Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA₉) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA₈) targeting a stem-loop. PNA₉, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA₉ (F-PNA₁₃). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA₉ and F-PNA₁₃, but not PNA₈ highlighting the importance of target site selection. Surprisingly, F-PNA₁₃ which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA₉ likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.