RICERCA ITALIANA     

RNA and microRNA targeting by peptide nucleic acids (PNAs) and their analogues

Università degli Studi di Parma

Abstract

RNA has been the target of the long-standing quest for molecules able to block gene expression in a sequence selective way, with the so called antisense strategy. During the past few years, molecular biologists have discovered hundreds of genes that encode small RNA molecules, the microRNAs (miRNAs), 21 to 25 nucleotides in length, which are involved in the post-transcriptional regulation of gene expression in plants and animals. Indeed, there are hints that the levels of some miRNAs are altered in cancer and there is also evidence that a miRNA regulates the cancer-promoting genes. Synthetic oligonucleotides or analogues acting as competitors by binding to miRNA have been proposed as novel potential drugs.
Moreover, molecules able to mimick small interfering RNA (siRNA) activity and targeting nuclear RNA precursors have been suggested to be of high therapeutic value for diseases like thalassemia, cancer and neurodegenerative disorders.
The interest for several foodborne diseases related to RNA viruses like the norovirus is also increasing, since they are difficult to detect and not cultivable in vitro.
Thus, it is very important to produce molecules able to target RNA either for diagnostic or therapeutic purposes .
Peptide nucleic acids (PNAs) are oligonucleotide analogues with a polyamidic backbone and are very promising tools for binding RNA, since they have a higher affinity for RNA than DNA, are stable to nucleases and are very specific. Their >>>

Principal Investigator

Rosangela Marchelli Università degli Studi di PARMA

Research Objectives

Aim of the present project is to develop PNA-based molecules able to target specifically RNA for diagnostic and therapeutic purposes.
In the last years microRNA (miRNA), small non-coding RNAs which are involved in viral infections and are associated with oncogenesis and can be inhibitors of apoptosis, have been discovered.
Thus, it is of particular interest to inhibit these RNAs or even to mimick their activity.
Peptide nucleic acids (PNAs) are ideal molecules for RNA recognition, since they show higher affinity for RNA than for DNA, are chemically very stable and resistant to nucleases and proteases.
The specific objectives are the following:

Objective 1. DESIGN OF PNA.

1.1 Description of the structural and steric requirements which make PNAs excellent binding agents for RNA.
By means of molecular design based on structural data and molecular modelling of PNA:RNA duplexes, the structural/steric requirements to increase the affinity of PNA for RNA and the selectivity will be evaluated. The design will be carried out by the Napoli Unit by “computer modelling” and energy minimization techniques also developing appropriate “ab inizio” force fields.

1.2 Selection of target RNA sequences and PNA design
mRNA and microRNA sequences of biological and diagnostic interest will be selected on the base of the experience of the Ferrara Unit. Accordingly, PNAs will be designed for: (a) targeting miRNA >>>

First Results

This project is very innovative, if compared to the current scientific background, since it deals with new issues of primary interest, both for basic research and for applications.
RNA recognition, a very timely issue in the biological and biomedical fields, has been scarcely studied by chemists, which so far concentrated their efforts mainly in the study of DNA.
From the research here proposed, an advancement in the basic knowledge of the structural characteristics which make PNAs able to selectively bind RNA or to mimic RNA is expected, as well as of the role played by RNA in relevant processes at the cellular level.
The applications of the new synthesized molecules for biomedical diagnostics and therapeutics seems to be very promising for opening new perspectives for the preparation of new diagnostic kits, for the identification of new therapeutic strategies and for the development of new drugs.

In particular, the following results can be expected:

1. OPTIMIZATION OF PNA STRUCTURAL PARAMETERS FOR DNA RECOGNITION AND IDENTIFICATION OF BIOLOGICAL TARGETS.

1.1. Knowledge of structural characteristics of PNAs.
Structural data and molecular modelling will allow to identify the structural and steric requirements for improving PNA affinity and selectivity towards RNA.

1.2. Identification of RNA target sequences.
The identification of mRNA and microRNA of biomedical and diagnostic >>>

Timescale

24 months

National and international background

Since non-viral gene therapy was developed and introduced as an effective way to control and modify gene expression, RNA has been considered as a molecular target of great relevance. Examples of RNA sequences to be targeted for therapeutic applications are mRNAs coding oncoproteins or RNA coding anti-apoptotic proteins for the development of anti-cancer therapy.
In the last years, progress in molecular biology has allowed to identify many genes coding for small RNA molecules, microRNA (miRNAs), non coding RNA molecules able to regulate gene expression at the translation level [1]. On account of their important role, the sequences of miRNAs are evolutionarily conserved. Recently reported data suggest that vertebrate genomes encode as many as 1000 unique miRNAs, which are predicted to regulate expression of at least 30% of genes.
On account of their specific roles, miRNAs have been demonstrated to be:
a) important for development, b) differentially expressed in tissues (for instance miRNA-221-222 are associated with erythropoiesis), c) involved in viral infection processes, d) associated with oncogenesis, e) inhibitors of apoptosis.
Like mRNAs, microRNAs are transcribed as regions of longer RNA molecules that can be as long as 1000 nucleotides, exhibiting heavy secondary structures. These RNA molecules are processed in the nucleus into hairpin RNAs of 70-100 nucleotides by the dsRNA-specific ribonuclease Drosha. The hairpin RNAs are transported to >>>