ECHO-LNA Conjugates: Hybridization-Sensitive Fluorescence and Its

Nov 19, 2010 - Lower-case and lower-case italic characters represent LNA and 2′-O-methyl .... Gray lines, probes before hybridization; black lines, ...
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Bioconjugate Chem. 2010, 21, 2276–2281

ECHO-LNA Conjugates: Hybridization-Sensitive Fluorescence and Its Application to Fluorescent Detection of Various RNA Strands Kaori Sugizaki† and Akimitsu Okamoto*,‡ RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, and PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Received June 30, 2010; Revised Manuscript Received August 23, 2010

Hybridization-sensitive fluorescent DNA probes containing the nucleotide units of locked nucleic acid (LNA) have been developed. Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes that incorporated LNA nucleotides achieved high thermostability of the hybrid with target RNA strands. The appropriately designed ECHO-LNA chimeric probes exhibited an effective on-off switching property of fluorescence depending on hybridization with RNA and facilitated fluorescent detection of the TAR RNA strand forming a hairpin structure and distinction of one base difference in PLAC4 RNA sequence.

INTRODUCTION Ribonucleic acid (RNA) is one of many essential biomolecules responsible for the expression, maintenance, and control of cell functions. RNA is highly diverse in terms of size, structure, mass, function, location, and expression timing, as well as in sequences (1). In particular, the structural and sequential diversity of RNA, such as higher-ordered structure formation and one-base mutations, plays an important role in characterizing the function of RNA. Therefore, effective detection of such RNA strands is very important for understanding the cell functions. Many fluorescent nucleic acid probes have been developed for observation of the functions of target nucleic acids (2-4). In particular, much effort has been invested in the rational design of hybridization-sensitive fluorescent probes to detect effectively the target nucleic acid using on-off switching of fluorescence depending on hybridization (5-9). However, the functions of these probes are insufficient for effective detection of a variety of RNA molecules, because accessing the RNA sequence that forms a higher-ordered structure and distinguishing the target RNA sequence from the one-base mutants are often thermodynamically difficult for fluorescent nucleic acid probes. Incorporation of the nucleotide units of locked nucleic acid (LNA) into hybridization-sensitive probes may provide one of the more effective methods for solving such problems (10). Chimeric probes having select positions modified with LNA nucleotides have been reported to enhance duplex stability (11). In addition, LNA trinucleotides enhance the thermostability of the duplex formed with the complementary sequences, whereas the duplex stability with mismatched sequences is much lower (12-14). Incorporation of LNA nucleotides into hybridization-sensitive fluorescent DNA probes would make fluorometric detection of a variety of RNA molecules more effective. However, the bridged sugar structure of LNA nucleotides may diminish the function of hybridization-sensitive on-off switching of fluorescence. To choose the correct course for design of the fluorescent probe sequence including LNA nucleotides, the * Phone: +81-48-467-9238. Fax: +81-48-467-9205. E-mail: [email protected]. † RIKEN Advanced Science Institute. ‡ PRESTO.

effect of LNA incorporation on the fluorescence intensity of probes needs to be estimated through several model experiments. In this paper, we report fluorescence properties of hybridization-sensitive fluorescent probes possessing LNA nucleotides. Incorporation of LNA nucleotides into hybridization-sensitive fluorescent DNA probes provided higher thermostability with the complementary RNA strand compared with probes consisting of only 2′-deoxyribonucleotides. In addition, fluorescent detection of the RNA strand forming a hairpin structure and distinction of a one-base difference in an RNA sequence were facilitated by LNA-incorporated fluorescent probes.

EXPERIMENTAL PROCEDURES General. Reverse-phase HPLC was performed on Chemcobond 5-ODS-H columns (10 × 150 mm) with a Gilson Chromatograph, model 305, using a Jasco multiwavelength detector, MD-2015Plus. MALDI-TOF mass spectra were measured with a Bruker Daltonics Reflex apparatus. Absorption and fluorescence spectra were recorded on a Shimadzu UV-2550 spectrophotometer and RF-5300PC spectrofluorophotometer, respectively. Probe Synthesis. Oligonucleotides containing both LNA nucleotides and a diamino-modified nucleotide, which is a precursor of the D514 nucleotide, were synthesized by Gene Design (Osaka, Japan). RNA was synthesized by Japan Bio Service. DNA oligomers were synthesized by a conventional phosphoramidite method on a DNA/RNA synthesizer. Oligonucleotides containing a diamino-modified nucleotide and a 2′O-methyl RNA nucleotide were synthesized through a conventional phosphoramidite method by using an H-8 DNA/RNA autosynthesizer (Nihon Techno Service). The diamino-modified nucleoside phosphoramidite was synthesized according to our previous report (15). The synthesized oligonucleotides were cleaved from the support with 28% aqueous ammonia and deprotected at 25 °C for 16 h. After removal of ammonia from the solution under reduced pressure, the DNA was purified by reverse-phase HPLC, followed by elution with a solvent mixture of 0.1 M triethylammonium acetate (TEAA) (pH ) 7.0) and linear gradient over 20 min from 5% to 30% acetonitrile at a flow rate of 3.0 mL/min. The concentration of the oligonucleotides was determined by absorption at 260 nm. A solution of the N-hydroxysuccinimidyl ester of thiazole orange pentanoic acid (15) (40 mM, 22 equiv to an active amino

10.1021/bc1002949  2010 American Chemical Society Published on Web 11/19/2010

ECHO-LNA Conjugates

group of an oligonucleotide) in DMF was added to a deprotected oligonucleotide solution (164 µM) in 100 mM sodium carbonate buffer (pH ) 9.0) and incubated at 25 °C for 30 min. The reaction mixture was diluted with ethanol. After centrifuging at 4 °C for 20 min, the supernatant liquid was removed. The residue was dissolved in a small amount of water, and then the solution was passed through a 0.45 µm filter. The product was purified by reverse-phase HPLC on a 5-ODS-H column, elution with a solvent mixture of 0.1 M TEAA (pH ) 7.0) and linear gradient over 30 min from 5% to 30% acetonitrile at a flow rate of 3.0 mL/min. The product was subsequently desalted by reverse-phase HPLC on a 5-ODS-H column, elution with a solvent mixture of deionized water, and linear gradient over 20 min from 0% to 100% acetonitrile at a flow rate of 3.0 mL/ min. The concentration of the oligonucleotides was determined by absorption at 260 nm. The oligonucleotides were identified by MALDI-TOF mass spectrometry. (Here, the molecular weight of the counteranions of dyes was not included in the value of M, and D514 denotes a doubly thiazole orange-labeled deoxynucleotide. Lower-case and lower-case italic characters represent LNA and 2′-O-methyl RNA nucleotides, respectively. The LNA nucleotide “c” denotes a 5-methylcytosine LNA nucleotide, and the 2′-Omethyl RNA nucleotide “u” denotes a uracil 2′-O-methyl RNA nucleotide.): DNA(0), CGCAATD514TAACGC, calcd for C180H217N56O78P12S2 ([M - H]+) 4848.8, found 4851.4; LNA(-4), CGcAATD514TAACGC, calcd for C182H220N56O79P12S2 ([M H]+) 4891.8, found 4891.3; LNA(-3), CGCaATD514TAACGC, calcd for C181H218N56O79P12S2 ([M - H]+) 4877.8, found 4878.9; LNA(-2), CGCAaTD514TAACGC, calcd for C181H218N56O79P12S2 ([M - H]+) 4877.8, found 4878.0; LNA(-1), CGCAAtD514TAACGC, calcd for C181H218N56O79P12S2 ([M - H]+) 4877.8, found 4880.0; LNA(+1), CGCAATD514tAACGC, calcd for C181H218N56O79P12S2 ([M - H]+) 4877.8, found 4879.8; LNA(+2), CGCAATD514TaACGC, calcd for C181H218N56O79P12S2 ([M - H]+) 4877.8, found 4878.9; LNA(+3), CGCAATD514TAaCGC, calcd for C181H218N56O79P12S2 ([M - H]+) 4877.8, found 4878.3; LNA(+4), CGCAATD514TAAcGC, calcd for C182H220N56O79P12S2 ([M - H]+) 4891.8, found 4893.9; OMe(-4), CGcAATD514TAACGC, calcd for C181H220N56O79P12S2 ([M - H]+) 4879.8, found 4879.1; OMe(-3), CGCaATD514TAACGC, calcd for C181H220N56O79P12S2 ([M - H]+) 4879.8, found 4879.2; OMe(-2), CGCAaTD514TAACGC, calcd for C181H220N56O79P12S2 ([M - H]+) 4879.8, found 4879.6; OMe(-1), CGCAAuD514TAACGC, calcd for C180H218N56O79P12S2 ([M - H]+) 4865.8, found 4865.5; OMe(+1), CGCAATD514uAACGC, calcd for C180H218N56O79P12S2 ([M - H]+) 4865.8, found 4865.3; OMe(+2), CGCAATD514TaACGC, calcd for C181H220N56O79P12S2 ([M - H]+) 4879.8, found 4880.6; OMe(+3), CGCAATD514TAaCGC, calcd for C181H220N56O79P12S2 ([M - H]+) 4879.8, found 4879.9; OMe(+4), CGCAATD514TAAcGC, calcd for C181H220N56O79P12S2 ([M - H]+) 4879.8, found 4879.5; LNA(anti-TAR), GCtCcCaGGCD514CAGaT, calcd for C213H256N67O101P15S2 ([M - H]+) 5899.4, found 5902.0; DNA(anti-TAR), GCTCCCAGGCD514CAGAT, calcd for C208H254N67O97P15S2 ([M - H]+) 5773.4, found 5771.4; LNA(anti-PLAC4-C), AGD514TAGacgA, calcd for C157H183N50O62P9S2 ([M - H]+) 4105.3, found 4107.3; DNA(anti-PLAC4-C), AGD514TAGACGA, calcd for C153H181N50O59P9S2 ([M - H]+) 4007.3, found 4008.5; LNA(antiPLAC4-T), AGD514TAGatgA, calcd for C157H182N49O63P9S2 ([M - H]+) 4106.3, found 4106.8; DNA(anti-PLAC4-T), AGD514TAGATGA, calcd for C154H182N49O60P9S2 ([M - H]+) 4022.3, found 4022.8. Melting Temperature. The melting temperature (Tm) values of duplexes (1.0 µM) were measured in 50 mM sodium phosphate (pH ) 7.0) containing 100 mM sodium chloride. The absorbance of the samples was monitored at 260 nm from 10

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Figure 1. Structures and functions of the fluorescent probes. (a) A hybridization-sensitive fluorescent nucleotide D514 and an LNA nucleotide. (b) Schematic representation of the energy levels of the singleand double-stranded states of the hybridization-sensitive fluorescent DNA probe controlled by excitonic interaction between the fluorescent dyes of D514. (c) Probe sequences in this study.

to 90 °C with a heating rate of 0.5 °C min-1. From these profiles, first derivatives were calculated to determine the value of Tm. Absorption and Fluorescence Spectra. Absorption and fluorescence spectra of the fluorescent probes (0.4 µM) in the absence or presence of the target RNA (0.4 µM) were measured in 50 mM sodium phosphate (pH ) 7.0) containing 100 mM sodium chloride using a cell with a 1 cm path length. The excitation and emission bandwidths were 1.5 nm. A water circulator EYELA NCB-1200 was connected to the spectrofluorophotometer for programmed temperature control in Figures 5 and 6. CD Spectra. CD spectra of the fluorescent probes (2.5 µM) in the absence or presence of the target RNA (2.5 µM) were measured in 50 mM sodium phosphate (pH ) 7.0) containing 100 mM sodium chloride using a cell with a 0.1 cm path length.

RESULTS AND DISCUSSION ECHO-LNA Chimeric Probe. We prepared a hybridizationsensitive fluorescent probe including an LNA nucleotide. Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes have been developed for the function of hybridization-sensitive on-off switching of fluorescence (Figure 1) (15, 16). This DNA probe is a new type of hybridization-sensitive probe, which has a doubly fluorescencelabeled nucleotide to achieve high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching of a single-stranded probe (17-22). An excitonic interaction between the fluorescence dyes linked covalently to a single

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Figure 2. Absorption and emission spectra of ECHO probes. Gray lines, probes before hybridization; black lines, probes hybridized with the corresponding complementary RNA. Emission spectra were recorded with excitation at 488 nm.

nucleotide in a probe was produced by the formation of an H-aggregate between dyes, and as a result, emission from the probe was suppressed through splitting of the excited state (23). Dissociation of dye aggregates by hybridization with the complementary RNA resulted in disruption of the excitonic interaction and a strong emission from the hybrid. We initially synthesized a probe with an LNA nucleotide incorporated next to the thiazole orange dye-labeled nucleotide (D514). The chimeric probe LNA(+1) showed hybridizationsensitive fluorescence emission at 531 nm when hybridized with the complementary RNA, but the fluorescence emission of the probe itself was much weaker (Figure 2a). A shift in the absorption band, which is characteristic for H-aggregation of dyes, was also observed. The absorption shift and fluorescence switching suggest that the fluorescence emission of ECHO probes modified by an LNA nucleotide was controlled by excitonic interaction between fluorescence dyes. However, the fluorescence intensity of the LNA(+1)-RNA hybrid at 531 nm was only about half that of the DNA(0)-RNA hybrid, which includes no LNA nucleotides (Figure 2b), although the Tm of the LNA(+1)-RNA hybrid (52 °C) was 5 °C higher than that of the DNA(0)-RNA hybrid. In addition, the absorbance of LNA(+1) was lower than that of DNA(0). These weak photophysical signals of LNA(+1) may suggest that the intercalation or aggregation of thiazole orange dyes linked to LNA(+1) was incomplete, as reported in the studies on the photophysics of free thiazole orange dye (24, 25). The reason for these photophysical behaviors of LNA(+1) was not the existence of an LNA nucleotide in the probe, but the location of LNA incorporation. Another LNA probe LNA(+4) showed high fluorescence intensity after hybridization with RNA (Figure 2c), as well as formation of a highly thermostable hybrid (58 °C). The photophysical behavior of LNA(+4) was close to that of the DNA(0). Incorporation of an LNA nucleotide into a

Sugizaki and Okamoto

Figure 3. Effect of site incorporating LNA on the intensity of fluorescence emission and induced CD. (a) Intensity of fluorescence emission at 532 nm and induced CD at 520 nm. The fluorescence intensities of LNA-incorporated probes (open circles), and their hybrids with complementary RNA (closed circles), 2′-O-methyl RNAincorporated probes (open squares), and their hybrids with complementary RNA (closed squares) are presented, together with the fluorescence intensity of the probe with an unmodified DNA backbone, DNA(0) (an open triangle and an accompanying dot line), and its hybrid with the complementary RNA (a closed triangle and an accompanying dotted line). The intensities of the induced CD of LNA-incorporated probes are shown by the height of the bars at each LNA incorporation site. (b) Typical CD spectra showing a strong or negligible induced CD signal. Gray and black lines are CD spectra of LNA(+1) and LNA(+4) hybridized with the complementary RNA, respectively.

different site in the probe resulted in different absorption and fluorescence spectra. Effect of Location of the Incorporated LNA Nucleotide. In the case of LNA(+1), incorporation of an LNA nucleotide into ECHO probes induced a decrease in the fluorescence intensity of the hybrid with complementary RNA, although the hybrid showed higher stability and maintained its hybridizationsensitive emission property. The design of the probe containing LNA nucleotides, i.e., determination of where we should insert LNA nucleotides, is a key point for obtaining an effective fluorescent probe. We next prepared several chimeric probes with LNA nucleotides incorporated at different sites in the probe sequence (Figure 1c). The Tm values of the hybrids formed by the ECHO-LNA probes and the complementary RNA were 4-11 °C higher than that of the DNA(0)-RNA hybrid. The incorporation of an LNA nucleotide made the duplex with the complementary RNA more stable, regardless of the incorporation site. In contrast, the fluorescence intensities of the hybrids of chimeric probes and complementary RNA strongly depended on the LNA insertion site. The fluorescence intensities of the hybrid with RNA by LNA(-2), LNA(-1), LNA(+2), and LNA(+3) were low, and likewise for the LNA(+1)-RNA hybrid (Figure 3a). On the other hand, LNA(-4), LNA(-3), and LNA(+4), where an LNA nucleotide was located at the site far from the dye nucleotide D514, showed high fluorescence intensity after hybridization with RNA, close to the fluorescence intensity of the DNA(0)-RNA hybrid. Fluorescence of ECHO probes has been reported to be induced by dissociation of H-aggregation and intercalation of dyes into stacking base pairs (15, 16). Incorporation of LNA nucleotides into the sites close to D514 may be unfavorable for the binding of two dyes of D514 to the hybrid structure.

ECHO-LNA Conjugates

LNA would slightly change the duplex structure from the structure of the DNA(0)-RNA hybrid, because the fixed puckering of LNA bridged ribose (C3′-endo) is close to that of RNA ribose but not DNA 2′-deoxyribose (C2′-endo) (26). However, sugar puckering may be unimportant for fluorescence intensity in the present case. The chimeric probes in which LNA nucleotides were replaced with a 2′-O-methyl RNA nucleoside (C3′-endo) kept the high fluorescence intensity of the hybrid with complementary RNA regardless of the site of 2′-O-methyl RNA nucleoside incorporation. Therefore, the effect of partial conformational change by sugar puckering on the fluorescence intensity in an ECHO probe was not large. The CD spectra of LNA-containing probes provided us with key information on the intercalation of D514 thiazole orange dyes. The CD spectra of the hybrid of LNA(+4) with RNA showed a strong positive Cotton effect at 520 nm (Figure 3b), which was also observed for the DNA(0)-RNA hybrid. On the other hand, the hybrid formed by RNA and LNA(+1), containing an LNA nucleotide located close to a D514 nucleotide, showed a very weak induced CD signal. These weak signals indicated that binding of D514 thiazole orange dyes was unsuccessful in the hybrid, suggesting that incorporation of an LNA nucleoside made the dye intercalation from the major groove side inefficient. The rigidity of the LNA sugar unit is disadvantageous for dye intercalation because dye intercalation requires the flexibility of sugar-phosphate backbones to extend the distance between base pairs (27). Actually, hybrids of the probes containing a lowly rigid 2′-O-methyl RNA nucleoside and complementary RNA showed an induced CD at 520 nm regardless of incorporation sites. Intercalation of D514 dyes into the hybrid structure is a key factor for dissociation of the dye aggregate and fluorescence emission. As shown in Figure 3a, the LNA incorporation sites of the probes showing a weak CD signal were consistent with the LNA incorporation sites of the probes showing low fluorescence intensity. Therefore, the rigidity of the LNA sugar unit resulted in a decrease in the fluorescence intensity of the probe through intercalation inhibition. The LNA incorporation sites where the fluorescence intensity of the hybrid decreased were not distributed symmetrically around D514 (two nucleotides of 5′ side of D514 and three of 3′ side) (Figure 4). This asymmetry can be explained by considering the distance that the dyes can reach through a linker in the right-handed double helical structure of the hybrid. To obtain an effective ECHO-LNA conjugate with high fluorescence intensity, LNA nucleotides should be incorporated into the probe, avoiding the positions marked with closed triangles in Figure 4. Fluorescence Detection of RNA with Higher-Ordered Structure: The Example of HIV-1 TAR RNA. Insertion of LNA nucleotides into the hybridization-sensitive fluorescent probes would facilitate analysis of higher-ordered structures (28). HIV-1 TAR RNA, which is the transactivation responsive element located at the 5′-end of HIV-1 mRNA (29), is a small RNA hairpin consisting of a stem-loop structure with a threenucleotide bulge, which is essential for Tat protein recognition and activity (30, 31). We designed the 16-mer ECHO probe (LNA(anti-TAR)) for fluorescent detection of the TAR RNA stem-loop structure (Tm ) 57 °C) (Figure 5). Four LNA nucleotides were incorporated into the probe to gain high binding affinity to TAR RNA. These LNA nucleotides lay at least three nucleotides apart from D514 to not lose the strong fluorescence of hybrid by LNA incorporation as described above. LNA(antiTAR) before hybridization showed low fluorescence intensity, which was at the same level as that of a DNA probe DNA(antiTAR). On the other hand, LNA(anti-TAR) showed more effective on-off switching of fluorescence emission, and the fluorescence intensity of LNA(anti-TAR) was higher after

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Figure 4. Binding site of D514 thiazole orange dyes proposed from fluorescence intensities and induced CD spectra of the hybrid with RNA. Thiazole oranges are represented by gray rectangles. The replacement by LNA nucleotides in the positions indicated with closed triangles resulted in hybrids with low fluorescence intensity and low induced CD signals.

Figure 5. Fluorescence intensities of ECHO probes binding to TAR RNA. The fluorescence intensities at 531 nm at different temperatures are plotted with open circles for LNA(anti-TAR), closed circles for its hybrid with TAR RNA, open triangles for DNA(anti-TAR), and closed triangles for its hybrid with TAR RNA.

hybridization with TAR RNA than that of DNA(anti-TAR). The Tm of the hybrid of LNA(anti-TAR) and TAR RNA was 56 °C, i.e., 11 °C higher than that of the hybrid of DNA(antiTAR) and TAR RNA (45 °C). Thus, the largest difference in their fluorescence intensities was observed for the fluorescence measurement at 60 °C, which was close to the Tm values observed for a stem-loop TAR RNA and an LNA(anti-TAR)TAR hybrid. The higher fluorescence intensity of the hybrid formed by a well-designed LNA-incorporating probe suggested that use of LNA nucleotides and temperature control was

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clear fluorescence detection of SNP. When the fluorescence intensity was measured at 50 °C, i.e., between the Tm values of the matched and mismatched hybrids of LNA-containing probes and PLAC4 RNA, the emission at 531 nm was selective for matched hybrids (Figure 6). LNA-containing probes distinguished a one-nucleotide difference of the target RNA more sensitively compared with DNA probes. Combination of a hybridization-sensitive fluorescent probe and an SNP-recognizing LNA trinucleotide was effective for mix-and-read fluorescent SNP typing. In conclusion, we have designed ECHO probes showing sequence-selective fluorescence for higher-ordered RNA and RNA SNPs through the incorporation of LNA nucleotides into appropriate sites of the probes. This probe achieved high thermostability of the hybrid with target RNA strands and fluorescence detection of higher-ordered RNA and single base alteration. Although further aspects remain to be examined toward an easier-to-use intracellular RNA analysis, such as molecular design for higher sensitivity and selectivity of a small amount of the target RNA, we anticipate that this new concept of the hybridization-sensitive fluorescent probe supported by the nucleic acid chemistry and photochemical basis will be the starting point for the development of a practical assay for detection of RNAs in vivo.

ACKNOWLEDGMENT We thank Dr. Takehiro Suzuki (RIKEN) for the MALDITOF mass spectrometry and Dr. Shuji Ikeda (RIKEN) for valuable discussions on the present study. Supporting Information Available: Photophysical data (three tables). This material is available free of charge via the Internet at http://pubs.acs.org. Figure 6. Fluorometric detection of the single nucleotide polymorphism (SNP) of PLAC4 RNA. (a) Sequences of a PLAC4 RNA fragment including a G/A SNP site and ECHO probes for detection of PLAC4 RNA. (b) Fluorescence spectra of LNA(anti-PLAC4-C). The temperature was 50 °C, and excitation wavelength was 488 nm. (c) Ratio of the fluorescence intensities of hybrids (Ids) to that of probe alone (Iss). The temperature was 50 °C for LNA probes and 25 °C for DNA probes.

effective for clearer on-off fluorescence observation of an RNA strand with a higher-ordered structure. Fluorescence Detection of Single Nucleotide Alteration of RNA: The Example of PLAC4 RNA. LNA is also effective for mismatch discrimination. A one-base difference in a genome sequence is well-known as a single-nucleotide polymorphism (SNP), and this sequence variation is among the very important research targets for progress of gene diagnosis and personalized medicine. LNA trinucleotides have been reported as being useful for recognition of one-base alteration (32). We next designed the ECHO probe for fluorescent SNP detection. The target SNP was placenta-specific 4 (PLAC4) rs130833, which is an SNP located in the transcribed regions of placental-expressed mRNA in maternal plasma (33, 34). We designed ECHO-LNA conjugates, in which the LNA trinucleotide recognizes the target SNP site of PLAC4 RNA, and compared the SNP selectivity of fluorescence emission with DNA probes. ECHO-LNA conjugate probes, LNA(anti-PLAC4-C) and LNA(anti-PLAC4-T), showed high Tm values for hybridization with matched PLAC4 RNA sequences (61 and 55 °C, respectively) but much lower Tm values with mismatched sequences (34 and 44 °C, respectively). On the other hand, the gap in Tm values of DNA probes for hybridization with matched and mismatched RNA was less than 3 °C, although the matched hybrid was only slightly more thermostable. The large gap between Tm values of matched and mismatched hybrids observed for LNA-incorporating probes meant that appropriate temperature control was effective for

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