γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue.

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.

DNA polymerase δ stalls on telomeric lagging strand templates independently from G-quadruplex formation.

Previous evidence indicates that telomeres resemble common fragile sites and present a challenge for DNA replication. The precise impediments to replication fork progression at telomeric TTAGGG repeats are unknown, but are proposed to include G-quadruplexes (G4) on the G-rich strand. Here we examined DNA synthesis and progression by the replicative DNA polymerase δ/proliferating cell nuclear antigen/replication factor C complex on telomeric templates that mimic the leading C-rich and lagging G-rich strands. Increased polymerase stalling occurred on the G-rich template, compared with the C-rich and nontelomeric templates. Suppression of G4 formation by substituting Li(+) for K(+) as the cation, or by using templates with 7-deaza-G residues, did not alleviate Pol δ pause sites within the G residues. Furthermore, we provide evidence that G4 folding is less stable on single-stranded circular TTAGGG templates where ends are constrained, compared with linear oligonucleotides. Artificially stabilizing G4 structures on the circular templates with the G4 ligand BRACO-19 inhibited Pol δ progression into the G-rich repeats. Similar results were obtained for yeast and human Pol δ complexes. Our data indicate that G4 formation is not required for polymerase stalling on telomeric lagging strands and suggest that an alternative mechanism, in addition to stable G4s, contributes to replication stalling at telomeres.

Hybridization of G-quadruplex-forming peptide nucleic acids to guanine-rich DNA templates inhibits DNA polymerase η extension.

The guanine quadruplex (G-quadruplex) is a highly stable secondary structure that forms in G-rich repeats of DNA, which can interfere with DNA processes, including DNA replication and transcription. We showed previously that short guanine-rich peptide nucleic acids (PNAs) can form highly stable hybrid quadruplexes with DNA. We hypothesized that such structures would provide a stronger block to polymerase extension on G-rich templates than a native DNA homoquadruplex because of the greater thermodynamic stability of the PNA-DNA hybrid structures. To test this, we analyzed the DNA primer extension activity of polymerase η, a translesion polymerase implicated in synthesis past G-quadruplex blocks, on DNA templates containing guanine repeats. We observed a PNA concentration-dependent decrease in the level of polymerase η extension to the end of the template and an increase in the level of polymerase η inhibition at the sequence prior to the G-rich repeats. In contrast, the addition of a complementary C-rich PNA that hybridizes to the G-rich repeats by Watson-Crick base pairing led to a decrease in the level of polymerase inhibition and an increase in the level of full-length extension products. The G-quadruplex-forming PNA exhibited inhibition (IC50=16.2±3.3 nM) of polymerase η DNA synthesis on the G-rich templates stronger than that of the established G-quadruplex-stabilizing ligand BRACO-19 (IC50=42.5±4.8 nM). Our results indicate that homologous PNA targeting of G-rich sequences creates stable PNA-DNA heteroquadruplexes that inhibit polymerase η extension more effectively than a DNA homoquadruplex. The implications of these results for the potential development of homologous PNAs as therapeutics for halting proliferating cancer cells are discussed.

Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis.

GammaPNA oligomers having one or two repeats of the sequence AATCCC were designed to hybridize to DNA having one or more repeats of the complementary TTAGGG sequence found in the human telomere. UV melting curves and surface plasmon resonance experiments demonstrate high affinity and cooperativity for hybridization of these miniprobes to DNA having multiple complementary repeats. Fluorescence spectroscopy for Cy3-labeled miniprobes demonstrate increases in fluorescence intensity for assembling multiple short probes on a DNA target compared with fewer longer probes. The fluorescent γPNA miniprobes were then used to stain telomeres in metaphase chromosomes derived from U2OS cells possessing heterogeneous long telomeres and Jurkat cells harboring homogenous short telomeres. The miniprobes yielded comparable fluorescence intensity to a commercially available PNA 18mer probe in U2OS cells, but significantly brighter fluorescence was observed for telomeres in Jurkat cells. These results suggest that γPNA miniprobes can be effective telomere-staining reagents with applications toward analysis of critically short telomeres, which have been implicated in a range of human diseases.

Polymeric cGAMP microparticles affect the immunogenicity of a broadly active influenza mRNA lipid nanoparticle vaccine.

Influenza outbreaks are a major burden worldwide annually. While seasonal vaccines do provide protection against infection, they are limited in that they need to be updated every year to account for the constantly mutating virus. Recently, lipid nanoparticles (LNPs) encapsulating mRNA have seen major success as a vaccine platform for SARS-CoV-2. Herein, we applied LNPs to deliver an mRNA encoding a computationally optimized broadly active (COBRA) influenza immunogen. These COBRA mRNA LNPs induced a broadly active neutralizing antibody response and protection after lethal influenza challenge. To further increase the immunogenicity of the COBRA mRNA LNPs, we combined them with acetalated dextran microparticles encapsulating a STING agonist. Contrary to recent findings, the STING agonist decreased the immunogenicity of the COBRA mRNA LNPs which was likely due to a decrease in mRNA translation as shown in vitro. Overall, this work aids in future selection of adjuvants to use with mRNA LNP vaccines.

Loop and backbone modifications of peptide nucleic acid improve g-quadruplex binding selectivity.

Targeting guanine (G) quadruplex structures is an exciting new strategy with potential for controlling gene expression and designing anticancer agents. Guanine-rich peptide nucleic acid (PNA) oligomers bind to homologous DNA and RNA to form hetero-G-quadruplexes but can also bind to complementary cytosine-rich sequences to form heteroduplexes. In this study, we incorporated backbone modifications into G-rich PNAs to improve the selectivity for quadruplex versus duplex formation. Incorporation of abasic sites as well as chiral modifications to the backbone were found to be effective strategies for improving selectivity as shown by UV-melting and surface plasmon resonance measurements. The enhanced selectivity is due primarily to decreased affinity for complementary sequences, since binding to the homologous DNA to form PNA-DNA heteroquadruplexes retains high affinity. The improved selectivity of these PNAs is an important step toward using PNAs for regulating gene expression by G-quadruplex formation.

Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts.

Ultraviolet light induces cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts, which interfere with DNA replication and transcription. Nucleotide excision repair (NER) removes these photoproducts, but whether NER functions at telomeres is unresolved. Here we use immunospot blotting to examine the efficiency of photoproduct formation and removal at telomeres purified from UVC irradiated cells at various recovery times. Telomeres exhibit approximately twofold fewer photoproducts compared with the bulk genome in cells, and telomere-binding protein TRF1 significantly reduces photoproduct formation in telomeric fragments in vitro. CPD removal from telomeres occurs 1.5-fold faster than the bulk genome, and is completed by 48 h. 6-4PP removal is rapidly completed by 6 h in both telomeres and the overall genome. A requirement for XPA protein indicates the mechanism of telomeric photoproduct removal is NER. These data provide new evidence that telomeres are partially protected from ultraviolet irradiation and that NER preserves telomere integrity.

Kinetic discrimination in recognition of DNA quadruplex targets by guanine-rich heteroquadruplex-forming PNA probes.

Guanine-rich peptide nucleic acid probes hybridize to DNA G quadruplex targets with high affinity, forming PNA-DNA heteroquadruplexes. We report a surprising degree of kinetic discrimination for PNA heteroquadruplex formation with a series of DNA targets. The fastest hybridization is observed for targets folded into parallel morphologies.