Demonstrating specificity of bioactive peptide nucleic acids (PNAs) targeting microRNAs for practical laboratory classes of applied biochemistry and pharmacology

Abstract

Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis.

Fig 1. Biological effects of a PNA targeting miR-221-3p and outline of the practical laboratory program.

A. Scheme of the background available in the literature on the biological effects of the R8-PNA-a221 on human glioma cell lines (Fabbri et al, 2017). More detailed information is shown in Figure A in S1 File. B. Timing of the laboratory practice, depending on the starting activity (identified by the blue arrows). The key activity is shown as the segment (c). Alternatively, U251 cell culture and RNA extraction/characterization (a) or only RNA extraction (b) might be considered. C. Scheme of the laboratory practice finalized to verify the specificity of the biological activity of the PNA-a221. The extracted U251 RNA is used for RT-qPCR in the presence of the PNA-a221 and the PNA-a221-MUT. The amplified miRNAs are indicated. Expected results (blue: inhibition; green: no inhibition) when PNA-a221 and PNA-a221-MUT are employed and miR-221, miR-222, miR-210 and let-7 sequences amplified by RT-qPCR. Specificity can be demonstrated if inhibition of the RT-qPCR product is obtained amplifying miR-221-3p in the presence of PNA-a221.

Fig 2. Effects of the PNA-a221 on the RT-PCR amplification of miRNA sequences.

A,B. Effects of increasing amounts of PNA-a221 (A) and mutated PNA-a221-MUT (B) on the amplification of miR-221-3p sequences. C-E. Effects of increasing amounts of PNA-a221 on the amplification of miR-222-3p (C), miR-210-3p (D) and let-7c-5p (E) sequences. F. Summary of the effects of 25 nM PNA-a221 on amplification of the indicated miRNA sequences. The comparison of the effects of 50, 100 and 200 nM PNA-a221 are presented in Figure E in S1 File.

Fig 3. Biological effects of a PNA targeting miR-145-5p and outline of the practical laboratory program.

A. Scheme of the background available in the literature on the biological effects of the R8-PNA-a145 on the Calu-3 cell line (Fabbri et al., 2017). More detailed information is shown in Figure D in S1 File. B. Scheme of the laboratory practice finalized to verify the specificity of the biological activity of the PNA-a145. The extracted Calu-3 RNA is used for RT-qPCR in the presence of the PNA-a145 and the PNA-a145-MUT (green: no inhibition; blue: inhibition). The amplified miRNAs (miR-145-5p, let-7c-5p and miR-155-5p) are indicated. Specificity can be demonstrated if inhibition of the RT-qPCR product is obtained amplifying miR-145-5p in the presence of PNA-a145.

Fig 4. Effects of the PNA-a145 on the RT-PCR amplification of miRNA sequences.

A,B. Effects of 25 nM PNA-a145 (A) and mutated PNA-a145-MUT (B) on the amplification of miR-145-5p sequences. C,D. Effects of 25 nM PNA-a145 on the amplification of miR-155-5p (C) and let-7c-5p (D) sequences. A summary of increasing concentrations of PNA-a145 are presented in Figure F in S1 File.

Fig 5. Effects of R8-PNA-a221 on miR-221-3p sequence detection by RT-ddPCR.

A. 1D RT-ddPCR plot obtained after the addition of incremental concentration of R8-PNA-a221 or R8-PNA-a221-MUT to the reaction mix. B. miR-221-3p content in 1:50 diluted cDNA obtained from U251 cells: 2D plots. C. miR-221-3p content detected after the addition of incremental R8-PNA-a221concentrations. D. miR-221-3p content detected after the addition of incremental R8-PNA-a221-MUT concentrations.

Fig 6. Effects of R8-PNA-a221 on miR-222-3p sequence detection by RT-ddPCR.

A. 1D RT-ddPCR plot obtained after the addition of incremental concentration of R8-PNA-a221, miR-222-3p is amplified. B. miR-222-3p content in 1:50 diluted cDNA obtained from U251 cells: 2D plots. C. miR-222-3p content detected after the addition of incremental R8-PNA-a221concentrations.