Targeting pre-miRNA by Peptide Nucleic Acids

Avitabile, Concetta; D’Andrea, Luca Domenico; Bianchi, Nicoletta; Fabbri, Enrica; Brognara, Eleonora; Gambari, Roberto; Romanelli, Alessandra

doi:10.4161/adna.20911

Cited by 21 publications

(10 citation statements)

References 47 publications

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“…RNAs are emerging as important biomarkers and therapeutic targets, due to the recent advances in the discoveries of the RNAs' roles in the regulation and catalysis of diverse biological processes 1 2 3 . Traditionally, antisense strands have been used to bind to ssRNAs through Watson-Crick duplex formation 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 . Recently, triplex-forming peptide nucleic acids (TFPNAs) have been designed to bind to dsRNAs via Hoogsteen hydrogen bonding ( Figure 1 ) 3 28 29 .…”

Section: Introductionmentioning

confidence: 99%

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

Toh

Patil

Chen

2017

JoVE

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RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

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Section: Introductionmentioning

confidence: 99%

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

Toh

Patil

Chen

2017

JoVE

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“…In this context the application of oligonucleotide analogues, such as Peptide Nucleic Acids (PNAs), showing resistance to degradation in vivo, high affinity and specificity of binding toward complementary DNA and RNA, is widespread (1)(2)(3). Peptide Nucleic Acids, containing a pseudo-peptide backbone in place of the sugar-phosphate backbone, have been tested as antisense, antigene molecules and recently as inhibitors of the function of miRNAs (4)(5)(6)(7). The limited efficiency of transfection of Peptide Nucleic Acids in cells severely hampers their application in vivo.…”

Section: Introductionmentioning

confidence: 99%

The influence of liposomal formulation on the incorporation and retention of PNA oligomers

Ringhieri

Avitabile

Morelli

et al. 2016

Colloids and Surfaces B: Biointerfaces

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Liposomal formulations composed of phospholipids with different unsaturation degrees, head groups and at different cholesterol content have been tested for the encapsulation of Peptide Nucleic Acid (PNA) oligomers. The best loading capability (177μg, ER%=87.2) was obtained for pure liposomes of phosphatidylglycerol (DOPG) with negatively charged head group. The insertion of a 10-20% of cholesterol in DOPG based liposomes provides a slight decrease (∼160μg) of the PNA loading. On the other hand, the cholesterol addition (20-30%) slows down the PNA's release (∼27%) in fetal bovine serum from the liposomal formulation. Based on the encapsulation and the release properties, PEGylated DOPG liposomes with a percentage of cholesterol of 10-20% are the optimal formulation for the loading of PNA-a210.

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“…Next the PNA oligomer was assembled, as described above. We conjugated the fluorescent dye TO following previously described procedures [ 36 , 37 ]. All PNA oligomers were purified and then characterized by liquid chromatography–mass spectrometry using a linear gradient of acetonitrile (0.05% trifluoroacetic acid) in water (0.05% trifluoroacetic acid) from 5% to 50% over 30 min.…”

Section: Methodsmentioning

confidence: 99%

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

et al. 2016

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Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies.

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Targeting pre-miRNA by Peptide Nucleic Acids

Cited by 21 publications

References 47 publications

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

The influence of liposomal formulation on the incorporation and retention of PNA oligomers

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

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