Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

Abstract

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the BRAF V600E oncogene, with a library of rationally designed peptides employing CuAAC "click" chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.

Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes

“…To challenge this even further, we used probes of same sequence but containing a different modification, a hydrophilic peptide, instead of the fluorophores (see Supplementary Information, chapter 5). 20 These peptideoligonucleotide conjugates contained LNA as well, in the same positions as the fluorescence probes. The resulting ∆Tm values for these new peptide-oligonucleotide controls were similar to those of the fluorescent probes.…”

Antibodies to synthetic citrullinated peptide epitope correlate with disease activity and flares in rheumatoid arthritis

“…Peptide epitopes were purchased from a commercial supplier (synthesized by solid-phase peptide synthesis [22,23]; HPLC purified fractions with purity > 90%). The initial screening a fib = fibrinogen; vim = vimentin; His = histone; col = collagen; fil = filaggrin.…”

“…Peptide antigens were synthesized and characterized as described in our recent papers [21][22][23]. In house ELISA has been carried out as described, using sera samples from patient groups and controls [21].…”

Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

“…Recently, we synthesized doubly labeled 21‐mer DNA/LNA oligonucleotides that targeted the BRAF oncogene containing the V600E mutation . Through CuAAC click chemistry, the precursors were labeled internally with a library of rationally designed peptides (Scheme ).…”

“…Recently,w es ynthesized doubly labeled 21-mer DNA/LNA oligonucleotides that targeted the BRAF oncogene containing the V600E mutation. [74] Through CuAACc lick chemistry,t he precursors were labeled internally with al ibrary of rationally designed peptides (Scheme 7). Our synthetic approach provided 30 POCs in at ime-and cost-effective manner,g iving good yields (43-83%)a nd high purity of the products (90 %).…”