C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration

Sequence-Specific Recognition of Double-Stranded DNA by Peptide Nucleic Acid Forming Double-Duplex Invasion Complex

Peptide nucleic acid (PNA) is an analog of natural nucleic acids, where the sugar-phosphate backbone of DNA is replaced by an electrostatically neutral N-(2-aminoethyl)glycine backbone. This unique peptide-based backbone enables PNAs to form a very stable duplex with the complementary nucleic acids via Watson–Crick base pairing since there is no electrostatic repulsion between PNA and DNA·RNA. With this high nucleic acid affinity, PNAs have been used in a wide range of fields, from biological applications such as gene targeting, to engineering applications such as probe and sensor developments. In addition to single-stranded DNA, PNA can also recognize double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is hard to achieve with other artificial nucleic acids and is expected to be a promising method to recognize dsDNA in cellula or in vivo since the invasion does not require the prior denaturation of dsDNA. In this paper, we provide basic knowledge of PNA and mainly focus on the research of PNA invasion

Investigation of the Characteristics of NLS-PNA: Influence of NLS Location on Invasion Efficiency

Peptide nucleic acid can recognise sequences in double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is a promising method for the recognition of dsDNA in cellula because peptide nucleic acid (PNA) invasion does not require the prior denaturation of dsDNA. To increase its applicability, we developed PNAs modified with a nuclear localisation signal (NLS) peptide. In this study, the characteristics of NLS-modified PNAs were investigated for the future design of novel peptide-modified PNAs

Sequence-Specific Recognition of Double-Stranded DNA by Using Only PNAs in Parallel with Natural Nucleobases

The sequence-specific recognition of double-stranded DNA (dsDNA) is a key property for the control of DNA function. Peptide nucleic acid (PNA) can be utilised for the direct recognition of dsDNA via the formation of a unique invasion complex. Strand invasion by PNA induces local changes in the structure of dsDNA and is useful for the regulation of gene expression and genome editing. However, the fact that nucleobases modification is required for efficient invasion, has stymied the wide-spread application of PNA. Herein, we succeeded in the efficient recognition of target dsDNA sequences via formation of invasion complex by utilising only parallel-stranded and unmodified PNAs. This approach also streamlines synthesis by permitting the use of a peptide synthesiser rather than the manual synthesis we had been dependent upon for nucleobase-modified PNAs. Our new method also exhibited high sequence specificity and flexibility for target dsDNA sequences