Visualizing RNA is critical for understanding RNA expression patterns and spatial organization within cells, offering valuable insights into gene regulation and cellular functions. High-resolution RNA imaging techniques are therefore indispensable for revealing the complexities of cellular pathways and physiological processes. Traditional RNA imaging methods, however, face significant limitations, such as high background noise resulting from labeling or cell fixation, which can impede the accurate tracking of RNA dynamics in live cells. Fluorescent light-up RNA aptamers (FLAPs) have emerged as a powerful alternative, distinguished by their capacity for enhanced fluorescence activation, reduced background interference, and advantages such as label-free imaging, small molecular size, and customizable structures. In this review, we provide an overview of the development of FLAPs, explore recent advancements in FLAP-based RNA imaging strategies, and discuss both the challenges and future directions in the field. Through this analysis, we aim to facilitate the further development and application of FLAPs in RNA research, fostering innovation and offering new perspectives in the study of RNA biology.

X-ray imaging technologies have developed rapidly in bioimaging due to their unparalleled qualities such as nano-spatial resolution, three-dimensional panoramic observation, and element specificity, making up for the shortcomings of the traditional optical, electron, and super-resolution microscope. However, it has insufficient signal for specific imaging of cell biomolecules or structures. Hence, it is increasingly crucial to develop X-ray-sensitive probes. The targeted signal can be enhanced by integrating high-Z elements in probes to exploit the differential absorption and phase contrast with cellular components, leveraging the advantages of X-ray microscopy. In this review, we focus on the recent progress of X-ray probe-based imaging approaches from molecules to organ scales. We assess the advantages and limitations faced by these probes in X-ray imaging. Some future demands and improvements were suggested for further development of X-ray probes. These advancements are expected to inspire innovative bioimaging tools and new discoveries in cell biology and precise medicine.

N6-Methyladenosine (m6A) stands out as the predominant internal modification in mammalian RNA, exerting crucial regulatory functions in the metabolism of mRNA. Currently available methods have been limited by an inability to quantify m6A modification at precise sites. In this work, we screened a Bst 2.0 warm start DNA polymerase with the capability of discriminating m6A from adenosine (A) and developed a robust m6A RNA detection method that enables isothermal and ultrasensitive quantification of m6A RNA at single-base resolution. The detection limit of the assay could reach about 0.02 amol, and the quantitative accuracy of the assay was verified in real cell samples. Furthermore, we applied this assay to single-cell analysis and found that the coefficients of variation of the MALAT1 m6A 2611 site in glioblastoma U251 cells showed over 20% higher than in oligodendrocytes MO3.13 cells. This method provides a highly sensitive analytical tool for site-specific m6A detection and quantification, which is expected to provide a basis for precise disease diagnosis and epigenetic transcriptional regulation.

Gliomas are histologically and genetically heterogeneous tumors. However, classical histopathological typing often ignores the high heterogeneity of tumors and thus cannot meet the requirements of precise pathological diagnosis. Here, proximity-anchored in situ spectral coding amplification (ProxISCA) is proposed for multiplexed imaging of RNA mutations, enabling visual typing of brain gliomas with different pathological grades at the single-cell and tissue levels. The ligation-based padlock probe can discriminate one-nucleotide variations, and the design of proximity primers enables the anchoring of amplicons on target RNA, thus improving localization accuracy. The DNA module-based spectral coding strategy can dramatically improve the multiplexing capacity for imaging RNA mutations through one-time labelling, with low cost and simple operation. One-target-one-amplicon amplification confers ProxISCA the ability to quantify RNA mutation copy number with single-molecule resolution. Based on this approach, it is found that gliomas with higher malignant grades express more genes with high correlation at the cellular and tissue levels and show greater cellular heterogeneity. ProxISCA provides a tool for glioma research and precise diagnosis, which can reveal the relationship between cellular heterogeneity and glioma occurrence or development and assist in pathological prognosis.

The RNA contained in exosomes plays a crucial role in information transfer between cells in various life activities. The accurate detection of low-abundance exosome RNA (exRNA) is of great significance for cell function studies and the early diagnosis of diseases. However, their intrinsic properties, such as their short length and high sequence homology, represent great challenges for exRNA detection. In this paper, we developed a dual-signal isothermal amplification method based on rolling circle amplification (RCA) coupled with DNAzyme (RCA-DNAzyme). The sensitive detection of low-abundance exRNA, the specific recognition of their targets and the amplification of the detection signal were studied and explored. By designing padlock probes to specifically bind to the target exRNA, while relying on the ligation reaction to enhance recognition, the precise targeting of exosome RNA was realized. The combination of RCA and DNAzyme could achieve a twice-as-large isothermal amplification of the signal compared to RCA alone. This RCA-DNAzyme assay could sensitively detect a target exRNA at a concentration as low as 527 fM and could effectively distinguish the target from other miRNA sequences. In addition, this technology was successfully proven to be effective for the quantitative detection of miR-21 by spike recovery, providing a new research approach for the accurate detection of low-abundance exRNA and the exploration of unknown exRNA functions.

Differential expression of RNA splice variants among individual cells accounts for cell heterogeneity of gene expression, which plays a key role in the regulation of the immune system. However, currently available techniques face difficulties in achieving single-cell analysis of RNA splice variants with high base resolution, high spatial resolution and accurate quantification. Herein, we constructed DNA-templated dual-functional nanocluster probes to achieve in situ imaging and accurate quantification of RNA splice variants at the single-cell level. By designing ultrasmall nanocluster labeled probes to directly target the splicing junction sequence of RNA splice variants, the base recognition resolution is significantly improved. Benefit from the controllable fluorescence of nanoclusters, in situ imaging and genotyping of RNA splice variants are achieved. Due to the atom-precise nanocluster, RNA splice variants can be accurately quantified by laser ablation inductively coupled plasma mass spectrometry at the single-cell level. We further applied the probes to explore the function of MyD88 splice variants in mononuclear macrophages under immune activation. This strategy provides a novel single-cell analysis tool for studying the functional diversity of the immune system and splicing-related immune diseases.