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© 1995 Oxford University Press 4992-4997

Footnote

Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucleotides. Homogeneous quantitative monitoring of in vitro transcription

Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucleotides. Homogeneous quantitative monitoring of in vitro transcription Takahiko Ishiguro*, Juichi Saitoh, Hideo Yawata, Masami Otsuka1, Teruhiko Inoue1 and Yukio Sugiura1

Tokyo Research Laboratories, Tosoh Corporation, 2743-1 Hayakawa, Ayase-shi, Kanagawa 252, Japan and 1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611, Japan

Received July 30, 1996; Revised and Accepted November 6, 1996

ABSTRACT

We have developed a fluorescent DNA probe, oxazole yellow (YO)-linked oligonucleotide complementary to a target DNA/RNA, which can enhance the fluorescence on hybridizing with a target nucleotide. We demonstrated the applicability of the YO-linked oligonucleotide probe to real-time monitoring of the in vitro transcription process of a plasmid DNA constructed containing the 5'-terminus non-coded region of hepatitis C virus RNA. In the process of in vitro transcription in the presence of YO-linked complementary oligonucleotide, the fluorescence of the reaction mixture showed a time-dependent linear increase corresponding to the generated target RNA product.

INTRODUCTION

An easy and simple method has been required to detect specific base sequences of nucleic acids in clinical diagnostics or molecular pathology as well as for the elucidation of various biochemical processes associated with the expression and transmission of genetic information (1 ,2 ).

Most of the current standard methods require hybridization on solid supports, electrophoretic migration, solid-phase capture or HPLC separation for the detection of the specific nucleic acid sequences (3 ). These separation steps are tedious and time- consuming in a clinical setting and, in particular, can be one of the main obstacles for the application of PCR-based assays in laboratory diagnosis. It is necessary to control the contamination of PCR products in post-PCR processes, such as sampling PCR products, to apply them to gel electrophoresis (4 -6 ).

A homogeneous format can be a strategy to relieve the obstacles due to those separation process in practice and so far many researchers have challenged the development on several approaches (7 -8 ). Helene and co-workers reported the fluorescent detection of a double-stranded DNA in forming triple helix with donor- and acceptor-linked DNA probes in terms of their energy transfer (9 ). Sixou recently demonstrated fluorescent detection of intracellular oligonucleotide hybridization in living cells by fluorescence energy transfer (10 ).

It is known that a DNA intercalator, oxazole yellow (YO), shows marked enhancement of fluorescence on binding to double-stranded DNA while YO itself is virtually non-fluorescent in the absence of DNA (11 ).Recently, we were successful in the fluorescence monitoring of the polymerase chain reaction by adding fluorescent DNA intercalator, oxazole yellow, in the reaction mixture and the quantification of starting number of a target template without any post-PCR analysis such as electrophoresis of PCR products (12 ).

We attempted to design a fluorescent DNA probe to enable us to construct an easy and specific homogeneous method to detect a nucleic acid sequence. We report herein the property of YO-linked oligonucleotide, which can emit enhanced fluorescence by binding to a complementary oligomer, and its application to in situ fluorescence detection of a specific sequence of RNA produced by in vitro transcription.

MATERIALS AND METHODS

Preparation of YO linked DNA probe

Thiol-modified oligonucleotide 5'-AAAAA*AAAAAAAA-3' (named DAL-13) and 5'-CTCGC*GGGGGCTG-3' (named YPF-271), were supplied from Yuki Gosei Yakuhin Kogyo, Co. Ltd, Japan, in which A*and C* are adenine and cytidine having a -(CH2)2NHCO(CH2)2SH appendage at 3' phosphorus, respectively. The nucleic acid sequence, 5'-CTCGC*GGGGGC-3', of YPF-271 is complementary to the positions 223-233 of the 5'-terminus non-coded region of hepatitis C virus RNA (HCV 5'NCR) (13 ). Two bases from the 3' terminus of YPF-271, TG, are intentionally mismatched with the target RNA to avoid the extension by a polymerase.

Oligo dT (30mer) and oligo dA (30mer) were purchased from Yuki Gosei Yakuhin Kogyo, Co. Ltd, Japan.

Synthetic oligonucleotides, complementary to YPF-271, were purchased from Yuki Gosei Yakuhin Kogyo, Co. Ltd, Japan, for DNA (named YPF-271+D), and from TaKaRa, Japan, for RNA (named YPF-271+R). Sequences of each oligomer are as follows: YPF-271+D: 5'-GTGCCCCCGCGAG-3', and YPF-271+R: 5'-GUGCCCCCGCGAG-3'.


Figure 1.Synthesis of YO-linked oligonucleotide probe. The details are described in the text.

YO-(dA)13. (Fig. 1 ) Aqueous dithiothreitol (0.01 M, 20 [mu]l) was added to the fraction containing purified DAL-13 by HPLC and shaken by avortex mixer (solution A). YO(CH2)3I (14 ) was saturated in a mixture of DMF (200 [mu]l), 1.0 M phosphate buffer (pH 10.0, 300 [mu]l), and H2O (500 [mu]l) under argon (solution B). Solutions A and B were mixed under strictly unaerated argon atmosphere (solution A:solution B = 1:2 ~ 1:3).The mixture stood for 2 h and was purified by Sephadex G-25 (5% acetonitrile/0.1 M triethylamine-acetic acid, pH 7.0). The purified fraction was concentrated in vacuo, and dried. The resultant was dissolved by distilled water, and further purified by HPLC (column: VX-Nucleotide, Shinwa Chemical, Japan/linear gradient mode from buffer (a) to buffer (b) [(a) 5% acetonitrile, (b) 50% acetonitrile, both contain 0.1 M triethylamine-acetic acid (pH 7.0)]. The fraction obtained was also concentrated in vacuo and dried to remove triethylamine-acetic acid thoroughly so as to give the YO derivative of DAL-13 [named YO-(dA)13] finally.

YO-YPF-271. Thiol-modified oligonucleotide (YPF-271) was reacted with YO(CH2)3I and purified, according to the same procedure as described above, to give the corresponding YO derivative (named YO-YPF-271).

DNA fragment bearing RNA polymerase promoter region

We constructed a plasmid DNA SKP/SR1-P2-6 which contains a T7 promoter, XhoI site, HCV 5' noncoding region (290 bp), HindIII site and T3 promoter in the order and orientation shown in Figure 2 . The details of the construction of the plasmid were described in our previous report (12 ).


Figure 2. Plasmid DNA SKP/SR1-P2-6 contains HCV 5' NCR sequences (290 bp).

SKP/SR1-P2-6 was digested with HindIII and XhoI to result in linear DNAs of HCV 5' NCR sequence containing T7 promoter and T3 promoter, respectively, followed by purification with HPLC.

Hybridization and fluorescence measurement

Each of dT (30mer) and dA (30mer), final concentration 5 nM, was added into a hybridization buffer of 40 mM Tris-HCl, pH 8.0, containing dithiothreitol (5 mM), MgCl2 (8 mM), BSA (50 [mu]g/ml) and YO-(dA)13 (5 nM).

Each of YPF-271+D and YPF-271+R, final concentration 50 nM, was added into a hybridization buffer (1* SSC, 1 mM EDTA), containing YO-YPF-271 (50 nM).

A volume of 500 [mu]l of the resultant mixture was transferred into a quartz cuvette (1 * 1 * 4.4 cm, 3.5 ml) in temperature control module supplied with the spectrometer, model FP-777, followed by the measurement of the fluorescence spectra (excitation wavelength 490 nm/HW 5 nm, fluorescence spectrometer model FP-777, Jasco, Japan).

Melting temperature

Each of YPF-271 and YO-YPF-271, final concentration 1.5 [mu]M, was added into a hybridization buffer (1* SSC, 1 mM EDTA, pH 7.0), containing a synthetic complementary sequences as a target (YPF-271+D: 5'-GTGCCCCCGCGAG-3').

Tm values were obtained as the temperature of half dissociation on the basis of the measurement of the hyperchromic effect of UV absorption at 260 nm (UV spectrometer model U-2000, Hitachi, Japan).


Figure 3.Models of YO-linked oligonucleotide (A) and the hybridized complex with oligo dT (B), calculated and drawn by HyperChem Nucleic Acids Databases (Hypercube, Inc., Canada). The bold lines indicate YO moiety.

In vitro transcription

In vitro transcription of SKP/SR1-P2-6/HindIII and SKP/SR1-P2-6/XhoI were performed by T7 (TaKaRa, Japan) or T3 (Toyobo, Japan) RNA polymerase (0.1 U/[mu]l), respectively, in a transcription buffer of 40 mM Tris-HCl, pH 8.0, containing dithiothreitol (5 mM), MgCl2 (8 mM), rNTP (0.4 mM), template DNA (0.015 mg/ml), YO-YPF-271 (0.025 pmol/[mu]l), RNase inhibitor (2 U/[mu]l).

As shown in Figure 2 , the resultant of the digestion with HindIII (SKP/SR1-P2-6/HindIII) affords to produce RNA complementary to YO-YPF-271 by in vitro transcription using T7 RNA polymerase. On the other hand, in vitro transcription of the digested fragment with XhoI (SKP/SR1-P2-6/XhoI) using T3 RNA polymerase does not produce RNA complementary to YO-YPF-271.


Figure 4.Fluorescence spectra (excitation at 490 nm) of YO-linked oligo dA probe in presence of oligo dT 30mer (A), dA 30mer (B) and recombinant HCV RNA (C), respectively. In (A), the broken curves indicate the fluorescence spectra of YO-linked oligo dA alone.

The present concentration of the template, 0.015 mg/ml, corresponds to ~7 * 10-6 nmol/[mu]l, or 4 * 109 copies/[mu]l. SKP/SR1- P2-6 contains 3251 bp, 2961 bp + 290 bp, of which the molecular weight is estimated to be 2.1 * 106 (660 * 3251).

The reaction was carried out in the quartz cuvette in 500 [mu]l of the transcription buffer at 40oC to be monitored the fluorescence intensity with the fluorescence spectrometer. The sample was excited at 490 nm and the fluorescence emission was measured at 510 nm (HW: 5 nm).

The reaction was also performed in 700 [mu]l of the transcription buffer in measuring the fluorescence intensity (ex: 490 nm, em: 510 nm, HW: 10 nm) . Aliquots of 16 [mu]l of the reaction solution were collected from the cuvette at 5 min intervals to be applied to gel electrophoresis to quantify the product with a densitometer (ACI Image Analysis System, ACI Japan).

RESULTS AND DISCUSSION

Fluorescence enhancement of the YO-linked DNA probe by hybridization

It is generally known that some DNA intercalators bind double- stranded DNA to enhance the fluorescence. Oxazole yellow (YO), one of the fluorescent DNA intercalative dyes, shows marked enhancement of the fluorescence on binding to a double stranded DNA (11 ,12 ). It is, therefore, thought that an oligonucleotide equipped with a YO would emit enhanced fluorescence on binding to a complementary oligomer.


Figure 5.Fluorescence spectra (excitation at 490 nm) of YO-YPF-271; (a) at 70oC, (b) at 50oC, (c) at 25oC, (d) in the presence of complementary DNA, YPF-271+D, at 25oC and (e) in the presence of complementary RNA, YPF-271+R, at 25oC.

We designed a YO-linked oligonucleotide, YO-(dA)13, 5'-AAAAA*AAAAAAAA-3', in which A* is adenine having YO through a linker, -(CH2)2NHCO(CH2)2S-YO, appendage at 3' phosphorus. As shown in Figure 3 , the model of the hybridized complex with oligoT suggests that the linker of YO-(dA)13 is readily to deliver the YO to adjacent base pairs of formed double-stranded DNA with the complementary oligo T. The hybridization, thus, would bring the enhanced fluorescence.

In Figure 4 , the presence of 30mer brought marked enhancement of the fluorescence of YO-(dA)13 while YO-(dA)13 itself is virtually non-fluorescent. Non-complementary nucleic acids, dA 30mer and rHCV RNA, did not give the fluorescent enhancement of YO-(dA)13 in the mixture.

The present results imply that YO-(dA)13 recognizes the complementary nucleic acids to hybridize with it and YO moiety intercalates into formed double-stranded DNA so as to enhance the fluorescence, as YO itself showed in dye-DNA intercalation complexes.

Fluorescence monitoring of in vitro transcription

YO-linked DNA probe described in the present work would be applicable to homogeneous fluorescence detection and quantification of specific nucleic acids.

We chose hepatitis C virus (HCV) RNA as our target sequence for the detection by a YO linked DNA probe.

We designed a YO linked DNA probe, YO-YPF-271, complementary to the 5'-terminus non-coded region of hepatitis C virus RNA (HCV 5'NCR). A YO moiety was inserted at an internal cytidine, C*, of a 13mer, 5'-CTCGC*GGGGGCTG-3'.

We investigated the property of YO-YPF-271 in forming duplex on a target DNA or RNA (Fig. 5 ).

YO-YPF-271 showed the enhancement of the fluorescence in the presence of the complementary deoxynucleotide sequences (Fig. 5 c and d).


Figure 6.Fluorescence monitoring of in vitro transcription of SKP/SR1-P2-6 in the presence of YO-YPF-271 (0.025 pmol/l). (A) Transcription with T7 RNA polymerase on digested fragment of SKP/SR1-P2-6 with HindIII; (B) digested fragment of SKP/SR1-P2-6 with HindIII omitting T7 RNA polymerase and (C) transcription with T7 RNA polymerase on digested fragment of SKP/SR1-P2-6 with XhoI .


Figure 7.Gel electrophoresis confirming the production of both the complementary RNA (third lane from the right) and the non-complementary RNA (first lane from the right) in the presence of YO-YPF-271 (0.025 pmol/l) (2% agarose gels stained with 0.5 [mu]g/ml of ethidium bromide). The transcription in the absence of YO-YPF-271 are shown in the third lane from the left.

It is noted that the fluorescence intensity of YO-YPF-271 itself decreases remarkably with the increase of the temperature of the solution (Fig. 5 a-c). The intrinsic fluorescence signal is thought to be caused mainly from the formation of the secondary structure of YO-YPF-271 itself, or self-assembling in the solution, and the intercalation of the YO moiety into the double-stranded domain, while YO-(dA)13 itself cannot form such a secondary structure so as to show marked enhancement of the fluorescence on binding to the target oligo T, as well as lower intensity in the target's absence.


Figure 8.Quantification of the in vitro transcription of SKP/SR1-P2-6 under T7 promoter (YO-YPF-271: 0.025 pmol/l). (A) Increase of the fluorescence of the transcription reaction mixture. Aliquots were collected every 5 min shown by arrows. (B) Agarose gel electrophoresis of each aliquot (2% agarose gels stained with 0.5 [mu]g/ml of ethidium bromide). (C) Plot of the RNA content in each aliquot.Figure 5 d and e also reveals that YO-YPF-271 gives the fluorescence enhancement for RNA target as well as for DNA. Marked difference of the enhancement was not found in each case.

Therefore, we carried out in vitro transcription in the presence of YO-YPF-271 and monitored the fluorescence intensity of the reaction mixture in the time course (Fig. 6 ). When the HindIII digestion product of SKP/SR1-P2-6 was subjected to in vitro transcription using T7 RNA polymerase, a time-dependent linear increase of the fluorescence intensity was observed (Fig. 6 A). In contrast, the fluorescence was not increased in the case of the transcription of the XhoI product of SKP/SR1-P2-6 with T3 RNA polymerase (Fig. 6 C), indicating that non-complementary RNA was not detected by YO-YPF-271. The same reaction without using T7 RNA polymerase resulted in no increase of fluorescence because RNA was not produced (Fig. 6 B). In Figure 7 , gel electrophoresis of the products at 30 min incubation shows the production of both the complementary RNA in antisense transcription and the non-complementary RNA in sense transcription.

In addition, there is no difference in the yield found between transcription in the presence or absence of YO-YPF-271 (Fig. 7 ). The present results indicate that YO-YPF-271 specifically binds the target RNA in the course of the reaction to give rise to the fluorescence enhancement, and the presence of YO-YPF-271 in the in vitro transcription has no effect on the activity of RNA polymerase.

We assessed further the relationship between the increase of the fluorescence and the production of RNA. In the course of transcription, aliquots were collected at 5 min intervals to be applied to gel electrophoresis to quantify the product with a densitometer, in measuring the fluorescence intensity of the reaction mixture (Fig. 8 ). The amount of the RNA product also increased time-dependently corresponding to the enhancement of the fluorescence of the reaction mixture. The yield of RNA after 30 min incubation reached 0.3 pmol/[mu]l, as shown in Figure 8 C. One unit of the activity of T7 RNA polymerase used in our present work is specified in manufacturer's data as the capability of uptake of 1 nmol of GMP in 1 h incubation at 37oC to lead to the estimation of the yield, 0.2 pmol/[mu]l (1 nmol * 0.1 U/[mu]l * 0.5 h/290 bases), which corresponds well with the above experimental value. The CV of the fluorescence increase was ~8%.

On the basis of the study demonstrated herein, it is said that increase of the fluorescence intensity corresponds to the RNA products of the transcription, and the rate of the increase of the fluorescence intensity can be a marker for the detection and quantification of target nucleic acid sequences with excellent reproducibility.

In the fluorescence profile of monitoring the in vitro transcription by 0.025 pmol/ml (Fig. 9 A) and 0.1 pmol/ml (Fig. 9 B) of YO-YPF-271, the increase of the fluorescence intensity at 30 min on the initial fluorescence intensity, F(30 min) - F(0 min), was 570 and 150, respectively. The former is mostly four times higher than the latter, corresponding to the ratio of the amount of added probe in the reaction mixture. In other words, most of the added probe in the initial reaction mixture was consumed to form the complex with the product RNA in the transcription.


Figure 9.Influence of the concentration of YO-YPF-271 on the detection of in vitro transcription. (A) 0.025 pmol/ml; (B) 0.1 pmol/ml.

To evaluate the affinity of the probe in question, we investigated the melting temperature of the YO-linked oligo DNA probe-target oligo DNA complex (15 ). According to the report of Helene and co-workers (16 ), Tm of the intercalator-labeled oligomer tends to be higher than the unmodified one. In our experimental conditions, the YO-linked oligo DNA probe-target oligo DNA complex (Tm = 66oC ) was also slightly more stable than the corresponding unmodified duplex (Tm = 63oC).

Those results imply that rapid equilibrium is established for the formation of the probe-target complex under the present incubation temperature (40oC) so as to ensure the capability of the probe to monitor the dynamic process, such as the production of target RNA in the course of the transcription.

On the basis of the work described here, the authors can readily suggest that the oxazole yellow (YO)-linked oligonucleotide probe presented herein brings a specific and simple homogeneous strategy for fluorescence detection and quantification of a target nucleic acid sequence without any separation steps such as electrophoresis.

The present success of the applicability of the probe to real-time monitoring of the in vitro transcription showed that YO-linked DNA probe can be a powerful and versatile tool with which to construct a new methodology to study the dynamics of gene expression, and also to provide a more practical way of detecting and quantifying a target sequence in a clinical specimen specifically in a homogeneous format.

ACKNOWLEDGEMENTS

The authors are indebted to Mr Yoshiharu Toida, Ms Naho Mitsui and Mr Masahito Uchida, analytical laboratory of Tokyo Research Center of Tosoh Corporation, for their help in computer aided molecular modeling.

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*To whom correspondence should be addressed. Tel: +81 467 77 2219; Fax: +81 467 70 1178; Email: isiguro@tosoh.co.jp
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