Quantitative mRNA Imaging with dual channel qFIT probes to Monitor

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Quantitative mRNA Imaging with dual channel qFIT probes to Monitor Distribution and Degree of Hybridization Imre Gaspar, Felix Hövelmann, Jasmine Chamiolo, Anne Ephrussi, and Oliver Seitz ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01007 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on February 1, 2018

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Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization Imre Gaspar,† Felix Hövelmann,‡ Jasmine Chamiolo,‡ Anne Ephrussi† and Oliver Seitz*,‡ †

European Molecular Biology Laboratory (EMBL) Heidelberg, 69117 Heidelberg, Germany

‡

Institut für Chemie der Humboldt-Universität zu Berlin, 12489 Berlin, Germany

Abstract. Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signalling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here we introduce qFIT (quantitative forced intercalation) probes that allow the detection both of probe-target complexes and of unbound probe on separate, independent channels. A surrogate nucleobase based on thiazole orange (TO) probes the hybridization status. The second channel involves a non-responsive near-IR dye which serves as a reporter of concentration. We show that the undesirable perturbation of the hybridization reporter TO is avoided when the near-IR dye Cy7 is connected by means of short triazole linkages in ≥ 18 nucleotides distance. We used the qFIT probes to localize and quantify oskar mRNA in fixed egg chambers of wild-type and mutant Drosophila melanogaster by wash-free fluorescence in situ hybridization. The measurements revealed a relative 400-fold enrichment of oskar within a 3000 µm3 large volume at the posterior pole of stage 8−9 oocytes, which peaked at a remarkably high 1.8 µM local concentration inside 0.075 µm3 volume units. We discuss detection limits and show that the number of oskar mRNA molecules per oocyte is independent of the oocyte size which suggests that the final levels are attained already during the onset of oskar localization at stage 8.

Introduction. The knowledge of gene expression levels is essential for our understanding of cellular processes. Numerous methods such as sequencing,1 northern blotting2 and quantitative PCR3, 4 give insight into the RNA-expression of an average of the population of the cells investigated. When spatial and temporal resolution in single cells or in tissue is needed, more advanced techniques are required. The ideal method provides quantitative information about localization and copy numbers of the mRNA target of interest. Frequently, the mRNA of interest is genetically tagged with repeats of recognition units (e.g. MS25 or λN226 hairpins) that recruit coexpressed autofluorescent fusion proteins. Because each mRNA target is loaded with tens of fluorophores, low abundance targets can be quantified by means of counting of single molecules. In a somewhat related approach, RNA aptamers that fluoresce upon binding of profluorescent dyes have been used for staining of mRNA in bacterial cells and miRNA in eukaryotic cells; however, applying the method for imaging eukaryotic mRNA has proven challenging.7-10 Autofluorescent protein fusions with RNA-binding proteins based ACS Paragon Plus Environment

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on PUMILIO proteins11 or the CRISPR system12 avoid the need for RNA-based tag sequences. Though successful in many instances, the approaches call for genetic modification of cells and background from unbound fluorescent protein fusions can be high. For imaging of mRNA in non-modified wild-type cells, in situ hybridization with fluorescent oligonucleotide probes usually is the method of choice. Single-molecule fluorescence in situ hybridization (smFISH) is a powerful method that involves the use of tens of labelled oligonucleotide probes designed to bind to various regions of the mRNA target.13 Prior to analysis of fixed cells unbound probes must be removed by washing. Because each mRNA target is loaded with tens of fluorophores, low abundance targets can be quantified by means of counting of single molecules. The use of the hybridization chain reaction provides single molecule sensitivity in mRNA imaging with a set of two oligonucleotides.14, 15 Fluorogenic hybridization probes have been developed with the aim to speed up analysis times by enabling wash-free FISH.16-34 In addition, fluorogenic hybridization probes allow RNA imaging of live cells where separation of unbound from bound probes is impossible. Probes such as molecular beacons,18-20 FIT probes,21-24 binary probes,25-27 ECHO probes,28,

29

reactive probes30-34 and

others16, 17 exhibit enhancements of fluorescence upon sequence-specific hybridization owing to changes in i) the distance between a fluorophore and fluorescence quencher or ii) the environment of locally responsive fluorophores. Fluorescence-activating hybridization probes face a common challenge: differences in efficiency of delivery, in subcellular localization and nuclear sequestration, and false positive signalling can have dramatic effect on the fluorescence background in areas of low target concentration.22, 35 This poses a challenge to quantitative measurements because any fluorescence signal can be caused either by a low number of target bound probes or a high number of unbound probes. It is for this reason that the existing methods for counting RNA copy numbers typically involve wash steps or require repeat tags to increase contrast. We assumed that quantitative measurements of mRNA concentration should be feasible when self-reporting probes allow not only the detection of probe-target-complexes, but also of unbound probe on a separate, independent channel. Such a multichannel system has been introduced by Tsourkas and co-workers, who used 65 nucleotides long ratiometric bimolecular beacons (RBMBs) involving an extended, siRNA-like, double-stranded sequence and three labels to quantitatively assess the expression of engineered mRNA.36-38 Quantitative imaging of wildtype cells has not been demonstrated. We developed the so-called forced intercalation (FIT) probes that make use of a single, environmentally sensitive fluorophore of the thiazole orange (TO) family of dyes (Figure 1A).23, 39 We have demonstrated that FIT probes allow qualitative imaging of RNA in non-engineered cells.40-44 In FIT probes, the dye substitutes for a canonical nucleobase. The fluorescence emission remains low in absence of target because twisting around the methine bridge rapidly depletes the TO excited state.45 Twisting is hindered in the target-bound form, when hybridization enforces intercalation of the TO dye. As a result, FIT probes furnish marked increases of fluorescence upon hybridization. Owing to the requirement for intercalation, FIT probes have low vulnerability



to false positive signalling by nuclease degradation or protein binding.22 We reported methods to improve upon the hybridization-induced fluorescence enhancement and the brightness in the target-bound state.41, 42, 46 However, with signalling based on intensity changes it was impossible to distinguish a high concentration of unbound probes from a low concentration of target-bound probe.

Figure 1. A) FIT probes signal binding of complementary RNA by enhancement of fluorescence emitted from a thiazole orange (TO) nucleotide. B) qFIT probes provide information about concentration and hybridization status by means of two detection channels which include the environmentally sensitive TO dye and a hybridization independent near-infrared dye such as Cy7.

In this study, we introduce DNA qFIT-probes for quantitative RNA imaging (Figure 1B). The probes combine an environmentally sensitive TO nucleotide with a second, near-IR dye in a way that prevents fluorescence responses of the near-IR dye and leaves the brightness of the TO-nucleotide unaffected. While the TO nucleotide serves as a reporter of hybridization the non-responsive near-IR dye provides information about probe concentration. To demonstrate quantitative mRNA imaging in complete tissue we studied localization and determined the concentration of oskar-mRNA in specific compartments of Drosophila melanogaster oocytes.

RESULTS AND DISCUSSION Design and Synthesis. In an ideal scenario, the two reporter dyes in qFIT probes should function as independent modules. The hybridization reporter should be dark in absence of target and emit bright fluorescence upon binding of the RNA target. It is therefore desirable to minimize quenching of TO emission by the concentration reporter. This calls for a near-IR dye which minimizes spectral overlap between its absorbance and the emission of the TO dye. We furthermore sought for a means to prevent ground state interactions between the ACS Paragon Plus Environment

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concentration reporter and TO. Unintentionally, this could make the fluorescence properties of the concentration reporter susceptible to changes of the probe’s hybridization status. We envisioned that a short tether and a net positively charged concentration reporter would help avoid contact with the positively charged TO dye and based on the ease of synthesis and functionalization chose the cyanine-7 (Cy7) dye. The NIR dye was introduced to DNA FITprobes by means of alkyne-azide click chemistry.47, 48 Oligonucleotide synthesis was carried out according to previously reported methods.22,

41

A serinol-TO phosphoramidite24 was

introduced at three different positions (nt 6, 12 and 18) of a 24mer DNA-sequence which targets oskar-RNA in Drosophila melanogaster (Bp. 83−106 of CDS). The same segment was previously targeted with molecular beacon by Tyagi et al. for in vivo studies.49 The alkyne was introduced at the 5’ end by using butyne-1-ol-phosphoramidite 2 for copper catalysed click reaction. The conjugation of Cy7 azide 1 proceeded smoothly in nearly quantitative yield by applying a procedure developed by Brown et al. (see Figures S2 and S3 for HPLC-traces).50

A)

1 N

N

N3

I OCH2CH2CN P NiPr2 O 2

B)

O O

P

O FIT-DNA

O

Bu-FIT DNA

a) Cy7 N

N N

O O

P

O O

FIT-DNA

Cy7-FIT DNA

Figure 2. A) Building blocks used for the synthesis of qFIT probes. B) Cy7 is introduced via Cu-based click reaction with Cy7-N3 (1). Conditions: a) 100 µM Bu-FIT DNA, 500 µM 1, 7.5 mM sodium ascorbate, 3.75 mM THPTA, 0.75 mM copper(II)sulfate in buffer (100 mM TRIS, 100 mM NaOAc, 1 mM MgCl2, pH 8), 3 h, 55 °C, 40-60% overall yield.

Fluorescence with synthetic RNA. A comparison of fluorescence spectra obtained in absence and presence of synthetic RNA target showed that, upon hybridization, all tested FIT-probes provide enhancements of TO emission at 535 nm (Table 1). UV melt analyses revealed little, if any, differences in the thermal stability of the probe-target duplexes. We concluded that any change in fluorescence properties would solely be due to the type of Cy7 attachment and the Cy7-TO distance rather than by differential hybridization properties. The fluorescence response I/I0 furnished by qFIT-probes was higher than the response by TO-only FIT-probes provided that the TO-Cy7 distance exceeded 6 nucleotides (Table 1, see Figure S4 for UV and fluorescence spectra of all probes). For example, the TO-only probe osk1c showed a rather modest 5-fold intensification of fluorescence (Fig. 3A), which improved to 15-fold upon introduction of the 5’-terminal Cy7 in osk1c_Cy7 (Fig. 3B). The emission spectra revealed



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Förster energy transfer from TO to Cy7 (emission 750−850 nm) in the single stranded state, which - despite the very small spectral overlap - probably is feasible owing to the very short distance between TO and Cy7 in the unbound state. Of note, energy transfer and, thus, TO quenching was negligible in the target-bound qFIT-probe osk1c_Cy7 where Cy7 and TO were separated by an 18 nt distance. Importantly, the readout of the Cy7-channel revealed independent emission (I/I0 = 1.0−1.3), regardless of the presence or absence of target RNA and the brightness of the TO signal remained unaffected as long as the Cy7-TO distance was sufficiently large to avoid FRET in the double-stranded state. As an added advantage, the feasibility of FRET in the single stranded state increases the hybridisation-induced response of TO emission. Table 1: Sequences, melting temperatures and fluorescence properties of oskar probes with and without Cy7-labelling. name

sequence 5' → 3'

TM / °C

I0[a]

I[a]

I/I0

I/I0 (Cy7)

osk1a

Bu-GAC TTX AGA TAA TAG GTT TTG GCG

56

24

82

3.5

-

osk1b

Bu-GAC TTA AGA TAX TAG GTT TTG GCG

55

20

182

9.3

-

osk1c

Bu-GAC TTA AGA TAA TAG GTX TTG GCG

55

37

173

4.7

-

osk1a_Cy7

Cy7-Bu-GAC TTX AGA TAA TAG GTT TTG GCG

57

5

17

3.1

1.0

osk1b_Cy7

Cy7-Bu-GAC TTA AGA TAX TAG GTT TTG GCG

56

5

83

15.4

1.1

osk1c_Cy7 (osk1q)

Cy7-Bu-GAC TTA AGA TAA TAG GTX TTG GCG

56

11

166

15.2

1.1

osk2q

Cy7-Bu-AAA AGC GGA AAA GXT TGA AGA

48

8

107

13.4

1.1

osk5q

Cy7-Bu-CGG TTT TCT GGX TTT GGG T

60

4

66

16.5

1.3

[a] The experimental error due to inaccuracies of concentration determination is in the 10% range. Conditions: 0.5 µM probe and 5 eq. RNA-target, when added, in PBS (100 mM NaCl, 10 mM Na2HPO4, pH 7) at 25 °C. Red marked X denotes a Ser(TO) nucleotide (see Fig. 1). TO: λex = 485 nm, λem = 535 nm; Cy7: λex = 690 nm, λem = 774 nm.

Figure 3. Fluorescence emission spectra of A) osk1c and B) osk1c_Cy7. Excitation of TO (λex = 485 nm, λem = 500−900 nm) before (black) and after (red) addition of target; Excitation of Cy7 (λex = 690 nm, λem = 700−900 nm) before (grey) and after (blue) addition of target. Conditions: 0.5 µM probe and 5 eq. RNA-target, when added, in PBS (100 mM NaCl, 10 mM Na2HPO4, pH 7) at 25 °C.

For application in fluorescence microscopy, qFIT probes must tolerate biological matrix without loss of performance. We evaluated the probes in lysate from 106-107 MDCK cells. The


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turbidity of the mixture that contained all cellular compounds such as membrane components, proteins, fragments of organelles and nucleic acids showed a relatively high background signal. The responsiveness of TO and the Cy7-emission was determined upon stepwise addition of target (0.1 eq. steps) in order to simulate the absence target (0.0 eq. target), partial hybridization (0.1-0.9 eq. target) and full hybridization (≥ 1.0 eq. target). The TO emission increased until a 1:1 probe/target stoichiometry was reached (Figure 4A). Measurements in PBS buffer revealed that the TO quantum yield of osk-1c_Cy7 (φ0 = 0.05, φ = 0.29) was increased by a factor of 6 upon RNA binding. By contrast, Cy7 quantum yields remained unchanged (φ0 = φ = 0.29) and the independence of Cy7 emission persisted also in cell lysate over the complete range (0.0 to 1.6 eq.) of titration (Figure 4B). This should allow the calculation of a contrastfactor I(Cy7)/I(TO) which indicates the degree of probe-hybridization and could be used as a calibration for application in cells.

Figure 4: A) TO-emission and B) Cy7 emission spectra of osk-1c_Cy7 upon stepwise (0.1 eq) addition of target RNA. Conditions: 200 pmol probe in MDCK lysate (106-107 cells/mL), target RNA added in 0.1 eq steps (20 pmol). TO: excitation at 485 nm, integral of emission signal 500−700 nm; Cy7: excitation at 690 nm integral of emission signal 700−900 nm. All values are background corrected.

Quantitative mRNA imaging in Drosophila oocytes. Previously we used a combination of three fluorogenic hybridization probes (osk1, osk2 and osk5) for targeting different sites of oskar mRNA in Drosophila oocytes (see Figure S7).41, 42 This procedure increases the sensitivity of RNA imaging (vide infra). In addition to osk1q (identical to osk1c_Cy7) we converted the osk2 and osk5 FIT-probes to qFIT-probes osk2q and osk5q, respectively (Table 1). The probes showed, again, hybridization dependent TO emission while Cy7 emission remained virtually unaffected by hybridization (e.g. osk5q: φ0(TO) = 0.06, φ(TO) = 0.27, φ0(Vy7) = 0.22, φ(Cy7) = 0.23). We performed a rapid, wash-free FISH employing osk1q or a combination of osk1q, osk2q and osk5q in fixed, stage 8-9 Drosophila oocytes, in which oskar mRNA begins to accumulate in great quantities at the posterior pole of the oocyte. Confocal fluorescence microscopy was used to obtain the signal of Cy7 (Figure 5A) and TO (Figure 5B) dyes of the qFIT-probes, which report the local concentration and the hybridization status of the probe, respectively. In any given voxel, we assumed that the local concentration (cx,y) is a linear combination of the single-stranded and double-stranded probe species. By measuring Cy7 and ACS Paragon Plus Environment


TO emission and knowing the fluorescence of TO and Cy7 in cell-free background as well as the responsiveness of emission of the given qFIT probes in a microscope setting (Figure S6), we determined the concentration of probe-bound mRNA target (see SI for details). Figure 5E shows the distribution of the concentration of double stranded qFIT probes. The enrichment of oskar mRNA at the posterior pole of stage 8-9 Drosophila oocytes (Figure 5A and B, green dashed line) has frequently been observed.49 Our measurements (Figure 5E) revealed a surprisingly high 208±80 nM concentration of oskar mRNA (mean±SD, n=8, combined results of osk1q and osk1q,2q,5q) within a 3140±410 µm3 volume at the posterior pole (~2.4 % of total oocyte volume). Of note, we detected up to 1.8 µM local concentration per voxel in this posterior region, which exceeds the concentration of probes applied (0.5 µM osk1q, osk2q and osk5q each, see also Figure S11). We considered the feasibility of a local enrichment of probes due to a concentration gradient induced upon binding of target. Indeed, the concentration distribution measured by the Cy7 signal indicates the enrichment of probes in the posterior domain and depletion in the remaining regions of the oocyte (Figure 5A and C). For the estimation of oskar mRNA copy number in the oocyte we introduced a threshold value which was set to the 97.5th percentile of the cds,x,y distribution measured in follicle cells (where oskar is not expressed): voxels having less than 97.5 % of the maximum of the already low values found in this area were excluded from the downstream analysis (Figure S13A). To estimate the number of oskar mRNA molecules found within the oocyte the raw copy number (Nraw) was calculated by using Avogadro’s law (see SI for details). The residual, low intensity from TO remaining in the follicle cells after thresholding was used to estimate the mean falsepositive detection rate which was applied to correct the raw copy number. Using osk1q probes and an equimolar mixture of osk1q,2q,5q probes on wildtype oocytes, we obtained around 6.6x105 (3.9·105−1.1·106) and around 1.0·106 (5.9·105−1.6·106, p=0.0513) copies of oskar mRNA per oocyte (Figure 5F). In recent experiments, oskar mRNA copy number was assessed by means of qRT-PCR. Values around 3·105−1.2·106 in Drosophila embryos51 are within reasonable agreement with the values obtained by the qFIT probes. Oocytes that lacked half the amount of oskar mRNA (oskar0/+, see also Figure S10C) showed a significantly lower copy number of oskar mRNA ~3.5·105 (1.4·105−5.9·105, p

Quantitative mRNA Imaging with dual channel qFIT probes to Monitor

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