High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules

Abstract

Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.

SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution

“…Nevertheless, the low signal-to-noise ratio still remains a major technical challenge of these methods, especially for tissue sections with strong autofluorescence from structural extracellular matrix components like collagen and elastin [ 15 ]. New strategies for signal amplifications, such as branched-DNA amplification (RNAScope) [ 16 ] and hybridization chain reaction [ 17 , 18 ], have been recently combined with sophisticated probe design (high-fidelity amplified FISH [AmpFISH]) [ 19 ] to increase sensitivity and specificity of smFISH.…”

Methods for spatial and temporal imaging of the different steps involved in RNA processing at single‐molecule resolution

“…In another modification, called ClampFISH, a number of hybridization chain reaction (HCR) based modifications of Stellaris probes, along with a variety of click chemistries, have also been proposed to enhance signal strength (Rouhanifard et al, 2018). Another method of HCR‐dependent signal amplification based on Stellaris probes provides much higher signal strength, with as little as one probe, which is able to detect single‐nucleotide changes (Marras, Bushkin, & Tyagi, 2019). The use of techniques such as CLARITY (Shah et al, 2016) and expansion microscopy, known as ExFISH (Asano et al, 2018; Chen et al, 2016; Wang, Moffitt, & Zhuang, 2018), in conjunction with single‐molecule FISH are opening up new avenues for the generation of clean signals from tissue sections hybridized with FISH probes.…”

“…However, the quantum yield of the currently available fluorophores used to label Stellaris probes is low, and a large number of fluorophores often need to be accumulated at one site to enable the generation of a distinct, diffraction-limited signal. This drawback is now being overcome by signal amplification through the use of hybridization chain reactions, in addition to various adaptations of the basic method (Marras et al, 2019). These modifications not only enable the imaging of shorter RNAs, but also allow the detection of single-nucleotide polymorphism in target RNAs.…”

Comparison of Bias and Resolvability in Single-Cell and Single-Transcript Methods

“…6a-g). HCR offers several potential advantages over FISH such as background suppression, amplifiable signal, and the ability to discern single nucleotide variants 20,31 . Because HCR had not previously been used for transcript-counting in bacteria, we first optimized several aspects of the HCR sample preparation protocol, including starting cell culture volume, permeabilization agent, permeabilization temperature, permeabilization time, and amplification time (Supplementary Note).…”