Defects in the functions of RNA binding proteins (RBPs) are at the origin of many diseases; however, targeting RBPs with conventional drugs has proven difficult. PROTACs are a new class of drugs that mediate selective degradation of a target protein through a cell's ubiquitination machinery. PROTACs comprise a moiety that binds the selected protein, conjugated to a ligand of an E3 ligase. Herein, we introduce RNA‐PROTACs as a new concept in the targeting of RBPs. These chimeric structures employ small RNA mimics as targeting groups that dock the RNA‐binding site of the RBP, whereupon a conjugated E3‐recruiting peptide derived from the HIF‐1α protein directs the RBP for proteasomal degradation. We performed a proof‐of‐concept demonstration with the degradation of two RBPs—a stem cell factor LIN28 and a splicing factor RBFOX1—and showed their use in cancer cell lines. The RNA‐PROTAC approach opens the way to rapid, selective targeting of RBPs in a rational and general fashion.

SummaryTris(2-aminobenzimidazole) conjugates with antisense oligonucleotides are effective site-specific RNA cleavers. Their mechanism of action is independent of metal ions. Here we investigate conjugates with peptide nucleic acids (PNA). RNA degradation occurs with similar rates and substrate specificities as in experiments with DNA conjugates we performed earlier. Although aggregation phenomena are observed in some cases, proper substrate recognition is not compromised. While our previous synthesis of 2-aminobenzimidazoles required an HgO induced cyclization step, a mercury free variant is described herein.

In this report, we investigate the efficiency and selectivity of a Zn2+-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7–8 h as compared to 11–12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50–60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21–24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7–8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site.

We have previously shown that PNA-neocuproine conjugates can act as artificial RNA restriction enzymes. In the present study we have additionally conjugated the PNA with different entities, such as oligoethers, peptides etc. and also constructed systems where the PNA is designed to clamp the target RNA forming a triplex. Some conjugations are detrimental for the activity while most are silent which means that conjugation can be done to alter physical properties without losing activity. Conjugation with a single oligoether close to the neocuproine does enhance the rate almost twofold compared to the system without the oligoether. The systems designed to clamp the RNA target by forming a triplex retain the activity if the added oligoT sequence is 5 PNA units or shorter and extends the arsenal of artificial RNA restriction enzymes. Changing the direction of a closing base pair, where the target RNA forms a bulge, from a GC to a CG pair enhances the rate of cleavage somewhat without compromising the selectivity, leading to the so far most efficient artificial nuclease reported.

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.