Peptide nucleic acids (PNAs) are high-affinity synthetic nucleic acid analogs capable of hybridization with native nucleic acids. PNAs synthesized having amino acid sidechains installed at the γ-position along the backbone provide a template for a single biopolymer to simultaneously encode nucleic acid and amino acid sequences. Previously, we reported the development of “bilingual” PNAs through the synthesis of an amphiphilic sequence featuring separate blocks of hydrophobic and hydrophilic amino acid functional groups. These PNAs combined the sequence-specific binding activity of nucleic acids with the structural organization properties of peptides. Like other amphiphilic compounds, these γ-PNAs were observed to assemble spontaneously into micelle-like nanostructures in aqueous solutions and disassembly was induced through hybridization to a complementary sequence. Here, we explore whether assembly of these bilingual PNAs is possible by harnessing the nucleic acid code. Specifically, we designed an amphiphile-masking duplex system in which spontaneous amphiphile assembly is prevented through hybridization to a nucleic acid masking sequence. We show that the amphiphile is displaced upon introduction of a releasing sequence complementary to the masking sequence through toehold mediated displacement. Upon release, we observe that the amphiphile proceeds to assemble in a fashion consistent with our previously reported structures. Our approach represents amore »
Modular self-assembly of gamma-modified peptide nucleic acids in organic solvent mixtures
Nucleic acid-based materials enable sub-nanometer precision in self-assembly for fields including biophysics, diagnostics, therapeutics, photonics, and nanofabrication. However, structural DNA nanotechnology has been limited to substantially hydrated media. Transfer to organic solvents commonly used in polymer and peptide synthesis results in the alteration of DNA helical structure or reduced thermal stabilities. Here we demonstrate that gamma-modified peptide nucleic acids (
- Publication Date:
- NSF-PAR ID:
- 10160881
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Publisher:
- Nature Publishing Group
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Peptide self-assembly, wherein molecule A associates with other A molecules to form fibrillar β-sheet structures, is common in nature and widely used to fabricate synthetic biomaterials. Selective coassembly of peptide pairs A and B with complementary partial charges is gaining interest due to its potential for expanding the form and function of biomaterials that can be realized. It has been hypothesized that charge-complementary peptides organize into alternating ABAB-type arrangements within assembled β-sheets, but no direct molecular-level evidence exists to support this interpretation. We report a computational and experimental approach to characterize molecular-level organization of the established peptide pair, CATCH. Discontinuous molecular dynamics simulations predict that CATCH(+) and CATCH(−) peptides coassemble but do not self-assemble. Two-layer β-sheet amyloid structures predominate, but off-pathway β-barrel oligomers are also predicted. At low concentration, transmission electron microscopy and dynamic light scattering identified nonfibrillar ∼20-nm oligomers, while at high concentrations elongated fibers predominated. Thioflavin T fluorimetry estimates rapid and near-stoichiometric coassembly of CATCH(+) and CATCH(−) at concentrations ≥100 μM. Natural abundance13C NMR and isotope-edited Fourier transform infrared spectroscopy indicate that CATCH(+) and CATCH(−) coassemble into two-component nanofibers instead of self-sorting. However,13C–13C dipolar recoupling solid-state NMR measurements also identify nonnegligible AA and BB interactions among a majoritymore »
-
Abstract Telomeres terminate with a 50–300 bases long single-stranded G-rich overhang, which can be misrecognized as a DNA damage repair site. Shelterin plays critical roles in maintaining and protecting telomere ends by regulating access of various physiological agents to telomeric DNA, but the underlying mechanism is not well understood. Here, we measure how shelterin affects the accessibility of long telomeric overhangs by monitoring transient binding events of a short complementary peptide nucleic acid (PNA) probe using FRET-PAINT in vitro. We observed that the POT1 subunit of shelterin reduces the accessibility of the PNA probe by ∼2.5-fold, indicating that POT1 effectively binds to and protects otherwise exposed telomeric sequences. In comparison, a four-component shelterin stabilizes POT1 binding to the overhang by tethering POT1 to the double-stranded telomeric DNA and reduces the accessibility of telomeric overhangs by ∼5-fold. This enhanced protection suggests shelterin restructures the junction between single and double-stranded telomere, which is otherwise the most accessible part of the telomeric overhang.
-
Although our understanding of the microbial diversity found within a given system expands as amplicon sequencing improves, technical aspects still drastically affect which members can be detected. Compared with prokaryotic members, the eukaryotic microorganisms associated with a host are understudied due to their underrepresentation in ribosomal databases, lower abundance compared with bacterial sequences, and higher ribosomal gene identity to their eukaryotic host. Peptide nucleic acid (PNA) blockers are often designed to reduce amplification of host DNA. Here we present a tool for PNA design called the Microbiome Amplification Preference Tool (MAPT). We examine the effectiveness of a PNA designed to block genomic Medicago sativa DNA (gPNA) compared with unrelated surrounding plants from the same location. We applied mitochondrial PNA and plastid PNA to block the majority of DNA from plant mitochondria and plastid 16S ribosomal RNA genes, as well as the novel gPNA. Until now, amplifying both eukaryotic and prokaryotic reads using 515F-Y and 926R has not been applied to a host. We investigate the efficacy of this gPNA using three approaches: (i) in silico prediction of blocking potential in MAPT, (ii) amplicon sequencing with and without the addition of PNAs, and (iii) comparison with cultured fungal representatives. When gPNAmore »
-
Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin–Watson–Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.
