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The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, has been shown to adopt higher-order structures, specifically G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition through chromosome maintenance and manipulation of oncogene expression featuring their G-rich promoter regions. Besides higher order structures, several regulatory roles are attributed to DNA epigenetic markers. In this work, we investigated how the structural dynamics of a G-quadruplex, formed by the telomeric sequence, is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)_ntelomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)₄, (IGG)₄, (GIG)₄, (GGI)₄, (IGI)₄, (IIG)₄, (GII)_4,and (III)₄, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na⁺and K⁺containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)₄and the terminal modified DNAs (IGG)₄and (GGI)₄in both Na⁺and K⁺containing buffers. Specifically, quadruplex hybrid was observed for (GGG)₄in K⁺buffer. Among the other modified sequences, (GIG)₄, (IGI)₄and (GII)₄show parallel features, while (IIG)₄and (III)₄show no detectable conformation in the presence of either Na⁺or K⁺. Our studies indicate that terminal lesions (IGG)₄and (GGI)₄may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)₄in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K⁺cation confers more thermodynamic stability compared to Na⁺. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with position dependent G-to-I mutations in different ion conditions. 

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.