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Construction of Intramolecular Luciferase Complementation Probe for Detecting Specific RNA Tamaki Endoh,† Masayasu Mie,† Hisakage Funabashi,† Tatsuya Sawasaki,‡ Yaeta Endo,‡ and Eiry Kobatake†,* Department of Biological Information, Graduate School of Bioscience and Biotechnology, 4259, Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan, and Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan. Received November 9, 2006; Revised Manuscript Received January 12, 2007
Intermolecular enzyme complementation assay is a useful method for detecting protein-protein interactions. Specifically, bioluminescent signals produced from reconstructed split luciferase fragments are powerful tools for in ViVo analysis because the bioluminescent signals have been visualized both in cultured cells and living animals. However, they are limited for detection and evaluation of biological events relevant to intermolecular protein-protein interactions. In this study, we constructed an intramolecular luciferase complementation probe for detecting target biomolecules other than protein-protein interactions. It consists of peptide-inserted firefly luciferase (PI-FLuc) containing a short peptide between internally divided firefly luciferase. The inserted short peptide triggers FLuc complementation or discomplementation and subsequent reactivation or inactivation of FLuc activity through its induced fit conformational changes. We chose RNA binding arginine rich motif (ARM) peptides, Rev and/or Tat, for model peptide insertion, and expressed constructed PI-FLuc probe variants using a wheat germ cell-free protein synthesis system. They showed FLuc activity changes, reactivation, or inactivation after binding to their specific RNA targets. Furthermore, to expand the versatility of the PI-FLuc RNA detection system, we designed split-RNA probes built to reform the ARM peptide binding site in the presence of arbitrarily selected target-RNA. As a result, the target RNA was homogeneously detected by FLuc luminescent signals mediated by a cooperative function of the PI-FLuc and split-RNA probe sets.
INTRODUCTION Enzyme complementation and fluorescent resonance energy transfer (FRET) are typical assay systems for the detection of protein-protein interactions. The FRET assay is useful as a high-resolution assay since fluorescent signals provide both temporal and spatial resolution (1, 2). In contrast, enzyme complementation is more precise than FRET, because enzymatic signals provide quantitative readout and signal amplification, resulting in more sensitive assays (3, 4). Several proteins, such as dihydrofolate reductase (DHFR), β-galactosidase, and β-lactamase, have been used as reporter proteins, indicating intermolecular complementation and activity reconstruction (4-8). Recently, it was discovered that firefly luciferase (FLuc) and renilla luciferase (RLuc) could correspondingly function as reporter proteins for intermolecular complementation (9, 10). Luciferase produces low background and highly sensitive bioluminescent signals simply by the administration of an appropriate substrate in the presence of cofactors. In addition, the bioluminescent signals produced from luciferase may be noninvasively visualized both in cultured cells and in living animals using cooled charge-coupled device (CCD) cameras (11, 12). As a result, they are now becoming one of the most useful reporter proteins for analyzing protein-protein interactions especially for in ViVo analysis. Using intermolecular luciferase complementation, previous studies have also reported direct protein-protein interactions or conditional protein-protein interactions mediated by chemical molecules in the living mouse (9, 13, 14). In the meantime, there have also been efforts to construct recombinant protein probes, that can detect target biomolecules * To whom correspondence should be addressed. Tel: +81-45-9245760, FAX: +81-45-924-5779, E-mail:
[email protected]. † Tokyo Institute of Technology. ‡ Ehime University.
and antibodies intramolecularly, using an enzyme complementation assay (15-18). These recombinant proteins have been constructed by inserting domains or epitopes into internally divided reporter proteins. The inserted domains function as a response unit, undergoing conformational changes in response to the target biomolecules such as specific ligand bindings and signal transductions. In turn, conformational changes of the response units induce activity changes in reporter enzymes through a structural reorientation of the internally divided reporter enzyme. Such constructs would provide an advanced tool for analyzing target biomolecules other than protein-protein interactions and could be applied to in ViVo analysis, especially in mammalian cells and living animals. However, until now it has seemed difficult to construct the intramolecular enzyme complementation probes via domain insertion as compared to similarly assembled intramolecular FRET probes (19-22). Only some enzymes are known to be available as reporter proteins, and only some domains have been successfully used for domain insertion (18). In the case of luciferase, just one group reported a successfully constructed intramolecular luciferase complementation probe (23, 24). We posit that this failure is due to the detection principle of enzyme complementation. An intramolecular enzyme complementation probe requires the rational structural reconfiguration of divided reporter enzymes following the conformational changes of the response units, while FRET protein probes can detect such alterations directly by distant changes in fluorescent proteins (25). Therefore, the construction of an intramolecular luciferase complementation probe would be quite difficult if the inserted response unit is a relatively large domain. In a previous report, we constructed an intramolecular FRET protein probe by using short peptides as response units for detecting specific RNAs (26). In that study, HIV-1 Rev-peptide (17 a.a.), which specifically binds to RRE-RNA and Rev-
10.1021/bc060351o CCC: $37.00 © 2007 American Chemical Society Published on Web 03/17/2007
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aptamer, was inserted between EYFP and ECFP. The resulting FRET protein probe indicated FRET increase and decrease as mediated by the peptide’s conformational change after binding to RRE-RNA and Rev-aptamer, respectively. Moreover, we were able to detect arbitrarily selected target-RNA by designing split-RNA probes containing variable sequence regions. On the basis of these results, we expected that the construction of an intramolecular luciferase complementation probe should be possible by inserting short peptides as response units. In this study, we constructed a novel peptide-inserted firefly luciferase (PI-FLuc) probe by using short RNA-binding peptides for insertion. We expected that the constructed PI-FLuc probe would retain FLuc activity since the inserted peptide was sufficiently small so as not to disturb the steric configurations of native FLuc. We also expected that the conformational change of the inserted peptide following specific RNA binding would induce FLuc activity changes through a structural reorientation of the internally divided FLuc domains, either by FLuc complementation or discomplementation.
EXPERIMENTAL SECTION Plasmid Construction. All PI-FLuc probe variants were constructed on cloning vector pBluescript II SK (+) (Stratagene). Gene fragments coding NFLuc (N437, N445), CFLuc (C438, C446) and Full-FLuc were respectively amplified by PCR from PicaGene Basic Vector 2 (Toyo Ink) using primers combinations. Forward primers were split-FLuc-s (5′-GCGACTAGTATGGAATTCGAAGACGCCAAAAACATAAAG-3′), split-CFLuc-s438 (5′-GCGAGATCTCTGAAGTCTCTGATTAAGTAC-3′) and split-CFLuc-s446 (5′-CTTAGATCTGGCTATCAGGTGGCTCCCGCT-3′). Reverse primers were split-FLucas (5′-CTTGGGCCCGCGGCCGCTTAAGGCCTCGCGACCACGGCGATCTTTCC GCCCTT-3′), split-NFLuc-as437 (5′GCGGGATCCGCGGTCAACGATGAAGAAGTG-3′) and splitNFLuc-as445(5′-GCGGGATCCTTTGTACTTAATCAGAGACTT3′). Underlined bases are the restriction enzyme sites for gene recombination. Rev-peptide-inserted PI-FLuc probe variants were constructed replacing the EYFP and ECFP regions of FRET protein probe (YRG0C-11ad) (26) by NFLuc and CFLuc, respectively. Tat-peptide gene fragment was obtained from annealed oligonucleotide pair (5′-CAGACCTAGAGGCACCAGAGGCAAGGGCAGAAGAATCAGAAGAGGCGGCA-3′ and 5′-GATCTGCCGCCTCTTCTGATTCTTCTGCCCTTGCCTCTGGTGCCTCTAGGTCTGGTAC-3′), and Tat-peptideinserted PI-FLuc probe variants were constructed replacing the Rev-peptide region by Tat-peptide coding annealed oligonucleotide. FLAG-tag gene fragment was obtained from annealed oligonecleotide pair (5′-CTAGTATGAGGCCTGATTACAAGGATGACGACGATAAGGAATTCTAAG CGGCCGCGGGCC3′ and 5′-CGCGGCCGCTTAGAATTCCTTATCGTCGTCATCCTTGTAATCAGGCCTCATA-3′), and Stu I cut C-terminus FLAG-tag sequence was inserted between Stu I and Apa I site of PI-FLuc variants or Full-FLuc. Then, the constructed probe variants were transferred to expression plasmid for wheat germ cell free protein synthesis system (pEU-E01-MCS) using Spe I and Not I. Protein Expression and RNA Synthesis. Constructed PIFLuc probe variants and Full-FLuc were expressed using a cellfree protein synthesis system with wheat germ extract (27). Transcriptional templates were amplified by PCR from the constructed plasmids to make DNA fragments containing SP6 promoter sequence. Then, mRNAs were transcribed using SP6 RNA polymerase and translated according to a previously reported bilayer cell-free protein synthesis system (28). Translated protein variants were confirmed by SDS-PAGE and Western blotting using anti-FLAG M2 antibody (Eastman Kodak).
All short RNA oligonucleotides, RNA-targets, and split-RNA probes were synthesized and purified according to a previously reported method (26). Native-PAGE Western Blotting. Five microliters of translated products in wheat germ extract and 30 pmol of synthesized RNA oligonucleotides were mixed in 15 µL of interaction buffer (10 mM HEPES-KOH, 100 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 10% glycerol, pH ) 7.4). After 30 min of incubation on ice, samples were resolved on a 10% nondenaturing polyacrylamide gel with Tris/glycine running buffer at 4 °C. Then resolved proteins were transferred to PVDF membrane with Tris/glycine transfer buffer for 80 min at 350 mA. The PI-FLuc probe variants in transferred membrane were labeled using anti-FLAG M2 antibody and Alexa-546 conjugated antimouse IgG antibody and visualized using FluorImager595 (Molecular Dynamics). FLuc Activity Assay. FLuc activities were evaluated using luminometer (Luminous CT-9000D: Dia-Iatron). One microliter of translated products were mixed with an aliquot amount of RNA-targets or constructed hybridized complexes in 50 µL of interaction buffer using 96-well black plates (Sumilon). After 30 min of incubation at room temperature, luminescent signals were measured by addition of 50 µL of substrate (Picagene Luciferase Assay Kit: Toyo Ink) and counting for 10 s. The luminescent signals were normalized by subtracting the signal obtained from nontranslated wheat germ extract. Target-RNA Homogeneous Assay. Two microliters of translated products and a pair of split-RNA probes (final concentration at 500 nM) were previously mixed in interaction buffer. Varied concentrations of specific or nonspecific targetRNA were immediately added to the premixed probes in total 100 µL of interaction buffer using nonadsorption 96-well black plates (Sumilon). Luminescent signals were measured by addition of 100 µL of substrate and counting for 10 s after 90 min of incubation at 37 °C followed by 30 min of incubation at room temperature. The luminescent signals were normalized by subtracting the signal obtained from nontranslated wheat germ extract.
RESULTS Design and Expression of PI-FLuc Probes. To construct an intramolecular luciferase complementation probe, two ARM peptides, HIV-1 Rev-peptide (17 a.a.) and BIV Tat-peptide (13 a.a.) peptide, were selected for insertion. These two peptides are both well-characterized RNA-binding peptides, showing induced fit conformational changes following specific RNA binding. NMR and circular dichroism experiments indicate that the BIV Tat-peptide undergoes a change to a β-sheet structure upon binding to TAR-RNA (29-31), while HIV-1 Rev-peptide takes on R-hilical and elongated conformation upon binding to RRE-RNA and Rev-aptamer, respectively (32-35). FLuc was selected as a reporter luciferase, and two dividing points after 437th arginine and 445th lysine were chosen for peptide insertion, referring the previously reported intermolecular FLuc complementation probes (9, 12, 14). Then, eight variants of PI-FLuc probes were constructed by combining NFLuc domains (N437 and N445), CFLuc domains (C438 and C446), and ARM peptides (Rev and Tat) (Figure 1a). A FLAG-tag sequence was connected to the C terminus of each for subsequent Western blotting. These variants were expected to alter FLuc activity after binding specific RNAtargets via conformational changes of the ARM peptides. If RNA binding broke the steric configuration of the connected FLuc domains, we should expect FLuc activity and luminescent signal to decrease. On the other hand, if RNA binding corrected the steric configuration of connected FLuc domains, we would expect FLuc activity and luminescent signal to increase (Figure
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Figure 1. Principle of intramolecular luciferase complementation assay and PI-FLuc probe construction. (a) Rev-peptide (-TRQARRNRRRRWRERQR-) or Tat-peptide (-RPRGTRGKGRRIRR-) was inserted between NFLuc (N437, N445) and CFLuc (C438, C446). Control FLuc (No. 9) was constructed as wild type FLuc. A FLAG-tag sequence (-DYKDDDDK-) was connected to the C terminus of constructed variants. (b) PI-FLuc probes specifically bind to RNA-targets reflecting the inserted ARM peptide characteristics. If RNA binding induce discomplementation of the divided FLuc domains, luminescent signal would decrease (left). If RNA binding induce complementation of the divided FLuc domains, luminescent signal would increase (right). (c) Constructed split-FLuc probe variants were translated using a wheat germ cell-free protein synthesis system. An aliquot amount of translated products was resolved on an 8% SDS-PAGE and analyzed by CBB staining (3 µL: left) or Western blotting (1 µL: right). Lane numbers correspond to the numbers in part a). Nontranslated wheat germ extract was resolved as negative control (Ex).
1b). All constructed variants (Nos. 1-8) and the wild type FLuc (Full-FLuc No. 9) were expressed using a wheat germ cell-free protein synthesis system and confirmed by both SDS-PAGE and Western blotting (Figure 1c). Characterization of Constructed PI-FLuc Probes. We next investigated whether the PI-FLuc probes have an ability to bind to specific RNA-targets (RRE-RNA, Rev-aptamer, TAR-RNA; see Figure 2a). The translated PI-FLuc probe variants and synthetic RNA were mixed in an interaction buffer and resolved on native-PAGE. After a transfer reaction to PVDF membrane, we analyzed gel-mobility shifted bands using anti-FLAG antibody (Figure 2b). In all constructed PI-FLuc probe variants, we found specific signal bands at the lanes mixed with their RNA targets, while there were no discriminative signal bands at control lanes (RNA -). In the case of Rev-peptide-inserted variants (Nos. 1-4), signal bands were observed at the lanes mixed with RRE-RNA and Rev-aptamer, which were the targets of the Rev-peptide. There were trends that the signal bands had clearly converged with RRE-RNA and widely spread with Revaptamer, except for variant No. 4. In constrast, in the case of Tat-peptide-inserted variants (Nos. 5-8), clearly converged signal bands were observed at the lanes mixed with TAR-RNA, the target of the Tat-peptide. Further, in all variants there were no distinctive signal bands at the lanes mixed with nonspecific RNAs corresponding to the control lanes. These results indicate that each inserted peptide has the ability to specifically bind to their corresponding RNA-targets. Next, to evaluate whether PI-FLuc probe variants showed appropriate FLuc activity changes following specific RNA binding, we measured the luminescent signal produced after the addition of the respective RNA (Figure 2c). The results confirmed that the PI-FLuc probe variants, containing the N437 domain (Nos. 1, 3, 5, 7), have almost lost FLuc activity. These probes had luminescent signals as weak as the background signals obtained from nontranslated wheat germ extract (Ex) and did not show any luminescent signal change with addition of the relevant RNAs. In contrast, other variants, containing the N445 domain (Nos. 2, 4, 6, 8), retained FLuc activity. In addition, three of these four variants showed FLuc activity changes after addition of specific RNA-targets. Interestingly,
Figure 2. Detection of specific RNA-target with PI-FLuc probe. (a) Secondary structure of respective RNA-targets (TAR-RNA, RRE-RNA, Rev-aptamer). (b) Specific RNA binding was confirmed by nativePAGE gel mobility shift assay. Five microliters of translated products was mixed with 30 pmol of RNAs (C: RNA -, T: TAR-RNA, R: RRE-RNA, A: Rev-aptamer) and resolved on a 10% nondenaturing polyacrylamide gel. Transferred PI-FLuc probes were detected using Western blotting. (c) Luminescent signals were evaluated after the binding reaction of 1 µL of translated products and 25 pmol of respective RNAs. Values were average with standard deviation (n ) 3).
in the case of the Rev-peptide-inserted variant No. 4, FLuc was inactivated after the addition of both RRE-RNA and Rev-
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Figure 3. RNA concentration dependency in intramolecular luciferase complementation assay. Luminescent signals were evaluated after binding reaction of 1 µL of translated products and varied concentrations of respective RNAs. Values were average with standard deviation (n ) 3).
aptamer. On the other hand, in the case of the Tat-peptideinserted variants No. 6 and No. 8, FLuc was reactivated after addition of the TAR-RNA. Moreover, no variants showed any change in their FLuc activity after the addition of nonspecific RNA. RNA Detection by Intramolecular FLuc Complementation. We also investigated the RNA concentration dependency of FLuc activity changes in three variants (Nos. 4, 6, 8) (Figure 3). Varied concentrations of the RNA-targets were mixed with translated PI-FLuc variants, and FLuc activity was evaluated after the binding reaction. Variants No. 6 and No. 8 showed dose-dependent FLuc reactivation with TAR-RNA, while there were no signal changes with other RNAs. The signal change ratios corresponded to Figure 2c. In contrast, variant No. 4 showed dose-dependent FLuc inactivation with RRE-RNA and/ or Rev-aptamer, while there were no signal changes with TARRNA. These results indicate that the RNA-targets were homogeneously detected by luminescent signal changes with sufficient specificity. Intramolecular Complementation Assay for ArbitrarilySelected Target-RNA. For detection of arbitrarily selected target-RNA other than specific RNA-targets, we designed splitRNA probes built to reform the ARM peptide binding site in the presence of the target-RNA. The split-RNA probes containing variable sequence regions respond to any target-RNAs. First, a split-RRE-RNA probe was constructed as previously described
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Figure 4. Principle of intramolecular luciferase complementation assay for arbitrarily selected target-RNA. (a) A pair of split-RNA probe hybridizes to an arbitrarily selected target-RNA and reforms the ARM peptide binding site. PI-FLuc probe binds to the ARM peptide binding site constructed on the hybridized complex. They indicate luminescent signal changes thorough the ARM peptide conformational change. (b) Secondary structure and sequence of designed hybridized complex. Surrounded region indicates minimal element necessary for the ARM peptide binding.
(26). Second, we designed a split-TAR-RNA probe according to our previous methodology. The TAR-RNA was split at its loop region, and RNA oligonucleotides containing complementary sequences of arbitrarily selected target-RNA were connected at the 5′ or 3′ end of the split-TAR-RNA. A minimal sequence for Tat-peptide recognition was retained for the split-TAR-RNA. Additionally, spacer-like sequences were inserted between the complementary sequence region and the split-TAR-RNA region. By this design, the constructed split-RNA probes hybridize to adjacent regions of the arbitrarily selected target-RNA, and the ARM peptide binding site is subsequently reformed on the hybridized complex. Next, the PI-FLuc probe binds to the hybridized complex with the conformational change of the ARM peptide. Therefore, following this last step, the arbitrarily selected target-RNA should be detectable by FLuc luminescent signals through the cooperative functions of such self-assembled probe sets as the PI-FLuc probe and the split-RNA probe (Figure 4a). The secondary structure and sequence of the hybridized complexes (split-RRE hybridized complex and split-TAR hybridized complex) are displayed in Figure 4b. Detection of Hybridized Complexes. To detect hybridized complexes, we first mixed split-RNA probes with arbitrarily selected target-RNA (specific-sequence) and confirmed hybridized formation by a polyacrylamide gel mobility shift assay (Figure 5a). Complementary sequence of the specific sequence was prepared as negative control (nonspecific-sequence). We found that there were new mobility shifted bands, indicative of
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Figure 5. Detection of hybridized complex by luminescent signal change. (a) Construction of the hybridized complexes was confirmed by gel mobility shift assay. One-hundred picomoles of respective splitRNA probes was mixed with 50 pmol of specific-sequence or nonspecific-sequence. After 60 min of incubation at 37 °C, they were resolved on a 15% polyacrylamide gel and stained by EtBr. (b) An aliquot amount of hybridized complexes constructed in part a (50 pmol of split-RNA probes and 25 pmol of target-RNA) was mixed with 1 µL of translated products. Luminescent signals were evaluated after the binding reaction.
the hybridized complex, in the sample lanes including all components, split-RNA probe pair and specific-sequence (lane No. 8). To check for valid reporter activity, we next investigated whether the PI-FLuc probe reflected FLuc activity changes after the addition of the hybridized complexes. Aliquot amounts of the constructed hybridized complexes were mixed with translated products of the PI-FLuc probe variants, and luminescent signals were analyzed after a 30-min incubation at room temperature (Figure 5b). Three PI-FLuc probe variants (Nos. 4, 6, 8) showed FLuc activity changes corresponding to the addition of just the appropriate RNA-target. The Rev-peptideinserted PI-FLuc probe variant showed FLuc inactivation with the addition of the hybridized complex constructed from the split-RRE-RNA probe (split-RRE hybridized complex). The Tatpeptide-inserted PI-FLuc probe variants showed FLuc reactivation with the addition of the hybridized complex constructed from the split-TAR-RNA probe (split-TAR hybridized complex). Further, neither showed FLuc activity changes with hybridized complexes constructed from nonspecific-sequence or nonspecific split-RNA probes. These results confirm that the observed FLuc activity changes were mediated by the binding of the correct hybridized complex, recognized by the inserted ARM peptide. Homogeneous Detection of Arbitrarily-Selected TargetRNA. Arbitrarily selected target-RNA can also be homogeneously detected by FLuc activity changes. Figure 6 shows the target-RNA concentration dependency in a homogeneous assay. All PI-FLuc probe variants (Nos. 4, 6, 8) decreased or increased their luminescent signals through the intramolecular FLuc discomplementation or complementation. These probes also showed good concentration dependency with the specificsequence, as well as no signal changes in the presence of nonspecific-sequence. Especially in the case of Tat-peptideinserted PI-FLuc probe variants, relatively small error bars were
Figure 6. Homogeneous detection of arbitrarily selected target-RNA by intramolecular luciferase complementation assay. Varied concentrations of the target-RNA were mixed with a pair of split-RNA probes (final concentration at 500 nM) and 2 µL of translated products. Luminescent signals were evaluated after binding reaction. Values were average with standard deviation (n ) 3).
observed at low target-RNA concentrations, and we obtained a significant increase of luminescent signal at 10 nM concentration. These results suggest that arbitrarily selected target-RNA was homogeneously and specifically detected by the cooperative function of PI-FLuc and split-RNA probes.
DISCUSSION This study attempted to construct peptide-inserted FLuc (PIFLuc) probes producing a high sensitivity luminescent signal mediated by intramolecular luciferase complementation in response to target biomolecules. Two essential factors were presumed to be required to achieve this objective. First, the PIFLuc probe must be able to recognize target molecules reflecting inserted peptide characteristics. Second, the PI-FLuc probe must partly retain luciferase activity after peptide insertion. Consequently, we decided to construct several PI-FLuc probe variant patterns. Previously reported crystal structure analysis had demonstrated that FLuc could be separated to two distinct
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domains (NFLuc and CFLuc) connected by a flexible linkerlike amino acid sequence (12, 36) Additionally, previously constructed intermolecular FLuc complementation probes had been divided into two parts around a linker-like amino acid sequence (9, 12, 14). This indicates that the linker-like region is not essential for enzymatic activity and can accept some degree of structural configuration variation. Making use of these results, we selected both ends of the linker-like amino acid sequence as the basis for FLuc dividing points, and eight variants of a PI-FLuc probe were constructed (Figure 1a). The binding ability of PI-FLuc probes was confirmed by native-PAGE Western blotting (Figure 2b). We proposed that the converged signal bands, especially in the lanes with RRERNA (Nos. 1-4) and TAR-RNA (Nos. 5-8), indicated structural stabilization of PI-FLuc probes after the binding of respective RNA-targets, and that the spread signal bands in the lanes with Rev-aptamer (Nos. 1-3) indicated structural ambiguity after the binding of Rev-aptamer. We suspect that the complexes efficiently migrated in native-PAGE and transferred to PVDF membrane due to the negative charge of bound RNA, while the monomeric PI-FLuc probes in control and nonspecific RNA lanes did not migrate and transfer due to the positive charge of the inserted ARM peptide. In contrast, the monomeric wild type FLuc migrated in native-PAGE and an obvious signal band was observed by Western blotting, although band patterns were not changed by the addition of RNA (data not shown). Retained FLuc activities and its alteration were evaluated via luminescent signal changes after RNA binding (Figure 2c and Figure 3). Since we could assess the expression levels of PIFLuc probes equal via Western blotting (Figure 1c), we estimated that the variants Nos. 2, 4, 6, 8 retained FLuc activities approximately 0.01-0.03% of wild type FLuc. Although it was relatively low compared to our expectations, variants Nos. 4, 6, 8 showed dose-dependent FLuc inactivation or reactivation after RNA addition. We hypothesize that FLuc inactivation in the Rev-peptide-inserted variant is due to the intramolecular discomplementaion of divided FLuc domains mediated by intruding RNA (Figure 1a, left). We also suggest that FLuc reactivation in the Tat-peptide-inserted variants is due to the intramolecular complementation of divided FLuc domains mediated by domain contiguity outside TAR-RNA, because Tatpeptide takes on a β-sheet-like turned conformation in conjunction with TAR-RNA (Figure 1a, right). In addition, in the case of Tat-peptide-inserted variants, it seems that variant No. 6 shows a more effective structural reconfiguration after TARRNA binding, because No. 6 had higher relative FLuc activity without TAR-RNA versus with TAR-RNA addition (∼10.8) as compared to variant No. 8 (∼4.4). Moreover, both Tat-peptideinserted variants displayed significant luminescent signal increases at 1.0 nM TAR-RNA concentration. Given these PIFLuc probe characteristics, we conclude that the Tat-peptideinserted PI-FLuc probes are potentially sensitive and useful RNA indicators, because their relative activity levels were higher and their RNA detection limits were lower than our previous FRET protein probe (26). To expand the versatility of the intramolecular FLuc complementation assay system, we attempted to detect an arbitrarily selected target-RNA through the formation of hybridized complexes. From Figure 5a, it was suggested that some expanded bands, observed in the case of the split-TAR-RNA probe, represented structural variations of the split-TAR hybridized complex. Then, the arbitrarily selected target-RNA was dose-dependently detected under homogeneous and biocompatible conditions, namely, simultaneous incubation of all probe components at 37 °C (Figure 6). Particularly in the case of Tatpeptide-inserted variants No. 6 and No. 8, the resulting luminescent signal saturated when it reached 100 nM target-
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RNA levels. This corresponds to the detection of TAR-RNA alone, because the mixed PI-FLuc probes were at twice the volume of Figure 3, whose signal saturations were observed to be at 50 nM TAR-RNA levels. Furthermore, the relative FLuc activities after saturation of the signal increase (2.0-2.5) were at lower levels compared to the detection of TAR-RNA alone. We believe that the reason for lower relative FLuc activity in this case is due to the tertiary structure of the hybridized complex, specifically the more bulky and rigid structure of the target-RNA hybridized complex. Even so, the 10 nM detection limit of the arbitrarily selected target-RNA is relatively lower than other intramoleculer FRET detection or intramolecular enzyme complementation probes (15, 16, 20, 21). In addition, we expect that this response could be improved by optimizing the design of hybridized complexes, as demonstrated in our previous report (26). In this RNA detection system, the increase or decrease of luminescent signals is due to the intramolecular complementation or discomplementation of internally divided FLuc domains mediated by the inserted peptide’s conformations changes. To extend this idea, recently peptides have drawn attention in the form of molecular recognition materials, such as peptide arrays and peptide aptamers (37, 38). Some of these peptides may also have the necessary characteristics to show induced fit conformational changes after binding to their target molecules (3941). If so, it is expected that the construction of PI-FLuc probes that target biomolecules other than RNA molecules would be possible by the same methodology. However, we suppose some more improvements are required in aspects of absolute activity and relative activity, because 10-s counting was needed to obtain sufficient luminescent signals and S/N values. It would require certain trial and error tests to optimize PI-FLuc variants. Even though these potential problems exist, we envisage that PI-FLuc probes could be an alternative strategy to the intramolecular FRET detection system for target biomolecules especially for in vivo detection, because PI-FLuc probes can be produced in living cells.
ACKNOWLEDGMENT We thank Takashi Masaoka for assistance with translation of PI-FLuc probe variants using wheat germ extract. This study was in part supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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