Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and non-specific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400x) signal amplification. ClampFISH probes form a “C” configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bioorthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.

Cellular plasticity describes cells’ ability to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR/Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a novel, plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In the context of cancer therapeutics, plasticity refers to transient fluctuations in the molecular state of tumor cells, driving the formation of rare cells primed to survive drug treatment and ultimately reprogram into a stably resistant fate. However, the biological processes governing this cellular plasticity remain unknown. We used CRISPR/Cas9 genetic screens to reveal genes that affect cell fate decisions by altering cellular plasticity across a range of functional categories. We found that cellular plasticity and cell fate decision making can be decoupled in that factors can affect cell fate decisions in both plasticity-dependent and independent manners. We discovered a novel mode of altering resistance based on cellular plasticity that, contrary to known mechanisms, pushes cells towards a more differentiated state. We further confirmed our prediction that manipulating cellular plasticity before the addition of the main therapy would result in changes in therapy resistance more than concurrent administration. Together, our results indicate that identifying pathways modulating cellular plasticity has the potential to alter cell fate decisions and may provide a new avenue for treating drug resistance.

Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes' transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.

Abstract:Non-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present a new method (click-amplifying FISH; clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that clampFISH is multiplexable, compatible with expansion microscopy, and works in tissue samples. Main text:Single molecule RNA fluorescence in situ hybridization (RNA FISH), which enables the direct detection of individual RNA molecules 1,2,3 , has emerged as a powerful technique for measuring both RNA abundance and localization in single cells. Yet, while single molecule RNA FISH is simple and robust, the total signal generated by single molecule RNA FISH probes is relatively low, thus requiring high-magnification microscopy for detection. This keeps the assay relatively low throughput and precludes the ability to combine it with flow cytometry. As such, high efficiency, high gain amplification methods for single molecule RNA FISH signal could enable a host of new applications for RNA FISH.A number of different signal amplification techniques are available for RNA FISH, but each suffers from particular limitations. Approaches such as tyramide signal amplification (TSA) 4 , or enzyme ligated fluorescence (ELF) 5 utilize enzymes to catalyze the deposition of fluorescent substrates near the probes. Alternatively, enzymes can catalyze a "rolling circle" nucleic acid amplification to generate a repeating sequence that can subsequently detected using fluorescent probes [6][7][8] . These methods can lead to large gains in fluorescence, but can suffer from poor sensitivity because of the difficulties in getting bulky enzymes through the fixed cellular environment to the target molecule. Meanwhile, there are a number of non-enzymatic amplification methods, most notably the hybridization chain reaction 9-11 and branched DNA methods [12][13][14] . These methods rely only on hybridization to amplify signal by creating larger DNA scaffolds to which fluorescent probes can attach. However, these methods have an inherent tradeoff between stringency of hybridization and wash conditions for specificity and maintaining hybridization to increase signal gain. Thus, our goal was to create a non-enzymatic, exponential . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/222794 doi: bioRxiv preprint first posted online Nov. 21, 2017; amplification scheme with high sensitivity (detection efficiency), gain (signal amplification), and specificity (low background).We first designed probes that would bind with high specificity and sensitivity; specifically, we ...