Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs

Abstract

Peptide conjugation of oligonucleotides and their analogs is being studied widely towards improving the delivery of oligonucleotides into cells. Amongst the many possible routes of conjugation, the disulfide linkage has proved to be the most popular. This reversible linkage may have advantages for cell delivery, since it is likely to be cleaved within cells, thus releasing the oligonucleotide cargo. It is straightforward to introduce thiol functionalities into both oligonucleotide and peptide components suitable for disulfide conjugation. However, severe difficulties have been encountered in carrying out conjugations between highly cationic peptides and negatively charged oligonucleotides because of aggregation and precipitation. Presented here are reliable protocols for disulfide conjugation that have been verified for both cationic and hydrophobic peptides as well as oligonucleotides containing deoxyribonucleosides, ribonucleosides, 2'-O-methylribonucleosides, locked nucleic acid (LNA) units, as well as phosphorothioate backbones. Also presented are reliable protocols for disulfide conjugation of peptide nucleic acids (PNAs) with peptides.

miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA–peptide conjugates

“…Oligonucleotides, miRNA mimics, and PNA peptides Human/rat miR-122 mimic duplex sequences were obtained from the miRBase Sequence Database (Release 9.2) and were purchased from Dharmacon; human/rat sense strand, 59-UGGAGUGUGA CAAUGGUGUUUGU-39; human antisense strand, 59-AAACGCC AUUAUCACACUAAAUA-39; rat antisense strand, 59-AAACGC (Turner et al 2006). Synthesized sequences contained approximately alternating LNA and 29-OMe residues, with an LNA/OMe ratio of z0.4.…”

“…These ON sequences corresponded either to the full-complement of wild type, mature miR-122, 59-aCaAaCaCcAuuGuCaCaCuCca-39 (LNA/OMe), or to a scrambled sequence, 59-uCaGcCauAaCuAaCcCauAcCa-39. PNA ONs were synthesized on an APEX 396 robotic peptide synthesizer as described previously (Turner et al 2006). PNA sequences contained an N-terminal Cys for post-synthetic modification and four Lys residues for improved solubility.…”

“…PNA oligonucleotides were conjugated to R 6 -Pen(Cys) as described previously (Turner et al 2006). Briefly, 10 nmol of the corresponding PNA (0.5-1 mM) were diluted in 6 mL of NH 4 Ac 1 M and 30 mL of formamide.…”

“…Such CPP-PNA conjugates were shown to inhibit Tat-dependent transactivation at the TAR RNA target in a luciferase reporter system with integrated plasmids (Turner et al 2005b) or to redirect splicing in a luciferase upregulation system involving an aberrant inserted b-globin intron (Abes et al 2007). We therefore decided to synthesize an N-terminal conjugate of K-PNA-K3 to the C terminus of the CPP R 6 -Penetratin through a disulfide linkage afforded by added Cys residues, in a similar way to that previously described (Turner et al 2005b(Turner et al , 2006Abes et al 2007). Incubation of the resultant R 6 Pen-K-PNA-K3 with Huh7 cells in the absence of any transfection agent led to only a low level detected of endogenous miR-122, compared with scrambled control, as observed by Northern blot (Fig.…”

Increased RNAi is related to intracellular release of siRNA via a covalently attached signal peptide

“…100 mL of an aqueous solution of the single-stranded RNA (170 nmol), 100 mL of an aqueous solution of bromoacetyl peptide (1000 nmol, 5.9 eq. ), and 50 mL 1M Naphosphate buffer (pH 7.0) were mixed and the reaction allowed to continue for 16 h. Conjugates were purified by a single injection on a Resource Q analytical anion exchange column eluting at 1 ml min À1 with salt gradient and other parameters as previously described, and characterized by MALDI-TOF mass spectrometry (Turner et al 2006). siLAM-gTOX: m/e expected 7977.4, found 7975.5; siLAM-pTOX: m/e expected 8025.6, found 8009.3.…”