ECHO-liveFISH:in vivoRNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues

Abstract

Elucidating the dynamic organization of nuclear RNA foci is important for understanding and manipulating these functional sites of gene expression in both physiological and pathological states. However, such studies have been difficult to establish in vivo as a result of the absence of suitable RNA imaging methods. Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks. Upon in vivo electroporation, exciton-controlled sequence-specific oligonucleotide probes revealed focally concentrated endogenous 28S rRNA and U3 snoRNA at nucleoli and poly(A) RNA at nuclear speckles. Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue. Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

A stem-less probe using spontaneous pairing between Cy3 and quencher for RNA detection

“…Similar localization of Cy3 emission was observed when a conventional FISH protocol involving washing procedures was used (Figure S5). The emission observed using probe YR2–2_28S coincided with the previous studies employing 28S rRNA-specific probes;[12,23,24] therefore, we concluded that the self-quenching probe detects target RNA in cells.…”

Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function

“…S2B). To have information about the spatial localization of ribosomes, we labeled the 28s ribosomal RNA of the ribosomes by using exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes (33). In the absence of magnetic localization of the mRNA-NPs, we observed a strong background noise and a few natural hotspots (SI Appendix, Fig.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Time‐resolved imaging showed that fluorescence was much more intense at the mitotic phase than at the interphase, implying that relatively large quantities of mRNA are expressed as the cell divides. Later studies have shown the capability of ECHO probes to detect other intracellular RNA targets, including 28S rRNA and U3 small nucleolar RNA …”