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1. Quantitative, super-resolution localization of small RNAs with sRNA-PAINT

   https://doi.org/10.1093/nar/gkaa623  

   Huang, Kun; Demirci, Feray; Batish, Mona; Treible, Wayne; Meyers, Blake C; Caplan, Jeffrey L (July 2020, Nucleic Acids Research)

   null (Ed.)

   Abstract Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and ∼10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called ‘Vetting & Analysis of RNA for in situ Hybridization probes’ (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction. 

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2. Single-molecule fluorescence detection of a tricyclic nucleoside analogue

   https://doi.org/10.1039/D0SC03903A  

   Samaan, George N.; Wyllie, Mckenzie K.; Cizmic, Julian M.; Needham, Lisa-Maria; Nobis, David; Ngo, Katrina; Andersen, Susan; Magennis, Steven W.; Lee, Steven F.; Purse, Byron W. (February 2021, Chemical Science)

   null (Ed.)

   Fluorescent nucleobase surrogates capable of Watson–Crick hydrogen bonding are essential probes of nucleic acid structure and dynamics, but their limited brightness and short absorption and emission wavelengths have rendered them unsuitable for single-molecule detection. Aiming to improve on these properties, we designed a new tricyclic pyrimidine nucleoside analogue with a push–pull conjugated system and synthesized it in seven sequential steps. The resulting C -linked 8-(diethylamino)benzo[ b ][1,8]naphthyridin-2(1 H )-one nucleoside, which we name ABN, exhibits ε 442 = 20 000 M −1 cm −1 and Φ em,540 = 0.39 in water, increasing to Φ em = 0.50–0.53 when base paired with adenine in duplex DNA oligonucleotides. Single-molecule fluorescence measurements of ABN using both one-photon and two-photon excitation demonstrate its excellent photostability and indicate that the nucleoside is present to > 95% in a bright state with count rates of at least 15 kHz per molecule. This new fluorescent nucleobase analogue, which, in duplex DNA, is the brightest and most red-shifted known, is the first to offer robust and accessible single-molecule fluorescence detection capabilities. 

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3. Detection of MicroRNA Expression Dynamics Using LNA/DNA Nanobiosensor

   Yuwen Zhao, Shue Wang (January 2023, Methods in molecular biology)

   The investigation of complex biological processes requires effective tools for probing the spatiotemporal dynamics of individual cells. Single-cell gene expression analysis, such as RNA in situ hybridization and single-cell PCR, has been demonstrated in various biological applications (Tautz and Pfeifle, Chromosoma 98(2):81–5, 1989; Stahlberg and Bengtsson, Methods 50(4):282–288, 2010; Sanchez-Freire et al., Nat Protoc 7(5):829–838, 2012). However, existing techniques require cell lysis or fixation. The dynamic information and spatiotemporal regulation of the biological process cannot be obtained with these methods. Real-time gene expression analysis in living cells remains an outstanding challenge in the field. Here, we described a single-cell gene expression analysis method in living mammalian cells using a locked nucleic acid/DNA (LNA/DNA) nanobiosensor. This LNA/DNA nanobiosensor consists of a fluorophore-labeled detecting strand and a quenching strand. The fluorophore-labeled LNA probe is designed to hybridize with the target microRNA (miRNA) specifically and displace from the quenching strand, allowing the fluorophore to fluorescence. Large-scale single-cell dynamic gene expression monitoring can be performed using time-lapse microscopy to study spatiotemporal distribution and heterogeneity in gene expression. Multiplex detection of miRNAs can be achieved using different fluorophore-labeled LNA/DNA nanobiosensors. This LNA/DNA protocol is fast, generally applicable, and easily accessible. 

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4. Fluorescent Platforms for RNA Chemical Biology Research

   https://doi.org/10.3390/genes13081348  

   Du, Jinxi; Dartawan, Ricky; Rice, William; Gao, Forrest; Zhou, Joseph H.; Sheng, Jia (August 2022, Genes)

   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. 

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5. Magic-angle spinning NMR spectroscopy provides insight into the impact of small molecule uptake by G-quartet hydrogels

   https://doi.org/10.1039/D0MA00475H  

   Manjunatha Reddy, G. N.; Peters, Gretchen; Tatman, Ben; Rajan, Teena S.; Kock, Si Min; Zhang, Jing; Frenguelli, Bruno G.; Davis, Jeffery; Marsh, Andrew; Brown, Steven P. (January 2020, Materials Advances)

   Small molecule guests influence the functional properties of supramolecular hydrogels. Molecular-level understanding of such sol-gel compositions and structures is challenging due to the lack of long-range order and inherently heterogeneous sol-gel interface. In this study, insight into the uptake process of biologically relevant small molecules into guanosine-quartet(G4) borate hydrogels is obtained by gel-state magic-angle spinning (MAS) NMR spectroscopy. G4∙K + borate hydrogel can absorb up to 0.3 equivalent of cationic methylene blue (MB) without a significant disruption of the G4 fibrils that make up the gel, whereas the addition of over 0.3 equivalents of MB to the same gel leads to a gel-to-sol transition. The gel-to-sol transition process is characterized ex situ by analyzing and comparing the 1 H and 11 B MAS NMR spectra acquired before and after the MB uptake. In particular, 11 B isotropic chemical shifts and quadrupole interactions were determined by analyzing the 11 B MAS NMR spectra acquired at different magnetic fields, 11.7 T, 14.1 T and 20 T, which enable the different local bonding environments of borate anions in sol- and gel domains to be distinguished and identified. By comparison, uptake of heterocyclic molecules such as adenine, cytosine and 1-methylthymine into G4∙Na + borate hydrogels lead to stiff and clear gels while increasing the solubility of the nucleobases as compared to the solubility of the same compounds in water. G4∙Na + gel can uptake one equiv. of adenine with minimal disruption to the sol-gel framework, thus enhancing the adenine solubility up to an order of magnitude as compared to water. Combined multinuclear ( 1 H, 11 B and 23 Na) NMR spectroscopy analysis and vial inversion tests revealed that the nucleobases are embedded into pores of the sol phase rather than being closely interacting with the G-4 fibrils that make up the gel phase. These results indicate that G-4 hydrogels have potential applications as carrier systems for small molecules. Gel-state MAS NMR spectroscopy can be used to gain insight into host-guest interactions in complex heterogeneous sol-gel systems, which is often difficult to obtain from the conventional techniques such as X-ray scattering, electron microscopy and optical spectroscopy. 

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