Transglutaminase-Mediated in Situ Hybridization (TransISH) System

Jun 19, 2012 - Transglutaminase-Mediated in Situ Hybridization (TransISH) System: A New Methodology for Simplified mRNA Detection. Momoko Kitaoka,...
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Transglutaminase-Mediated in Situ Hybridization (TransISH) System: A New Methodology for Simplified mRNA Detection Momoko Kitaoka,†,⊥ Masayuki Mitsumori,§ Kounosuke Hayashi,§ Yoshiyuki Hiraishi,§ Hisao Yoshinaga,† Koji Nakano,† Katsuyuki Miyawaki,∥,⊥ Sumihare Noji,∥ Masahiro Goto,†,‡ and Noriho Kamiya*,†,‡ †

Department of Applied Chemistry, Graduate School of Engineering, and ‡Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Japan § Hitachi Aloka Medical, Ltd., 3-7-19 Imai, Ome-shi, Tokyo, Japan ∥ Department of Life Systems, Institute of Technology and Science, The University of Tokushima, 2-1 minamijosanjima-cho, Tokushima, Japan S Supporting Information *

ABSTRACT: Detection and localization of specific DNA or RNA sequences in cells and tissues are of great importance for biological research, diagnosis, and environmental monitoring. However, the most common procedure for in situ hybridization employs laborious immunostaining techniques. In the present study, we report proof-of-concept for a new RNA-enzyme conjugated probe for the detection of mRNA on tissue sections with a simple procedure. An RNA probe modified with a specific dipeptide substrate of transglutaminase was prepared. Alkaline phosphatase was then covalently and site-specifically combined to the dipeptide-labeled RNA using microbial transglutaminase. The new RNA probe labeled with alkaline phosphatase was validated by in situ hybridization (ISH) and proved to be a sensitive and sequence specific probe for mRNA detection in tissues. The new transglutaminase-mediated ISH (TransISH) strategy is free from antigen−antibody reaction, leads to one-step signal amplification after hybridization, and thus will be widely applicable for highly sensitive nucleic acid detection. amplification strategies using enzymes, 3−7 fluorescent dyes,8−10 intercalators,11 and nanoparticles. 12 Of these strategies, enzymes are attractive because of their catalytic turnover and substrate specificity, leading to a stronger signal and higher signal-to-noise ratio. In ISH, immunological detection using hapten-labeled probes and antibody-enzyme conjugates in combination with fluorescent substrates or precipitating chromogens is the most widely accepted procedure because of its high sensitivity comparable to RI probes.13 With enzyme-catalyzed precipitation of chromogens, the localization of the target nucleic acids can be observed using a brightfield microscope and counterstaining of the sample is not always required. This technique is also suitable for routine diagnosis as the

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ith the successful application of genome sequencing in various organisms, determining gene function is becoming one of the next major scientific issues. In situ hybridization (ISH) techniques allow for the detection of spatial, developmental, and disease specific expression patterns of target nucleic acid sequences. Thus ISH is indispensable for studying biological processes and for routine diagnosis.1,2 In the common procedure of ISH, nucleic acid probes are hybridized to their naturally occurring complementary sequences, and visualization of the target nucleic acid is done with immunological detection or with signaling molecules directed at the probes, such as radioisotopes (RI) and fluorescent dyes. Recently, oligonucleotide-based highly sensitive detection assays using hybridization or aptamer recognition mechanisms have been developed for the diagnosis of viral and bacterial infections and certain genetic diseases. Although, not for use in cellular localization studies, the high sensitivity and selectivity of these assays is desirable; these assays employ signal © 2012 American Chemical Society

Received: December 22, 2011 Accepted: June 19, 2012 Published: June 19, 2012 5885

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TransISH system has the benefit of being able to advance to the staining reaction immediately after washing of the unhybridized probe (Scheme 2). This simplified mRNA detection protocol will facilitate more practical applications of the ISH technique in medical, food, and environment diagnosis.

chromogenic signals can be preserved and reanalyzed as needed.14,15 However, the immunological detection process uses thermally unstable antibodies, and the hapten-antibody reaction requires an extensive multistep procedure. Hybridization probes bearing signaling enzymes with covalent crosslinkages are more easily handled; however, the manufacturing process with chemical manipulation causes random crosslinkages between the probes and enzymes and may inactivate enzymes.16 To overcome these limitations, we have recently developed a new DNA hybridization probe to which the enzymes are covalently cross-linked in a site-specific manner by the aid of the enzyme transglutaminase (TGase). TGase posttranslationally modifies proteins by cross-linking glutamine to lysine or another primary amine.17,18 In the TGase family, we employed a microbial TGase (MTG) to cross-link the RNA probe and the signaling enzymes. MTG is a relatively small, stable, calcium-independent enzyme whose catalytic mechanism, using the dipeptide substrate Z-QG (benzyloxycarbonyl19−21 L-glutaminylglycine), is well-defined. Using the MTG cross-linking system, the successful application of the novel DNA-(enzyme)n conjugate probe was demonstrated using dotblot detection of target DNA.22 Here, we report a new ISH procedure, the TransISH. We show successful synthesis of the RNA-(enzyme)n conjugate, and the detection of mRNA in tissue sections using the new probe without an antigen−antibody reaction (Scheme 1). The TGase-mediated RNA-enzyme conjugation can be conducted in one step, and the resultant probe can be used for hybridization without further purification. Moreover, the

Scheme 2. Schematic Flowchart of the Common ISH Protocol and TransISH Protocol

Scheme 1. Illustration of Transglutaminase-Mediated in Situ Hybridization (TransISH) Procedure



EXPERIMENTAL SECTION Chemicals and Materials. Urea, formamide, trisodium citrate, sodium dodecyl sulfate (SDS), and tris(hydroxymethyl)aminomethane were from Nacalai Tesque (Kyoto, Japan). SYBR Green II (Molecular Probes) was purchased from Takara Bio (Shiga, Japan). NTPs (nucleoside triphosphates), digoxigenin-11-UTP, NBT/BCIP (p-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate), and RNA polymerases (T3, T7 and SP6) were purchased from Roche Applied Science (Basel, Switzerland). CHAPS (3[(3-cholamidopropyl)dimethylammonio]propanesulfonate) was obtained from Merck (Darmstadt, Germany). Triton X100 and polyvinyl alcohol (Mw 70,000−100,000) were from Sigma-Aldrich (St. Louis, MO). Sodium dextran sulfate (Mw 500,000) was from Wako pure chemical (Osaka, Japan). Z-QGUTP was custom synthesized from dipeptide Z-QG and 5-(3aminoallyl)-2′-uridine 5′-triphosphates (AA-UTP) at GeneACT, Inc. (Fukuoka, Japan) according to the previously described procedure.22 MTG was supplied by Ajinomoto Co., Ltd. (Tokyo, Japan). Recombinant alkaline phosphatase from Pyrococcus f uriosus which has optimized sequences for Escherichia coli bacterial expression and is fused with K-tag (Pf uAP) was expressed and purified at Yokohama BioResearch & Supply, Inc. (Kanagawa, Japan). PCR primers were synthesized at GeneNet (Fukuoka, Japan), and the sequences are as follows: 5′-CATACGATTTAGGTGACACTATAGATCAAAACTCCTGCGTGAGAA-3′ (Prm_SP6_Fw), 5′TAATTAACCCTCACTAAAGGGAGATCTGTACAGGTGGCTTGG-3′ (Prm_T3_Rev), 5′-AATTAACCCTCACTAAAGGGCGCAGCACAGAGTATGGTGT-3′ (Umod_5886

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Figure 1. (a) Denaturing polyacrylamide gel electrophoresis analysis of Z-QG labeled Prm RNA probe. An antisense RNA probe for mouse Prm mRNA was prepared by in vitro transcription using T3 RNA polymerase. In the transcription reaction mixture, 0% (Lane 1), 20% (Lane 2), 40% (Lane 3), 60% (Lane 4), and 80% (Lane 5) of UTP was substituted with Z-QG-UTP. M: 0.1−2 kb RNA Ladder (Invitrogen). (b−d) Agarose gel electrophoresis analysis of the MTG mediated conjugation of Pf uAP and Z-QG-RNA. A mixture of PfuAP and Z-QG(0%)-RNA (Lanes 1, 6), ZQG(20%)-RNA (Lanes 2, 7), Z-QG(40%)-RNA (Lanes 3, 8), Z-QG(60%)-RNA (Lanes 4, 9), or Z-QG(80%)-RNA (Lanes 5, 10) was incubated without MTG (Lanes 1−5) or with MTG (Lanes 6−10). MTG-mediated cross-linking procedure is described in the Experimental Section. M: 100 bp DNA Ladder (Invitrogen). RNA was stained with SYBR Green II (b), and then the Pf uAP was reacted with NBT/BCIP in the same gel. The image of NBT/BCIP staining was inverted to black and white (c) and overlaid on the image of SYBR Green II staining (d). The dashed boxes correspond to the bands of RNA (blue), PfuAP (yellow), and RNA-Pf uAP conjugates (green).

Pf uAP, 1 U/mL MTG, and 1 U/mL RNaseOUT. An aliquot of the MTG reaction mixture (10 μL) was subjected to agarose gel electrophoresis to confirm the presence of RNA-enzyme conjugates. The rest of the PfuAP conjugated probe was added to the hybridization mixture without purification. Electrophoresis Analysis. Denaturing polyacrylamide gel electrophoresis was performed in an 8% polyacrylamide gel in 1× TBE and 7 M Urea. Electrophoresis was carried out at 280 V for 50 min. RNAs were stained with SYBR Green II (1:10,000 dilution in water) for 30 min with gentle agitation. Agarose gel electrophoresis analysis of the RNA-enzyme conjugate probe was performed with a 1.5% agarose LE (Wako) gel in 1 × TAE. Electrophoresis was conducted at 100 V for 35 min, and then the gel was soaked in SYBR Green II solution (1:10,000 dilution in water) for 30 min with gentle agitation, followed by incubation in 0.1 M Tris-HCl (pH 9.5) containing 50 mM MgCl2, 350 μg/mL NBT, and 175 μg/mL BCIP, at 50 °C for 15 min. Atomic Force Microscopy (AFM) Imaging. Purified ZQG(0 and 40%)-RNA (10 ng/μL) was diluted 100-fold with Milli-Q water containing 1 mM MgCl2. MTG reaction mixtures were directly diluted 100-fold with Milli-Q water containing 1 mM MgCl2. A 10 μL droplet of each sample dilution was placed onto the surface of freshly cleaved mica (Nisshin EM, Tokyo, Japan). After 30 s, the surface was rinsed with Milli-Q water, desiccated under nitrogen, and then vacuum-dried for 2 h. AFM imaging was primarily performed in intermittent contact mode using JSPM-5410 scanning probe microscope (JEOL, Tokyo, Japan). Noncontact mode measurements with frequency modulation detection provided the higher-resolution images

T3_Fw), 5′-TAATACGACTCACTATAGGGTGGTGCCCACATACAGAAAA-3′ (Umod_T7_Rev). Template DNA for in Vitro Transcription. DNA for the in vitro transcription was amplified from protamine (Prm) cDNA cloned into a pGEM-T vector, using Prm_SP6_Fw and Prm_T3_Rev PCR primers. Theoretically, 285-mer sense and 287-mer antisense probes were transcribed using the Prm DNA template. Uromodulin (Umod) was also amplified from Umod cDNA cloned into a pGEM-T vector, using Umod_T7_Fw and Umod_T3_Rev PCR primers. Theoretically, 938-mer sense and antisense probes were transcribed using the Umod DNA template. Enzymatic Synthesis of Z-QG-RNA. RNAs were enzymatically synthesized by in vitro transcription using bacteriophage RNA polymerases. In vitro transcription was performed in 1× reaction buffer (Roche) containing 2 mM nucleoside triphosphates (NTP), 20 ng/μL template DNA, 40 U RNase OUT, and 20 U T3 RNA polymerase. The reaction mixture was incubated at 37 °C for 2 h, followed by additional incubation with 5 U of recombinant DNase I (Takara Bio) at 37 °C for 30 min. For the preparation of Z-QG(X%)-RNA, a percent of the UTP additive was substituted with Z-QG-UTP in the reaction mixture (X%: 0, 20, 40, 60, or 80). All RNA samples were purified using miniQuick Spin RNA Columns (Roche). Cross-Linking of Z-QG-RNA and PfuAP by MTG. Z-QGRNA (200 ng) was heat-denatured at 95 °C for 5 min, and then immediately cooled on ice for at least 5 min. The MTG reaction was performed at 40 °C for 2 h in 50 mM phosphate buffer (pH 6.0) containing 10 ng/μL Z-QG-RNA, 0.3 mg/mL 5887

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Figure 2. AFM images of Prm antisense RNA with and without conjugation to PfuAP. Z-QG(0%)-RNA (a) and Z-QG(40%)-RNA (b) following purification were heat-denatured and diluted with 1 mM MgCl2 before being cast on mica. A mixture of Pf uAP and Z-QG(0%)-RNA (c) or ZQG(40%)-RNA (d) was placed on mica after incubation with MTG. Presumed single RNA-enzyme conjugate molecules in the image (d) were outlined with white lines (e). Bars shown on the bottom left corner = 100 nm × 100 nm.

for the mixtures of Z-QG(40%)-RNA and PfuAP with and without MTG. Simple low-pass filtering reduced noise while height profile analyzed the surface morphology, both of which were provided by Winspm II data processing software (JEOL, Tokyo, Japan). Fundamental Procedure for TransISH. Paraformaldehyde fixed and polyester wax embedded mouse testis sections (6 μm) and paraformaldehyde fixed and paraffin embedded mouse kidney sections (6 μm) were dewaxed using ethanol and xylene, respectively. The dewaxed sections were air-dried, soaked in phosphate-buffered saline (PBS) for 5 min, and then treated with proteinase K (1 μg/mL in PBS). After 10 min of incubation, the proteinase K was removed by washing twice for 5 min in PBS. For prehybridization, 100 μL of a hybridization mixture (6 M urea, 20 mM sodium-phosphate buffer; pH 8.0, 0.5 M sodium chloride, 1 × Denhardt’s solution, 1 mg/mL torula yeast RNA, 10 mg/mL salmon sperm DNA) was applied to the sections, and the sections were incubated for 30 min at 50 °C. After removal of the hybridization mixture, 100 μL of the hybridization mixture containing 5% dextran sulfate (Mw 500,000) and the RNA-enzyme conjugate probe (1 μg/mL or otherwise denoted) were applied to the sections and incubated for 14 h in an incubation chamber (50 °C). Probe concentration represents the RNA concentration before PfuAP conjugation. The sections were washed for stringency by immersing three times in a primary wash buffer (2 M urea, 50 mM sodiumphosphate buffer; pH 8.0, 150 mM sodium chloride, 1% Triton X-100) for 20 min at 42 °C. Then, the sections were washed twice for 20 min with a secondary wash buffer (25 mM TrisHCl; pH 7.4, 140 mM sodium chloride, 2.7 mM potassium chloride, 0.1% Triton X-100, 0.1% CHAPS) at 65 °C. Subsequently the sections were washed twice for 5 min in a detection buffer (100 mM sodium chloride, 100 mM Tris-HCl; pH 9.5, 50 mM magnesium chloride, 0.1% CHAPS) at 50 °C. The signal was developed for 2 h to overnight at 50 °C in

detection buffer containing 5% polyvinyl alcohol (Mw 70,000− 100,000), 350 μg/mL NBT and 175 μg/mL BCIP. Photographs were taken with a microscope BX51 (Olympus) equipped with a CCD camera FX380 (Olympus).



RESULTS AND DISCUSSION In ISH, both DNA and RNA probes are employed. In this study, we focused on an RNA probe because it is easy to prepare a sense probe for negative control experiments. In addtion, RNA−RNA hybrids are shown to be generally more stable than DNA−DNA or DNA−RNA hybrids, which affords stringent conditions for hybridization.23 For the preparation of the new type of probe, RNA-(enzyme)n conjugate, an acyldonor substrate of the enzyme MTG, Z-QG, was incorporated in an RNA probe using in vitro transcription with RNA polymerase. The procedure is the same common procedure for the preparation of various RNA probes labeled with other tags, such as digoxigenin, biotin, 2,4-dinitrophenol, and fluorescent dyes.24 For the preparation of Z-QG-modified RNA, Z-QG was attached to the 5-position on the pyrimidine of UTP, and the UTP analog was used in the in vitro transcription mixture at various ratios. At first, we prepared an antisense RNA probe modified with Z-QG (at various ratios of labeled to unlabeled UTP: 0/100, 20/80, 40/60, 60/40, and 80/20) for the detection of mouse protamine (Prm) mRNA, using T3 RNA polymerase. Following purification, the Z-QG(X%)-RNA was analyzed by denaturing polyacrylamide gel electrophoresis (Figure 1a). Mobility of the Z-QG-RNA probe was retarded proportionately to the amount of incorporated Z-QG-UTP, which was consistent with previous studies analyzing Z-QGDNAs synthesized using DNA polymerases.22 Similar mobility shifts were observed with T7 and SP6 RNA polymerases (data not shown). An acyl-acceptor lysine tag that serves as a substrate for MTG, was introduced to the N-terminus of recombinant alkaline phosphatase from hyperthermophile Pyrococcus f uriosus 5888

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Figure 3. Detection of mouse Prm mRNA on mouse testis section using the antisense TransISH probes, prepared from Z-QG(0%)-RNA (i), ZQG(40%)-RNA (ii), and Z-QG(60%)-RNA (iv), and the sense TransISH probe prepared from Z-QG(40%)-RNA (iii). ISH was conducted for the same target with a digoxigenin-labeled antisense probe according to the manufacturer’s protocols (v). Bar = 200 μm.

(Pf uAP, Acc. No. AB479383). We chose this enzyme because it was expected to be resistant to the heating and chemical denaturation to which it would be exposed during the hybridization process. Pf uAP containing the K-tag (MKHKGSGGGSGGGS, the reactive lysine residue for the MTG-catalyzed cross-linking reaction is underlined) at the Nterminus was expressed in Eschelichia coli and purified using a hexahistidine-tag at its C-terminus. We then cross-linked the ZQG-RNA and the K-tagged PfuAP using MTG in a simple reaction. As described above, the efficiency of conjugation was assessed by a mobility shift in an agarose gel because the RNA(Pf uAP)n conjugates showed a slower mobility.22 The RNA and PfuAP were stained in the same gel with the a SYBR Green II intercalator dye and NBT/BCIP precipitating chromogen, respectively. Although the free Z-QG-RNA and PfuAP had different mobilities, the major portion of RNA staining and enzymatic activity staining were matched in the gel after incubation with MTG, indicating the tag-specific cross-linking between template RNA and Pf uAP (Figures 1b−d). For further characterization of the RNA-(enzyme)n probe, we attempted to visualize the probe by atomic force microscopy (AFM). The AFM images of the mouse Prm antisense RNA probes without PfuAP conjugation revealed relatively short but elevated strands, typically 30−60 nm in length and ∼2 nm in height, some of which were ribbon-like in shape. This suggests that the RNA strands tend to fold fully or partially during AFM sample preparation even when they are heat-denatured beforehand (Figure 2a, b).25 The AFM image of a mixture of Z-QG(0%)-RNA and Pf uAP incubated with MTG showed many partially extended RNA strands with other globular entities intermingled (Figure 2c). The specific height of these elements, either ropes or spheres, was ∼0.4 nm or ∼0.7 nm, respectively (Supporting Information). Therefore, the RNA probes did not aggregate with either of the enzymes in the solution but remained as discrete molecules, and these twotypes of materials independently adsorbed onto the mica to give distinct AFM images. Interestingly, the Z-QG(40%)-RNAPfuAP conjugated probe gave a totally different result in the high-resolution AFM image (Figure 2d); linear gatherings of globular molecules that were nearly uniform in size, 5 nm in height and 50 nm in length, covered the entire surface. As emphasized by the white line indications (Figure 2e), each

grouping possesses a specific contour length of 80−120 nm, which is considered reasonable for ∼290-mer ssRNA in an extended conformaion.26−28 Thus, we believe that the linear gatherings of globules are the enzyme-conjugated RNA molecules that fully extend rather than fold like unconjugated RNA. Precise quantification of the average number of Pf uAP molecules labeled with an RNA strand is rather difficult from the gel electrophoresis data (Figure 1b). AFM imaging also failed to give images for each Pf uAP molecule bound to the template RNA because of the limited spatial resolution. However, the marked contrast for the specific height, approximately 7 nm for the Z-QG(40%)-RNA-Pf uAP conjugate and 1 nm for the control sample, respectively, suggests that the entire length of the RNA template covered with the protein molecules. We therefore concluded that MTG directed specific cross-links between Z-QG and PfuAP as intended to form the RNA-(Pf uAP)n conjugates. We then employed the RNA-(Pf uAP)n probe (designated as TransISH probe hereafter) to detect mRNAs on tissue sections. Mouse Prm mRNA was targeted; sense and antisense probes were prepared from the Z-QG modified RNAs synthesized using T3 and SP6 RNA polymerases, respectively. Mouse testis sections were used for ISH. Hybridization of conventional digoxigenin-RNA probe is often performed in a buffer containing the detergent SDS to permeabilize tissue and to prevent nonspecific binding of the probes. Therefore, we first attempted to use an aqueous buffer solution containing SDS; however, no signal was obtained from our probe. On the other hand, a signal that was typical for Prm mRNA was observed when other detergents, such as Triton X-100, Tween 20, or CHAPS were used, suggesting that the SDS inactivated the Pf uAP.29 Another alkaline phosphatase, from the hyperthermophilic Archaea Pyrococcus abyssi, has also been shown to be sensitive to SDS. A Prm signal was observed only when using an antisense TransISH probe prepared from Z-QG(40 and 60%)-RNA (Figure 3), and we found that with a probe prepared from Z-QG(60%)-RNA showed a more intense signal in 2 h. The TransISH probes prepared from a sense ZQG(40%)-RNA or from antisense Z-QG(0%)-RNA did not generate signals, indicating that the signals were specific to the antisense conjugate probes. The expression patterns found were in good agreement with those obtained by conventional 5889

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signal (Figure 5). Signal intensity increased by extending the staining time, and it required 4 h to discriminate the signal

procedures using a digoxigenin (DIG) probe (Figure 3v), validating the TransISH procedure. We next used a longer TransISH probe to detect mouse uromodulin (Umod) mRNA in kidney tissue. According to the preceding results, both antisense and sense TransISH probes were prepared from corresponding Z-QG(60%)-RNAs. As shown in Figure 4a, expression of the Umod mRNA in the distal

Figure 5. Comparison of RNA probes labeled with different ratios of Z-QG groups. Antisense TransISH probes for mouse Umod mRNA were prepared from Z-QG(40%)-RNA (a), Z-QG(60%)-RNA (b), and Z-QG(80%)-RNA (c) and applied to a mouse kidney section at the probe concentration of 0.2 μg/mL. Hybridization was performed at 55 °C. Bar = 200 μm.

Figure 4. Detection of mouse Umod mRNA in mouse kidney section using the antisense TransISH probe prepared from Z-QG(60%)-RNA. (a) Optimization of probe concentration. Probe concentration of 0.1 (i), 0.25 (ii), 0.5 (iii), and 1.0 (iv) μg/mL, based on RNA input, were applied to detect mouse Umod mRNA. A sense TransISH probe did not generate a signal at the probe concentration of 1.0 μg/mL (v). Hybridization was performed at 50 °C, and signal development time was 3 h. (b) ISH conducted with a digoxigenin-labeled antisense (i) and sense (ii) probes according to the manufacturer’s protocols. Signal development time was 1 h. Bar = 100 μm.

derived from the target mRNA. By contrast, the conventional DIG system generated a clear signal for the same mRNA after 1 h staining (Figure 4b,i). The longer detection time required for the TransISH probe is possibly due to the difference in specific activity of enzymes employed for each procedure. The catalytic activity of Pf uAP at ambient temperature is much lower than calf intestine alkaline phosphatase used in a DIG system, and this drawback could be overcome by protein engineering of Pf uAP. The probe prepared from the Z-QG(80%)-RNA did not generate a signal at all. Similarly in the ISH analysis of mouse Prm mRNA, we could not detect a signal when the TransISH probe prepared from Z-QG(80%)-RNA (data not shown). One possible reason for this observation is that hybridization of the TransISH probe to target nucleic acid on tissue sections may be physically hindered when too many enzymes are conjugated to the probe. In addition, the increment of a large number of ZQG moieties into an RNA strand may decrease the stability of RNA−RNA hybrids. To mitigate any potential steric hindrance, and to increase the flexibility of the probe, the introduction of a longer linker group between the RNA and Pf uAP might be effective.

straight tubule of the nephron was clearly detected using an antisense TransISH probe. No signal was observed when using the sense TransISH probe, again showing the probe specificity (Figure 4a,v). For conventional probes like radioisotopes-, haptens-, and fluorescent dye-labeled probes, relatively high probe concentration is known to cause an intense background signal due to the nonspecific hybridization.30 After the signal development reaction for 3 h, a strong background signal was observed with a probe concentration of 0.5 μg/mL or more (Figure 4). On the other hand, when using a shorter TransISH probe for mouse Prm mRNA, a probe concentration of 1.0 μg/ mL generated a clear signal (Figure 3). These results suggest that optimal probe concentration depends on the length of TransISH probes and the type of tissues. Pretreatment and decolorization procedures of tissue sections also affected the signal detection with TransISH probes. An antisense DIG probe exhibited the comparable signal with a TransISH probe in 1 h (Figure 4b). To better understand the probe’s properties, we investigated various hybridization conditions using the TransISH probes. To test if the number of Pf uAP molecules in a single RNA strand could affect signal generation, mouse Umod antisense TransISH probes were prepared from Z-QG(40, 60 and 80%)RNAs, and we examined the impact of Z-QG modification on the target detection in tissue sections with a probe concentration of 0.2 μg/mL. As a result, the TransISH probe prepared from the Z-QG(60%)-RNA showed the most intense



CONCLUSIONS Here, we have described the first application of a new RNA(enzyme)n conjugate, the TransISH probe, in ISH applications. The TransISH probe is readily prepared using conventional techniques by MTG catalysis. Thermally stable Pf uAP withstands the hybridization processes at 50−55 °C in buffers containing denaturants, and the enzymatic staining reaction can be done immediately after stringency washes. Our novel system is therefore easy to handle, can reduce the protocol length, and offers more versatile opportunities for ISH-based diagnosis. 5890

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(22) Kitaoka, M.; Tsuruda, Y.; Tanaka, Y.; Goto, M.; Mitsumori, M.; Hayashi, K.; Hiraishi, Y.; Miyawaki, K.; Noji, S.; Kamiya, N. Chem. Eur. J. 2011, 17, 5387−5392. (23) Jin, L.; Lloyd, R. V. J. Clin. Lab. Anal. 1997, 11, 2−9. (24) Itzkovitz, S.; van Oudenaarden, A. Nat. Method 2011, 8, S12− S19. (25) Kuznetsov, Y. G.; Dowell, J. J.; Gavira, J. A.; Ng, J. D.; McPherson, A. Nucleic Acids Res. 2010, 38, 8284−8294. (26) Henn, A.; Medalia, O.; Shi, S. P.; Steinberg, M.; Franceschi, F.; Sagi, I. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5007−5012. (27) Hamon, L.; Pastré, D.; Dupaigne, P.; Le Breton, C.; Le Cam, E.; Piétrement, O. Nucleic Acids Res. 2007, 35, e58. (28) Hansma, H. G.; Revenko, I.; Kim, K.; Laney, D. E. Nucleic Acids Res. 1996, 24, 713−720. (29) Zappa, S.; Rolland, J.-L.; Flament, D.; Gueguen, Y.; Boudrant, J.; Dietrich. J. Appl. Environ. Microbiol. 2001, 67, 4504−4511. (30) Nuovo, G. J.; Elton, T. S.; Nana-Sinkam, P.; Volinia, S.; Croce, C. M.; Schmittgen, T. D. Nat. Protoc. 2009, 4, 107−115 11.

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: +81-92-802-2807. E-mail: [email protected]. kyushu-u.ac.jp. Author Contributions ⊥

M. Kitaoka and K. Miyawaki contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported by the Science and Technology Incubation Program in Advanced Regions from the Japan Science and Technology Agency (JST) (N.K.) and in part by Nanotechnology Network Project (Kyushu-area Nanotechnology Network) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.



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dx.doi.org/10.1021/ac2034198 | Anal. Chem. 2012, 84, 5885−5891