Supplementary information S2 (Table ... - · PDF fileMOLECULAR CELL BIOLOGY Supplementary ......

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Supplementary information S2 (Table) | Visualizing single mRNAs in fixed and live cells

Method:

Summary: Delivery Advantages: Disadvantages: Ref:

Protein-RNA interactions

Phage coat proteins

(MCP and PCP, λN-

boxB)

Multiple binding sites of phage coat

proteins are cloned to the mRNA of

choice (usually at the 3’UTR). The

coat proteins are fused to fluorescent

proteins. See figure 2a, b.

Genetically

encoded

Enables live imaging. Requires the expression of the coat

protein fused to a fluorescent protein

and may require exogenous mRNA

expression. The addition of multiple

coat protein binding sites to the

mRNA may affect its behaviour.

1-3

Organic dye labelling of

MCP-bound mRNAs

Multiple binding sites of coat

proteins are cloned to the mRNA of

choice (usually at the 3’UTR). The

coat proteins are fused to

Escherichia coli dihydrofolate

reductase (DHFR) or Snap tag

proteins, which can bind small-

molecule fluorophores.

Genetically

encoded &

membrane

permeable

dye

Enables live imaging. May be

brighter than fluorescent

proteins. Enhanced signal to

noise ratio.

Requires the expression of the coat

protein fused to DHFR or Snap tag

and may require exogenous mRNA

expression.

The addition of multiple coat protein

binding sites to the mRNA may

affect its behaviour.

4

Human U1A Multiple binding sites of U1A are

cloned to the mRNA of choice

(usually at the 3’UTR). U1A is

fused to fluorescent proteins.

Genetically

encoded

Enables live imaging. Requires expression of the U1A

protein fused to a fluorescent

protein.

Cannot be used in mammalian

systems.

The addition of multiple U1A



5

Bimolecular

fluorescence

complementation of

MS2 and PP7 with a

split fluorescent protein

Multiple alternate MS2 and PP7

binding sites are cloned to the

mRNA of choice (usually at the

3’UTR). Coat proteins from both

systems, each fused to half of a

Genetically

encoded

Enables live imaging,

Free of fluorescent

background.

May require exogenous mRNA

expression.

The addition of multiple coat protein



6



fluorescent protein, are expressed.

When the two coat proteins bind the

fluorescent protein is made whole

and fluoresces. See figure 2c.

Requires the coordinated expression

of two RBPs.

Pumilio homology

domain (PUM-HD)

Two RNA binding domains of

human Pumilio1, each genetically

modified to recognize a specific

target sequence, are fused to half of

a GFP. When the two domains bind

the mRNA, GFP is made whole and

fluoresces.

Genetically

encoded

Enables live imaging.

Target mRNA remains un-

altered.

Binding of only one GFP molecule

per mRNA.

Requires the coordinated expression

of two RBPs.

Requires total internal

reflection fluorescence (TIRF)

imaging.

7

DNA oligonucleotides

Tiled oligos (Biosearch) Multiple, specific, fluorescently

tagged antisense DNA

oligonucleotides that hybridize

along an mRNA of interest. See

supplementary information S1a,b.

DNA

oligos

permeate

fixed cells.

High signal to background

ratio.

Low false-positive signal.

Enables the study of

endogenous mRNAs.

Enables colour barcoding for

multiple mRNA analysis.

Cells are fixed: lacks information on

single cell and single molecule

dynamics.

8, 9

Branched DNA (bDNA)

(Affymetrix)

RNAScope (Advanced

Cell Diagnostics)

The mRNA is hybridized to two

target-specific, adjacent, non-

fluorescent probes with overhangs.

These primary probes are then

hybridized to secondary probes,

which only hybridize if both primary

probes are bound, which in turn are

hybridized to tertiary and then to

quaternary, fluorescent probes. See

supplementary information S1d.

DNA

oligos

permeate

fixed cells.


ratio.

Low false-positive signal.


endogenous mRNAs. High-

throughput.

Strong (bright) signal.

Applicable to miRNAs.

Can be detected and

quantified by flow cytometry.



dynamics.

Requires multiple rounds of

hybridization.

10-12

Fluorescence In Situ

Hybridization with

The mRNA is hybridized to one

target-specific, non-fluorescent

DNA

oligos


ratio.



13



Sequential Tethered and

Intertwined ODN

Complexes (FISH-

STICs)

probe with a unique overhang. This

primary probe is then hybridized to

secondary probes, which in turn are

hybridized to tertiary fluorescent

probes. See supplementary

information S1c.

permeate

fixed cells


endogenous mRNAs.

Affordable.

dynamics.

Requires multiple rounds of

hybridization.

Possibility of false-positive signals.

Hairpin nucleic acid probes

Molecular beacons;

Multiply labeled

tetravalent RNA

imaging probes

(MTRIPs); 2' O-Methyl

RNA probes

Hairpin structured-antisense probes

are labelled with a fluorophore and a

quencher at each end, juxtaposed by

the hairpin. Hybridization to the

target mRNA opens the hairpin and

separates the fluorophore from the

quencher, thereby allowing a

fluorescence signal to be emitted

upon excitation.

RNA

oligos

permeate

fixed cells

Study of endogenous mRNAs.

Reversible binding.

High cellular accessibility.

Possibility of false-positive signals.

Accumulation of nuclear signals

generates high nuclear background,

thus masking data on transcription

sites and nuclear RNA.

Many unique beacons are needed to

visualize a single mRNA.

Cyanine-conjugated probes

accumulate in mitochondria14

and

generate high cytoplasmic

background.

14, 15-17

Aptamer RNA labels

Spinach, Spinach2 Long (98nt) RNA aptamers that bind

and activate small fluorophores.

Genetically

encoded


Does not require expression

of an RNA binding protein.

Requires optimization for correct

folding of aptamer. So far, was not

demonstrated for single-molecule

imaging. Requires the addition of

exogenous fluorogenic compounds.

18, 19

RNA-Mango RNA aptamers that bind and activate

small fluorophores.

Genetically

encoded


Does not require the

expression of an RNA binding

protein. Shorter sequence than

Spinach (39nt). Aptamer-dye

affinity higher than for

Spinach.

Requires optimization for correct

folding of aptamer.

Requires the addition of exogenous

fluorogenic compounds.

20

RNA Microinjection



RNA microinjection Fluorescently tagged RNAs are

injected into a cell of interest.

microinject

ion


Use of bright, stable

fluorophores.

Enables the visualization of

miRNA.

Amount of injected RNA may

exceed physiological levels,

resulting in aberrant behaviour.

RNA lacks interactions with

regulatory RBPs that bind the RNA

co- or post-transcriptionally,

resulting in abnormal behaviour of

RNA.

21-23

In situ sequencing

Fluorescent in situ

sequencing (FISSEQ)

Random reverse transcription is

performed in intact cells. cDNA in

situ are amplified and sequenced.

See supplementary information S1e.

Enzymatic

reactions

and short,

fluorescentl

y labelled

non-

specific

DNA

probes.

Unbiased measure of mRNA

diversity in a cell, including of

unidentified mRNAs.

Allows visualization of only a

fraction (albeit, a large one) of the

RNA molecules in the cell

24

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3. Lange, S. et al. Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9, 1256-67 (2008).

4. Carrocci, T.J. & Hoskins, A.A. Imaging of RNAs in live cells with spectrally diverse small molecule fluorophores. Analyst 139, 44-7

(2014).

5. Brodsky, A.S. & Silver, P.A. Identifying proteins that affect mRNA localization in living cells. Methods 26, 151-5 (2002).

6. Wu, B., Chen, J. & Singer, R.H. Background free imaging of single mRNAs in live cells using split fluorescent proteins. Sci Rep 4, 3615

(2014).

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Encoded Fluorescent Probes. Analytical Chemistry 83, 5708-5714 (2011).

8. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585-90 (1998).



9. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly

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10. Battich, N., Stoeger, T. & Pelkmans, L. Image-based transcriptomics in thousands of single human cells at single-molecule resolution.

Nat Methods 10, 1127-33 (2013).

11. Taylor, A.M. et al. Axonal mRNA in uninjured and regenerating cortical mammalian axons. J Neurosci 29, 4697-707 (2009).

12. Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14, 22-9

(2012).

13. Sinnamon, J.R. & Czaplinski, K. RNA detection in situ with FISH-STICs. RNA 20, 260-6 (2014).

14. Rhee, W.J. & Bao, G. Slow non-specific accumulation of 2'-deoxy and 2'-O-methyl oligonucleotide probes at mitochondria in live cells.

Nucleic Acids Research 38 (2010).

15. Molenaar, C. et al. Linear 2 ' O-Methyl RNA probes for the visualization of RNA in living cells. Nucleic Acids Research 29, art. no.-e89

(2001).

16. Santangelo, P.J. et al. Single molecule-sensitive probes for imaging RNA in live cells. Nature Methods 6, 347-U46 (2009).

17. Simon, B., Sandhu, M. & Myhr, K.L. Live FISH: Imaging mRNA in Living Neurons. Journal of Neuroscience Research 88, 55-63

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18. Strack, R.L., Disney, M.D. & Jaffrey, S.R. A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.

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19. Paige, J.S., Wu, K.Y. & Jaffrey, S.R. RNA mimics of green fluorescent protein. Science 333, 642-6 (2011).

20. Dolgosheina, E.V. et al. RNA Mango Aptamer-Fluorophore: A Bright, High-Affinity Complex for RNA Labeling and Tracking. ACS

Chem Biol (2014).

21. Barbarese, E. et al. Conditional knockout of tumor overexpressed gene in mouse neurons affects RNA granule assembly, granule

translation, LTP and short term habituation. PLoS One 8, e69989 (2013).

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23. Tubing, F. et al. Dendritically Localized Transcripts Are Sorted into Distinct Ribonucleoprotein Particles That Display Fast Directional

Motility along Dendrites of Hippocampal Neurons. Journal of Neuroscience 30, 4160-4170 (2010).

24. Lee, J.H. et al. Highly multiplexed subcellular RNA sequencing in situ. Science 343, 1360-3 (2014).

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