Telomerase RNA gene paralogs in plants - the usual pathway to unusual telomeres.

Telomerase, telomeric DNA and associated proteins together represent a complex, finely tuned and functionally conserved mechanism that ensures genome integrity by protecting and maintaining chromosome ends. Changes in its components can threaten an organism's viability. Nevertheless, molecular innovation in telomere maintenance has occurred multiple times during eukaryote evolution, giving rise to species/taxa with unusual telomeric DNA sequences, telomerase components or telomerase-independent telomere maintenance. The central component of telomere maintenance machinery is telomerase RNA (TR) as it templates telomere DNA synthesis, its mutation can change telomere DNA and disrupt its recognition by telomere proteins, thereby leading to collapse of their end-protective and telomerase recruitment functions. Using a combination of bioinformatic and experimental approaches, we examine a plausible scenario of evolutionary changes in TR underlying telomere transitions. We identified plants harbouring multiple TR paralogs whose template regions could support the synthesis of diverse telomeres. In our hypothesis, formation of unusual telomeres is associated with the occurrence of TR paralogs that can accumulate mutations, and through their functional redundancy, allow for the adaptive evolution of the other telomere components. Experimental analyses of telomeres in the examined plants demonstrate evolutionary telomere transitions corresponding to TR paralogs with diverse template regions.

Connecting telomere maintenance and regulation to the developmental origin and differentiation states of neuroblastoma tumor cells.

A cardinal feature that distinguishes clinically high-risk neuroblastoma from low-risk tumors is telomere maintenance. Specifically, neuroblastoma tumors with either active telomerase or alternative lengthening of telomeres exhibit aggressive growth characteristics that lead to poor outcomes, whereas tumors without telomere maintenance can be managed with observation or minimal treatment. Even though the need for cancer cells to maintain telomere DNA-in order to sustain cell proliferation-is well established, recent studies suggest that the neural crest origin of neuroblastoma may enforce unique relationships between telomeres and tumor malignancy. Specifically in neuroblastoma, telomere structure and telomerase activity are correlated with the adrenergic/mesenchymal differentiation states, and manipulating telomerase activity can trigger tumor cell differentiation. Both findings may reflect features of normal neural crest development. This review summarizes recent advances in the characterization of telomere structure and telomere maintenance mechanisms in neuroblastoma and discusses the findings in the context of relevant literature on telomeres during embryonic and neural development. Understanding the canonical and non-canonical roles of telomere maintenance in neuroblastoma could reveal vulnerabilities for telomere-directed therapies with potential applications to other pediatric malignancies.

Cyclic ferrocenylnaphthalene diimides as a probe for electrochemical telomerase assay.

Novel cyclic naphthalene diimides, 8 and 12, containing ferrocene in the cyclic linker were synthesized as G-quartet (G4) specific electrochemical ligands via the reaction of 1,1'-ferrocenedipropanoic acid and the terminal amine moieties of naphthalene diimides with varying linker lengths. The redox potentials of 8 and 12 were ca. 0.2 V (vs. Ag/AgCl), and the background current in an electrolyte was successfully suppressed. Both 8 and 12 bound to TA-core, representing human telomere G4, with K = 4.4 and 38 × 105 M-1, respectively. The current response of 12 to an electrode immobilized with G4 was the highest among the acyclic derivatives, suggesting its potential application in electrochemical telomerase assays.

Detection of Tetraplex DNA and Detection by Tetraplex DNA.

G-quardruplex (G4) DNA forms through the gathering together of G-quartet planes formed with four guanine (G) bases. G4 DNA stabilizes with potassium ions (K+) by coordination with the G-quartet center. Fluorometric G4 DNA carrying the fluorescence resonance energy transfer (FRET) chromophore pair at both termini has been applied for the fluorometric sensing or imaging of K+ under a homogeneous aqueous medium. This system has realized non-conventional K+ selectivity over the sodium ion (Na+). The selectivity of the fluorescence G4 was converted to Na+ from K+ with a modification of its sequence. On the other hand, G4 DNA detection has been achieved in terms of cancer diagnosis because of a strong relationship of G4 DNA and cancer development. Ligands interacting with G4 are expected to have anti-cancer potential. In addition, fluorometric G4 ligands have been developed and tested as tools for the dynamic monitoring of G4 in living cells. Moreover, fluorometric G4 DNA has been utilized to evaluate the G4 ligand performance.

Artificial Restriction DNA Cutter Using Nuclease S1 for Site-Selective Scission of Genomic DNA.

By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.

Molecular and cellular basis of mammary gland fibrosis and cancer risk.

Mammary gland luminal cells are maintained by the proliferation of ER- luminal progenitor (LP) cells. Human breast LP cells exhibit telomere DNA damage, which is associated with mammographic density and increased cancer risk. Telomeric repeat factor 2 (TRF2) protects telomeres from DNA damage response. TRF2 expression is reduced in human breast cancers. We deleted TRF2 expression in mammary gland epithelium. Mammary glands lacking TRF2 expression exhibited increased telomere DNA damage response, histopathological and functional degeneration, and prominent ductal fibrosis. TRF2-deficient mammary tumors exhibited rapid onset and increased proliferation. Tumor derived LP cells failed to form tumors after transplantation. The MSC population was highly tumorigenic and maintained telomeres via the ALT mechanism. Telomere DNA damage response in mammary tumors resulted in p53 dependent ER+ cellular differentiation and sensitivity to anti-estrogen therapy. Our results provide a new in vivo model of mammographic density, stem cell differentiation, cancer risk, and therapeutic sensitivity.

Relationship between telomere DNA length of peripheral leukocytes and tumor type and prognosis in patients with non?small cell lung cancer / 中国综合临床

Objective To investigate the relationship between telomere DNA length of peripheral leukocytes and tumor type and prognosis in non-small cell lung cancer ( NSCLC) . Methods From June 2016 to April 2017,the thoracic surgery department of Baoding First Central Hospital,Hebei Province was included in the study. The patients with lung cancer and TNM stage were confirmed by pathological examination,excluding the patients with unknown general records and lost visits. Finally,55 patients were included,including 35 patients in adenocarcinoma group and 20 patients in other non-small cell lung cancer group. The telomere DNA length of peripheral blood leukocytes in patients with non-small cell lung cancer was detected by real-time fluorescence quantitative PCR, and the survival of patients was followed up to analyze the relationship between telomere DNA length of peripheral blood leukocytes and tumor pathological type and prognosis. Results The telomere DNA length (T／s) (1. 98±0. 69) of adenocarcinoma group was larger than that of other non-small cell lung cancer group (1. 43±0. 67),P＝0. 007; there was no significant difference in the telomere DNA length of peripheral blood leukocytes in patients with different TNM stages. According to the length of telomere DNA in peripheral blood, the average survival time of short telomere group was 17. 149 months(95%CI＝14. 696～19. 602),which was significantly lower than that of long telomere group (30. 857 months,95%CI＝28. 515～33. 199),the difference was statistically significant (P＝0. 001); Cox proportional risk regression analysis showed that: telomere DNA length (P＝0. 041), tumor stage (P＝0. 007) were independent risk factors for prognosis of patients. Conclusion Telomere DNA length of peripheral blood leukocytes in patients with lung adenocarcinoma is longer than that of other non-small cell lung cancer, and TNM stage and telomere DNA length of peripheral blood leukocytes are independent risk factors for prognosis of non-small cell lung cancer.

A resonance Rayleigh scattering sensor for sensitive differentiation of telomere DNA length and monitoring special motifs (G-quadruplex and i-motif) based on the Ag nanoclusters and NAND logic gate responding to chemical input signals.

BACKGROUND: Differentiation of telomere length is of vital importance because telomere length is closely related with several deadly diseases such as cancer. Additionally, G-quadruplex and i-motif formation in telomeric DNA have been shown to act as a negative regulator of telomere elongation by telomerase in vivo and are considered as an attractive drug target for cancer chemotherapy. RESULTS: In this assay, Ag nanoclusters templated by hyperbranched polyethyleneimine (PEI-Ag NCs) are designed as a new novel resonance Rayleigh scattering (RRS) probe for sensitive differentiation of telomere length and monitoring special motifs (G-quadruplex and i-motif). In this assay, free PEI-Ag NC probe or DNA sequence alone emits low intensities of RRS, while the formation of PEI-Ag NCs/DNA complexes yields greatly enhanced RRS signals; however, when PEI-Ag NCs react with G-quadruplex or i-motif, the intensities of RRS exhibit slight changes. At the same concentration, the enhancement of RRS signal is directly proportional to the length of telomere, and the sensitivity of 64 bases is the highest with the linear range of 0.3-50 nM (limit of detection 0.12 nM). On the other hand, due to the conversion of telomere DNA molecules among multiple surrounding conditions, a DNA logic gate is developed on the basis of two chemical input signals (K+ and H+) and a change in RRS intensity as the output signal. CONCLUSION: Our results indicate that PEI-Ag NCs can serve as a novel RRS probe to identify DNA length and monitor G-quadruplex/i-motif through the different increasing degrees of RRS intensity. Meanwhile, the novel attributes of the nanoprobe stand superior to those involving dyes or labeled DNA because of no chemical modification, low cost, green, and high efficiency.

A colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA based on silver nanoclusters and unmodified gold nanoparticles.

Human telomere DNA plays a vital role in genome integrity control and carcinogenesis as an indication for extensive cell proliferation. Herein, silver nanoclusters (Ag NCs) templated by polymer and unmodified gold nanoparticles (Au NPs) are designed as a new colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA. Ag NCs can produce the aggregation of Au NPs, so the color of Au NPs changes to blue and the absorption peak moves to 700nm. While the telomere DNA can protect Au NPs from aggregation, the color turns to red again and the absorption band blue shift. Benefiting from the obvious color change, we can differentiate the length of telomere DNA by naked eyes. As the length of telomere DNA is longer, the variation of color becomes more noticeable. The detection limits of telomere DNA containing 10, 22, 40, 64 bases are estimated to be 1.41, 1.21, 0.23 and 0.22nM, respectively. On the other hand, when telomere DNA forms G-quadruplex in the presence of K+, or dsDNA with complementary sequence, both G-quadruplex and dsDNA can protect Au NPs better than the unfolded telomere DNA. Hence, a new colorimetric platform for monitoring structure conversion of DNA is established by Ag NCs-Au NPs system, and to prove this type of application, a selective K+ sensor is developed.

Cyclic perylene diimide: Selective ligand for tetraplex DNA binding over double stranded DNA.

Synthesized cyclic perylene diimide, cPDI, showed the binding constant of 6.3 × 106 M-1 with binding number of n = 2 with TA-core as a tetraplex DNA in 50 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl using Schatchard analysis and showed a higher preference for tetraplex DNA than for double stranded DNA with over 103 times. CD spectra showed that TA-core induced its antiparallel conformation upon addition of cPDI in the absence or presence of K+ or Na+ ions. The cPDI inhibits the telomerase activity with IC50 of 0.3 µM using TRAP assay which is potential anti-cancer drug with low side effect.

Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand.

To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (106M-1 order) with diminished binding to dsDNA (104M-1 order) in 100mM AcOH-AcOK buffer (pH 5.5) containing 100mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K=3.4×106M-1 and a 2:1 stoichiometry) than dsDNA (K=7.5×104M-1) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.4µM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.

p53 binds human telomeric G-quadruplex in vitro.

The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe.

Comparative binding of antitumor drugs to DNA containing the telomere repeat sequence

Telomeres are the ends of the linear chromosomes of eukaryotes and consist of tandem GT-rich repeats in telomere sequence i.e. 500-3000 repeats of 5'-TTAGGG-3' in human somatic cells, which are shortened gradually with age. The G-rich overhang of telomere sequence can adopt different intramolecular fold-backs and tetra-stranded DNA structures, in vitro, which inhibit telomerase activity. In this report, DNA binding agents to telomere sequence were studied novel therapeutic possibility to destabilize telomeric DNA sequences. Oligonucleotides containing the guanine repeats in human telomere sequence were synthesized and used for screening potential antitumor drugs. Telomeric DNA sequence was characterized using spectral measurements and CD spectroscopy. CD spectrum indicated that the double-stranded telomeric DNA is in a right-handed conformation. Polyacrylamide gel electrophoresis was performed for binding behaviors of antitumor compounds with telomeric DNA sequence. Drugs interacted with DNA sequence caused changes in the electrophoretic mobility and band intensity of the gels. Depending on the binding mode of the anticancer drugs, telomeric DNA sequence was differently recognized and the efficiency of cleavage of DNA varies in the bleomycin-treated samples under different conditions. DNA cleavage occurred at about 1% by the increments of 1 mM bleomycin-Fe(III). These results imply that the stability of human telomere sequence is important in conjunction with the cancer treatment and aging process.