the substitution pattern of the anthraquinone. The insertion of anthraquinone was found to stabilize the i-motif structure when replacing any one of the positions of the central TAA loop and the thermal stabilities were typically higher than those previously found for i-motifs containing pyrene-modified uracilyl unlocked nucleic acid monomers or twisted intercalating nucleic acid. The 2,6-disubstituted anthraquinone linker replacing T10 enabled a significant increase of i-motif thermal melting by 8.2 °C. A substantial increase of 5.0 °C in i-motif thermal melting was recorded when both A6 and T16 were modified with a double replacement by the 2,6-isomer into the TAA loops in the outer regions. The largest destabilization is observed for the 1,5-disubstituted anthraquinone linker upon the replacement of A18. CD curves of anthraquinone-modified variants imply no structural changes in all cases under potassium buffer conditions compared with those of the native i-motif. Molecular modeling studies explained the increased thermal stabilities of anthraquinone-modified i-motifs.

Nucleic Acid Targeted Therapy: G4 Oligonucleotides Downregulate HRAS in Bladder Cancer Cells through a Decoy Mechanism.

In a previous study we have demonstrated that two neighboring G-quadruplexes, hras-1 and hras-2, located immediately upstream of the major transcription start site of HRAS, bind MAZ, a nuclear factor that activates transcription (Cogoi, S.; et al. Nucl. Acid Res. 2014, 42, 8379). For the present study we have designed G4 oligonucleotides with anthraquinone insertions and locked nucleic acids (LNA) modifications mimicking quadruplex hras-1. Luciferase, qRT-PCR, and Western blot data demonstrate that these constructs efficiently down regulate HRAS in T24 bladder cancer cells. The inhibitory efficiency of the G4 oligonucleotides correlates with their nuclease resistance in the cell environment. By chromatin immunoprecipitation we show that the association of MAZ to the HRAS promoter is strongly attenuated by the designed G4 oligonucleotides, thus suggesting that these constructs behave with a decoy mechanism.