The development of pyrene-conjugated oligonucleotide
probes for RNA analysis

TAKAKO UEDA
2013
Kyoto Institute of Technology

Contents
Abbreviations

General Introduction:
1 Background and objectives

1

2. Fluorescence in situ hybridization

2

3. Oligonucleotide probes

2

4. FRET probes

4

5. 2’-O-methyl RNA, LNA, PNA

5

6. Molecular beacons

6

7. The developments of Drosophila embryo

8

References

12

Chapter 1:
The detection of RNA expressed in Drosophila embryo with pyrene
modified RNA probes

1.1 Introduction

19

1.2 Materials and Methods
1.2.1. Probe preparation

22

1.2.2. Extraction of total RNA from Drosophila embryos andpurification of the mRNA

22

1.2.3. Fluorescence measurements

23

1.2.4. Whole mount in situ hybridization

23

1.2.5. Microscopic measurement and photography

23

1.3 Results and Discussion
1.3.1. Probe preparation25

1.3.2. The RNA recognition specificity and sequence specificity of
OMUpy236

1.3.3. The linear relationship between fluorescence intensity at 480 nm of
OMUpy2 and the concentration of cORN

40

1.3.4. The detection of target RNA in the total RNA exracted from
Drosophila embryos by OMUpy2

41

1.3.5. The detection of target RNA in the fixed Drosophila embryo

46

References

52

Chapter 2:
The quantitative analysis of RNA expression with RNA-specific probes

2.1 Introduction

55

2.2 Materials and Methods
2.2.1. Cell line

57

2.2.2. Extraction of total RNA from HeLa cells

57

2.2.3. Fluorescence measurements of OMUpy2 hybridized with cORN
and OMUpy2 hybridized with target RNA in total RNA extract

57

2.3 Results and Discussion
2.3.1. Probe preparation

58

2.3.2. The calibration plots of the fluorescence intensity at 480 nm of
OMUpy2 corresponding to cORN concentration

59

2.3.3. The quantitative analysis of RNA expression levels in a HeLa cell
calculated from fluorescence intensity of OMUpy2
References

61
64

Chapter 3:
The development of multi-colored FRET RNA probes

3.1 Introduction673.2 Materials and Methods
3.2.1. Probe preparation

70

3.2.2. Fluorescence measurements of the hybridization solutions withcyanine dye conjugated RNA probes
3.2.3. Tm values of probes with cORN7070

3.2.4. Visualization of three fluorescent dye-conjugated RNA probes
in the presence of cORN

70

3.3 Results and Discussion
3.3.1. Probe preparation

72

3.3.2. The absorption and fluorescence spectra of Cy3-conjugated ORN
and Cy5-conjugated ORN

74

3.3.3. Visualization of three fluorescent dye-conjugated RNA probes
in the presence of cORN
References

80
81

Summary

83

List of Publication

85

Acknowledgements86Abbreviations
bcd

bicoid mRNA

cDNA

complementary oligodeoxyribonucleotide

cORN

complementary oligoribonucleotide

Cpy

2’-O-pyrenylmethylcytidineeveeven-skipped mRNA

FRET

Förster resonance energy transferftz

fushi-tarazu mRNAgt

giant mRNA

hsa- mir

Homo sapiens microRNA

Kr

Krüppel mRNA

LNA

locked nucleic acidmiRNA

microRNAnos

nanos mRNA

ODN

oligodeoxyribonucleotide

ORN

oligoribonucleotide

PNA

peptide nucleic acidRISC

RNA-induced silencing complex

SARS

severe acute respiratory syndrome

snRNA

small nuclear RNA

Upy

2’-O-pyrenylmethyluridine

General Introduction

General Introduction
1. Background and objectives
RNA has been thought to be a molecule that reads nucleotide sequences from
DNA and sends its information to the ribosome, when proteins are expressed from DNA
having genetic information. However, many functional non-coding (nc) RNA was foundin succession since miRNA was discovered in 1993 [1] and RNA came to be recognized
as a molecule which had various functions in bio-regulation systems [2]. Now it isapparent that RNA is essential molecule acting on regulation of the genetic informationand that it is deeply involved in development, differentiation, and the onset of diseases.
98 percent of the entire human genome is non-coding region and the blueprint for theprotein was also revealed only about two per cent by extensive study of non-coding
RNA [3, 4, 5]. It was estimated that each miRNA targets an average of 200 mRNA andmore than one miRNA can act on a single mRNA target, suggesting that it is necessary
to perform the study on the expression of the mRNA in more detail as well as miRNA [6,7].The tool for the detection of RNA with swiftness, accuracy, and RNA-specificity will
be very valuable to know the expression, localization, and movement of RNA. The
elucidation of temporal and spatial function of RNA leads to the discovery of a networkand bio-regulation system between molecules involved in biological phenomena which
are not given from the gene sequence information. These discoveries will contribute to
the development in the field of development, differentiation, regeneration, and medical
diagnosis. In this thesis, I first described that target RNA expressed in Drosophilamelanogaster embryo could be detected in the total RNA extracted from Drosophila
embryos and in the fixed embryo using RNA-specific probe, OMUpy2. The expression
levels of target RNA were also investigated with OMUpy2. In a multi-cellar tissue,
Drosophila embryo, some genes are expressed time, positional specifically. For that,
Drosophila embryo has caught my attention as an ideal system in which to evaluate theperformance of the fluorescence probes, because the morphology and the emitting
position make it possible to determine whether the fluorescence probe could detecttarget RNA. Second, I described that the average molecule numbers of RNA expressed1General Introduction

in a HeLa cell were estimated using OMUpy2. Third, the development of multicolored
FRET RNA probes for the simultaneous observation of multiple target RNAs in a cell
was described.

2. Fluorescence in situ hybridization
Fluorescence in situ hybridizationis (FISH) a method to detect the target genehybridized by a probe labeled with a fluorescent substance or an enzyme under afluorescent microscope. in situ hybridization (ISH) is a technique that was developed by
Pardue and Gall [8] and John et al [9] in 1969, however their technique contained
several problems because of utilizing probes labeled with radioisotopes. First, theisotope decays over time so the specific activity of a probe is not constant and unstable.
Second, the resolution is limited for the dispersion accompanied with radioactive decay
although sensitivity of radiography is generally high. Third, long exposure times are
required to detect signals. Fourth, radioisotope-labeled probe is relatively costly andhazardous material [10]. Therefore histochemical detection method became available
using alkaline-phosphatase-coupled antibodies that detect Digoxigenin (DIG) labeledprobes [11-13]. This method demands considerably less skill compared with ISH using
radioisotope-labeled probe. These developments have further advanced and the first
application of fluorescence in situ hybridization was reported in 1980. The RNA
directly labeled with fluorescent molecules to the 3’ end was used as a probe [14]. FISH
has developed as an alternative to previous methods for visualization of RNA in termsof resolution, speed, safety, simultaneous detection of multiple RNA targets,
quantitative analysis, and imaging in living cells..

3. Oligonucleotide probes
ISH detection of mRNA has been commonly performed using RNA probes
transcribed from cDNA copy of the mRNA target [15, 16]. Therefore probes are several
hundreds or thousands of nucleotides long and contain repeat sequences that cause highbackground fluorescence by nonspecifically binding [17]. ISH for the detection of RNA
in fixed specimen with long probes relies on the steps to open the RNA target sequence2

General Introduction

and allow specifically binding of the long probes by annealing. The available sequencesin RNA target are also limited because certain sequences are unfound in cDNA library,or certain cDNA is difficult to gain. Furthermore, the amount of long probes cannot be
determined quantitatively because labeling molecules such as fluorescent molecules and
DIG are incorporated into RNA probes every 10-15nt at random. RNA probes produced
from cDNA are also easily degraded by RNase. Though long probes have been
introduced into living cells using microinjection to visualize the localization of a
specific RNA [18-20], this approach cannot be applied to the detection of endogenous
RNA in living cells.
Oligonucleotide probes using modified nucleic acid bases have been resolved
many of these problems. Commonly chemical synthesized FISH probes employ 15–30
nucleotides long with a DNA, RNA, or modified nucleic acid backbone. Fluorescence isdirectly observed using a fluorophore attached to the 5’- or 3’-terminus, or internallylabeled probes. The sequences of oligonucleotide probes are able to be designed insilico, and easily synthesized compared with RNA probes made from cDNA. It is also
confirmed that oligonucleotide probes hybridize specifically to the complementary RNA
by the melting curve analysis. Methods based on the hybridization with a fluorescentlabeled oligonucleotides probe have been able to extend to the application in living cells
since linear probes enable to access to the target site of long RNA target without
annealing to open the target site for binding.
In 1989, fluorescein labeled oligonucleotide probes and rhodamine labeld
oligonucleotide probes hybridized to 16S ribosomal RNA (rRNA) in the fixed cells andvisualized its existence under a fluorescence microscope [21]. The Studies using
rhodamine labeld oligonucleotide probes targeting for ribosomal RNA (rRNA) werealso performed as a means of analysis [22, 23]. These studies with fluorescent labeledoligonucleotide probes led to studies on poly (A) RNA in living cells.
In 1995, first detection of RNA in living cells using fluorescent (Texas Red)labeled oligodeoxynucleotides was reported by Politz et al. [24]. In this report, it was
shown that phosphorothioate and fluorescent labeled phosphodiester oligo dT enteredcells rapidly and hybridized to poly (A) RNA within 30 min [24]. In 1998, intranuclear
3General Introduction

diffusion of poly (A) RNA was observed with fluoresein labeled oligodeoxynucleotide
in living cells [25]. The main problem accompanied with the use of these probes forvisualization of the distributed RNA is the low signal to noise ratio. That occurs by thereason why the emitting from unhybridized probes is strong and the signal from target
RNA is small. This problem can be resolved by the probe which emits only when itbinds to target RNA.

4. FRET probes
FRET (Förster Resonance Energy Transfer) is one of the fluorescence-related
phenomena. A photon from an energetically excited fluorophore, the donor, raises theenergy state of an electron in another molecule, the acceptor, to higher vibrational levelsof the excited singlet state. As a result, the energy level of the donor fluorophore returns
to the ground state, without emitting fluorescence. This mechanism is dependent on the
dipole orientations of molecules and is limited by the distance between the donor andthe acceptor molecules. Typical effective distances between the donor and acceptor
molecules are in the 10 to 100 Å range. This range is the distance between three to thirty
nucleotides located in the double helix of a DNA molecule. Another requirement is thatthe fluorescence emission spectrum of the donor must overlap the absorption spectrumof the acceptor. The acceptor can be another fluorophore or a non-fluorescent molecule.
If the acceptor is a fluorophore, the transferred energy can be emitted as fluorescence[26]. In 1988, it was reported that FRET was caused to rhodamine (acceptor) fromfluorescein (donor) covalently attached to the 5’ ends of oligonucleotides. These results
showed the hybridization of nucleic acid could be detected by FRET [27]. Various
studies on structural analysis [28-33] and oligonucleotide hybridization [34-37] by
FRET have been reported. These FRET techniques with further development proved
useful for detection and quantify of nucleic acid hybridization in living cells. In 2000,
Tsujii et al. observed, for the first time, human c-fos mRNA expressed at more than 104
molecules in a living transfected Cos7 cell by FRET probes (Bodipy493/503 and Cy5
was used as a donor and an acceptor) under a fluorescence microscope [38]. Then they
developed a method not affected by the cell autofluorescence to detect smaller number
4General Introductionof RNA with a time resolved fluorescence microscopy and visualized the presence of
c-fos mRNA in HeLa cells using FRET probes [39].

5. 2’-O-methyl RNA, LNA, PNA
In order to overcome the problems of the degradation of oligo nucleotide
probes by an enzyme and the sufficient affinity with the target RNA in the cell, nucleic
acid probes having a non-natural backbone such as, 2’-O-methyl RNA, locked nucleic
acid (LNA), and peptide nucleic acid (PNA) have been developed [40-45]. Because thethermal stability of the hybrids of the artificial nucleic acid probes and RNA is higher
than that of the base paring between the DNA-RNA, it is possible to suppress the false
signal by nonspecific binding.
2’-O-methyl RNA probes have been reported to form very stable hybrids withtarget RNA in terms of high melting temperature. Further, because of the low cost,2’-O-methyl RNA probes is most commonly used to detect RNA or to inhibit the target
gene expression. Majlessi et al. showed that 2’-O-methyl RNA probes exhibit higher
affinity for target RNA than the corresponding 2’-deoxyoligoribonucleotide (ODN)
probes resulting in higher melting temperatures [45]. The affinity of 2’-O-methyl RNA
probes and RNA was the highest in 2’-O-methyl RNA probes and DNA, ODN probes
and RNA, and ODN probes and DNA [45]. In addition to the enhancement of Tm value,
they revealed that 2’-O-methyl RNA probes hybridized 2- to 4-fold faster to RNA thanODN probes [45]. Therefore 2’-O-methyl RNA probes greatly have advantages overODN probes for detecting RNA. 2’-O-methyl RNA probes also have high resistance toenzymatic [46-49]. In 2001, Molenaar et al. confirmed that cells microinjected with the2’-O-methyl RNA probes could be monitored for several days [46]. The half-life of
DNA probes injected in the living cells was shown to be about 15 min [47]. Czauderna
et al. also reported that the effect of gene expression inhibition in RNAi experiments
using dsRNA containing 2’-O-methyl RNA remained for five days [48].
Locked nucleic acid (LNA) LNA is a nucleic acid analogue containing a 2’-O,
4’-C methylene bridge [50]. This methylene moiety causes a change in theconformation of nucleic acid helices and increases thermal stability to form the A-form
5

General Introduction

duplexes. LNA was independently synthesized by the group of Takeshi Imanishi in 1997[50], and by the group of Jesper Wengel in 1998 [51]. LNA oligonucleotides display
unprecedented hybridization affinity toward complementary RNA and complementary
DNA [52-55]. A new method for highly efficient detection of microRNAs by northernblot analysis using LNA (Locked Nucleic Acid)-modified oligonucleotides was shown
by Valoczi et al. [56]. They designed DNA probes containing LNA for microRNAs in
animals and plants. Using the DNA probes substituted by LNA at every three position innorthern blot analysis employing standard end-labeling techniques and hybridization
conditions, the sensitivity of detection signals increased at least 10-fold compared toconventional DNA probes [56]. In fixed cells, LNA probes detected target RNA with far
better efficiency than conventional DNA probes [57]. LNA probes have also been used
to detect 21-22-nt microRNAs by whole mount in situ hybridization [58, 59]. Darnell et
al. employed LNA modified DNA probes (12-24-nt), containing an LNA nucleotide at
every three position and labeld at the 5’ end with DIG, to visualize the localization ofmRNA in fixed chicken embyos [60]. They also showed the detection of alternatively
spliced exon using LNA probes [60]. The problems are high cost and unavailability
unlike 2’-O-methyl RNA to be able to freely design and synthesize.
The main bone of PNA consists from N- (2-aminoethyl) glycine linked by anamide bond instead of sugar in a nucleic acid. Purine ring and pyrimidine ringcorresponding to the nucleobase are bound to the main chain through a methylene group
and a carbonyl group. Since the charge of the phosphate site such as DNA or RNA is notpresent in PNA, the duplex of PNA/DNA and PNA/RNA are formed strongly than
DNA/DNA, RNA/DNA, and RNA/RNA by a decrease in electrostatic repulsion [61].
However, PNA is so rigid molecules that it is difficult to access to highly folded RNA
structures and therefore PNA have not been used as a hybridization probe in living cells[62].

6. Molecular beacons
Molecular beacons (MB) were first developed by Tyagi et al. in 1996 as a tool
for real-time PCR assays [63, 64]. Molecular beacons are ODNs that form a stem-loop
6General Introduction

hairpin structure and are dual-labeled with a reporter fluorophore at one end and aquencher at the other. In the absence of a complementary target, the molecular beacon isin a stem-loop configuration in which the fluorescence is quenched. Following
hybridization to a complementary target, the hairpin structure is changed to an open
configuration separating the fluorophore and quencher and restoring fluorescence [65].
The obstacle in monitoring the localization or movements of RNA transcribing in living
cells with the typical fluorescent probes is the emission of fluorescent labeled probes
before hybridizing to target RNA. This makes it difficult to trace the origin of
movements, and fate of the specific RNA. Molecular beacon has improved thisproblem.
In 1998, the first application of molecular beacons to the probe for mRNA
detection was reported [66, 67] and many studies to track the movement of RNA using
molecular beacons were performed [68-71]. Though the backbone of early molecularbeacons was DNA, molecular beacons came to be synthesized with 2’-O-methyl RNA
backbone in order to improve the lack of resistance enzyme and low reproducibility [68,72, 73]. The thermodynamic and kinetic properties of the hybridization between
2’-O-methyl RNA molecular beacons and RNA were improved more stable compared to
DNA molecular beacons. And the problem of false-positive signals caused by DNA
molecular beacons degraded by nuclease in living cells is resolved. However,2’-O-methyl molecular beacons is lower performance compared with linear
2’-O-methyl RNA probe and in particular the signal intensity observed with
2’-O-methyl molecular beacons was 2-3-fold lower compared with linear 2’-O-methyl
RNA probe [48, 68]. 2’-O-methyl molecular beacons also have time-lag until emitting
the maximum signal because they are in a closed and quenched form. Therefore initialsignal intensity is very low. In the report by Molenaar et al., from 5-10 min after
microinjection, the signal intensity increased steadily throughout the nucleus and similar
localization patterns were observed as obtained with linear 2’-O-methyl RNA probes[46].
To improve signal-to-noise ratio, molecular beacons using FRET have beendeveloped. If the optimal emission wavelength is only a few nanometers longer than the7General Introduction

optimal excitation wavelength (Stokes shift) a portion of the excitation light can reachthe detector by scattering and reflection, thus limiting detection sensitivity. On the other
hand the large Stokes shifts of wavelength-shifting molecular beacons allow more
effective filtering of the excitation light, thereby enhancing the sensitivity of target
detection. Large Stokes shifts are particularly significant in the detection of the hybridsof probes and target RNA in living cells, where autofluorescence of cellular components
introduces a background signal. This approach was first tried with wavelength shifting
molecular beacons in multiplex PCR assays [74]. Dual FRET molecular beacons werealso designed to detect high sensitively specific endogenous mRNA in living cells. [71,75] Dual FRET molecular beacons consisted of two molecular beacons. One beacon
was labeled with a donor fluorophore and second beacon was labeled with an acceptor
fluorophore. These two molecular beacons were designed to hybridize to adjacentregions on a target mRNA to occur an energy transfer between two molecular beacons
when both beacons hybridized to target mRNA. The fluorescence emission of acceptor
fluorophore cannot be observed unless donor fluorophore is excited, which enables todistinguish readily the positive signals and the false positive signals caused by probedegradation and nonspecific probe opening [76]. In 2004, Santangelo et al. showed thedetection of only a few hundred copies of an endogenous mRNA in a single living cellwould be feasible with dual FRET molecular beacons [71]. Using dual FRET molecularbeacons, the localization of oskar mRNA in Drosophila oocytes could be confirmed invivo [73]. Further by Mhlanga et al., a more refined model of oskar mRNA transport in
the Drosophila oocyte were revealed using molecular beacons designed by Bratu et al.
[73, 77]. However, molecular beacons have not yet been able to detect other mRNAs in
Drosophila embryos.

7. The developments of Drosophila embryo
The early development of the fruit fly, Drosophila melanogaster, is better
understood than that of any other animal. Astonishing discoveries in developmental
biology over the past 30 years have revealed that many of the genes that control thedevelopment of Drosophila are similar to those controlling development in vertebrates,
8General Introduction

and many other animals.
The Drosophila embryo is a multiple-cellar tissue and in which the genes
involved in early development show unique expression patterns. The Drosophila egg israther oblong and the anterior end is easily recognizable by the micropyle, anipple-shaped structure in the tough external coat surrounding the egg. Sperm enter theanterior end of the egg through the micropyle. After fertilization and fusion of the spermand egg nuclei, the zygote nucleus undergoes a series of rapid mitotic divisions, one
about every 9 minutes, but, unlike in most other animal embryos, there is initially nocleavage of the cytoplasm and no formation of cell membranes to separate the nuclei.
The result after 12 nuclear divisions is a syncytium in which around 6,000 nuclei arepresent in a common cytoplasm. Early development of a Drosophila embryo isextremely rapid. The first 13 nuclear divisions are completed in just over 3 hours andoccur in the absence of cytokinesis, producing an embryo of about 6,000 nuclei in acommon cytoplasm. During these syncytial divisions, the body axes and segments are
established. The dividing nuclei progress through an orchestrated series of migrations tosituate in a well-organized monolayer of nuclei at the actin-rich cortex. These eventsoccur on a time-scale of minutes, many of the nuclear cycles are less than 10 minutes. In
spite of the speed and crowding of the nuclei, the fidelity of these events is high[78-80].
The Drosophila embryo is patterned with a distinct polarity along its
anterior-posterior axis. The groundwork for this pattern is organized by the maternalmRNA and protein which is deposited in the egg during oogenesis [81]. The patterning
along the anterior-posterior axis which leads to the segmentation of a body depends on
the Bicoid and Nanos gradients [82, 83] (Figure 1.). Hunchback is distributed
throughout the embryo as a maternal mRNA. However, Bicoid activates Hunchbacktranscription anteriorly, and Nanos which is a mRNA-binding protein blocks thetranslation of Hunchback transcripts posteriorly. Therefore the concentration gradient of
Hunchback is formed from the anterior toward posterior [84]. The gradient of
Hunchback dictates where the gap genes, such as Krüppel, Knirps, and Giant, areexpressed by providing a series of concentration thresholds that regulate each gene
9General Introduction

independently. Thus Hunchback triggers several distinct responses as a function of its
graded distribution [84-88]. Based on the anterior-posterior axis which is defined by the
first gradient of Bicoid and Nanos, a body plan is formed more precisely bysegmentation genes. Segmentation genes are classified into three groups, gap genes,
pair-rule gene, and segmental polarity genes. First, the concentration gradient ofmaternal effect genes act on gap genes and their products roughly divide an embryo into
each part which are based on the next cascade. Then, pair-rule genes are expressed by
the effects of gap genes and form the segments. Further, segmental polarity genes
induced by a prepattern of pair-rule gene products are expressed and define domains inmore detail [89-92].

10

General Introduction

anterior

Maternal effect gene

Time

Body area

posteriorhunchback mRNAnanos mRNA

caudal mRNA

mRNA
concentration

bicoid
mRNA

nanos
mRNA

concentration

bicoid mRNA

Nanos

BicoidHunchbackProteinconcentration

HunchbackCaudalNanos

Bicoid＜Gap gene＞
HunchbackKrüppel mRNA

giant mRNA
Gap gene
expression＜Pair-rule gene＞

even-skippedmRNA

knirpsmRNA

Fushi-tarazu
mRNA

giant
mRNA

Krüppel
mRNA

giant
mRNA

Figure 1. A model of anterior-posterior pattern generation by Drosophila maternal
effect genes
The bicoid, nanos, hunchback, and caudal mRNAs are placed in the oocyte by theovarian nurse cells. The Bicoid protein gradient extends to from anterior to posterior,and Nanos protein gradient extends from posterior to anterior. Nanos inhibits thetranslation of hunchback mRNA, while Bicoid prevents the translation of the caudal
mRNA. This inhibition results in opposing Caudal and Hunchbask gradients. The gap
genes expression patterns are determined by the gradients of protein products of Bicoid,
Caudal, and Hunchback. Gap genes establish the segmented body plan of the embryo
along the longitudinal axis. The RNA of each of the gap genes has very distinct
distribution that defines abutting or slightly overlapping regions of expression. Theirprotein products control the more detail expression patterns of the pair-rule genes [92].

11General Introduction

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18

Chapter 1

Chapter 1
The detection of RNA expressed in Drosophila embryowith pyrene modified RNA probes

1.1 Introduction
Recent RNA research has revealed the close involvement of various RNAs incellular functions. Not only RNA itself, but also RNA protein complexes such as
miRISC are key players in cells [1]. RNA has been becoming the inevitable target
molecules for research of gene expression in details. In order to clarify the roles of RNA
related molecules, Northern blotting [2], RT-PCR [3], and DNA microarray [4] havebeen included in many conventional studies. However, those techniques cannot study
the differences between individual cells, tissues and embryos. In tissues, the individual
cells may present different gene expressions. In embryos, the cells may be in different
stages of differentiation. Therefore, new systems for the observation of gene expressions
in individual cells are necessary.
RNA and its related complexes are also promising targets for disease diagnosis[5, 6]. Multi-cellular specimens such as organ tissues, histopathological specimens, andembryos are among the possible targets of RNA-based diagnostic techniques. Proof ofthe presence of certain RNAs in a specimen could provide information about diseases
such as SARS [7] and newly emerging virus infections [8]. Rapid and possibly
quantitative analysis of a specific RNA could easily lead to rapid and precise diagnosis
of diseases. Taking this background into consideration, both in research into
fundamental biology and in clinical disease diagnosis, selective and rapid detection of
specific RNA in multi-cellular specimens is essential. The conventional analyticalmethod of RNA is in situ hybridization accompanied with an immune staining protocol,
which requires cDNA having a certain antigen such as digoxigenin and biotin [9].
Though this protocol has shown reliability to date, it requires skills and time-consuming
procedures. Recently, elegant in situ hybridization protocols to detect RNA and its
19Chapter 1

related substances have been reported [10-12]. That is, RNA imaging protocols havebeen intensively developed using fluorescent nucleic acid probes, and the rapid
development of imaging technology has accelerated this field, allowing the visualizationof RNA in cells, tissues and embryos. Several reports related to this new protocol
include molecular beacons [13, 14], FRET-probes [14, 15], quenched autoligation
probes [16], and fluorescent-protein based probes [17]. Though the probes used in those
detection protocols have presented promising results for gene imaging studies, they
were not RNA specific. For further development of RNA detection systems for the
diagnosis of diseases, detection systems that can clearly detect RNA in specimens areurgently required.
A uniquefluorescentRNA-specificprobehavingtwoconsecutive

pyrene-conjugated pyrimidine nucleosides has been developed. A pyrene wasintroduced at the 2’-position of a pyrimidine nucleoside denoted as Upy. Our probe,2’-O-methyloligoribonucleotides (2’OMe ORN) having two consecutive modified
nucleosides, denoted as OMUpy2, possesses two important characteristics. First, it ishighly RNA specific [18, 19]. The hybrids between OMUpy2 and its complementaryoligo RNA (cORN) are highly fluorescent, whereas those between OMUpy2 and its
complementary DNA are scarcely fluorescent. Even the hybrids between OMUpy2 andcORN containing a single mismatch cause significant quenching of the fluorescence.
The spectroscopic studies revealed that the fluorescent behavior of the pyrene attachedto the sugar was strongly affected by the surrounding environment of the pyrene [20].
And conformational studies using molecular dynamic simulations and 1H NMR
measurements revealed that the pyrene attached to the sugar in an RNA duplex was
located outside of the helix, whereas that in a DNA duplex was located inside of thehelix [21]. Therefore, the pyrene modified RNA provides a useful tool for RNA
detection. Secondly, OMUpy2 does not emit fluorescence alone when excited.
Therefore, a washing procedure to remove unbound probes from the detection system is
not necessary, which enables the application of the probes to in situ RNA detection inliving cells.
In the present study, we conducted in situ RNA detection in multi-cellular
20

Chapter 1

systems, Drosophila embryo, using OMUpy2. This system is suitable for detecting
RNA in its spatial and temporal aspects, because it is easy to evaluate whether the probe
was able to detect the RNA target from the position of RNA expression changing
according to the kinds of RNA and development time. In addition, the protocol
employed in this study makes whole mount in situ hybridization (WISH), which iscommonly used for embryo studies, easier and faster than ever due to the elimination ofwashing steps. We especially focused on Krüppel mRNA (Kr mRNA). The Krüppel
gene, a gap gene, is one of the first zygotic segmentation genes activated in early
Drosophila embryos [22, 23]. It is well known that the Kr mRNA is abundantly
expressed in the early stages of differentiation, and its temporal and spatial expression
patterns have been well characterized [24-26]. Transcription of Kr mRNA starts at thesyncytial blastoderm stage (stage-3) and lasts up to 8 h after fertilization [27]. At theonset of transcription, the Kr mRNA is detected mostly in the center domain of
Drosophila embryos during stage-3-5 [9, 25, 26]. Therefore, OMUpy2 for Kr mRNA is
expected to emit signals in the center region of Drosophila embryos if it specificallyhybridizes with Kr mRNA in the embryos.

21

Chapter 1

1.2 Materials and Methods
1.2.1. Probe preparation
The probes were synthesized according to previous reports using conventional
phosphoramidite chemistry and purified by reversed-phase HPLC [28]. The sequencesused in this study are listed in Table 1. Kr-F is a 5’-fluorescein labeled 2’-OMe ORN
which has the same nucleotide sequence as Kr-OMUpy2. Kr-OMUnonpy is a
non-pyrene-labeled 2’-OMe ORN which has the same sequence as Kr-OMUpy2.
Control-OMUpy2 consists of the same base composition as Kr-OMUpy2 with ascrambled sequence.

1.2.2. The extraction of total RNA from Drosophila embryos and purification ofmRNA
All embryos used in this study were from wild-type Drosophila flies (Canton
S). Embryos were collected at 2-hour intervals and aged until the desired developmental
stage on yeast juice agar plates at 25 °C. The embryos were dechorionated with 5 %sodium hypochlorite in water for 1.5 min and then frozen in liquid nitrogen. After 500µl of TRIzol® Reagent (Invitrogen) was added to the frozen embryos on ice, theembryos were homogenized quickly, and further 500 µl TRIzol® Reagent and 200 µl
chloroform/isoamyl alcohol (49/1, v/v) were added, followed by vigorous shaking. Afterincubation on ice for 5 min, the suspension was centrifuged at 12,000 rpm at 4 °C for 15
min. RNA in the aqueous phase was transferred to a sterilized tube. After addition of
500 µl of isopropanol to the tube, the solution was stored on ice for 5 min followed bycentrifugation at 12,000 rpm at 4 °C for 10 min. Isopropanol was exchanged with 75 %aq. ethanol and the ethanol solution was centrifuged at 7,500 rpm at 4 °C for 3 min threetimes. The precipitate, RNA, was suspended in 75 % aq. ethanol and stored at -20 °C for
more than 12 hours. The RNA suspension was collected by centrifugation and dissolved
in PBS buffer, followed by treatment with an oligo (dT)-cellulose column (GEHealthcare).

22

Chapter 1

1.2.3. Fluorescence measurements
Fluorescence spectra were measured with a spectrofluorophotometer(RF-5300PC, Shimadzu). The fluorescence spectra of Kr-OMUpy2 in the presence ofthe extracted mRNA were measured with a micro volume cell (Capillary Adaptor Cell;
HELIX Biomedical Accessories; cell volume, 3 µl). The concentration of the extractedmRNA solution used for measurements was 65 mg/ml and those of Kr-OMUpy2 andControl-OMUpy2 were each 30 µM.

1.2.4. Whole mount in situ hybridization
The collections of Drosophila melanogaster wild-type embryos, Canton S,
were dechorionated in a solution of 5 % sodium hypochlorite in water for about 1.5 minin a small basket. Embryos were thoroughly washed with 0.07 % NaCl/ 0.1 %TritonX-100. The embryos were then fixed with 4 % paraformaldehyde in 10 mM
phosphate buffer containing 0.1 M NaCl and heptane (1/4 : v/v) with gentle shaking for
20 min at 25 °C. After fixation, the aqueous phase was completely removed andembryos were vigorously shaken in a 1:1 mixture of heptane and methanol so that they
were devitellinized. Embryos were then washed with methanol twice and stored in
100 % methanol at -85 °C [29]. Prior to WISH using fixed embryos, the supernatant,methanol, was sequentially exchanged with PBT (10 mM phosphate buffer, pH 7.0/ 0.1
M NaCl/ 0.1 % Tween 20) /methanol solution (75, 50, and 25 % methanol) and thenwashed twice in PBT. Embryos were stored in PBT at 25 °C for 15 min. The fixed
embryos were then added in the probe solution (30 µM OMUpy2 in PBT), followed byincubation at 40 °C for 30 min, then at 4 °C for 2 hours. The embryos treated withprobes were then washed with PBT once. Embryos were mounted on a glass slide in
70 % glycerol in PBS and observed by fluorescent microscope using a 20 × objectivelens.

1.2.5. Microscopic measurement and photography

23

Chapter 1

Microscopic measurements and imaging of embryos treated with OMUpy2
were performed on a Nikon ECLIPSE TE300 (Nikon) microscope equipped with axenon lamp. Fluorescent images were obtained under the following conditions: Filter
sets: For OMUpy2, EX: 340/15 nm, DM: 380 nm, EM: 480/30 nm; For Kr-F, EX:
470/20 nm DM: 505 nm EM: 535/25 nm. The objective lens was a 20 ×/0.45 Plan Fluor(Nikon) for differential interference contrast (DIC) images. The merge images wereconverted to pseudo-color. Microscopic fluorescence spectra were measured with a
Hamamatsu photonic multi-channel analyzer (PN-100, Hamamatsu Photonics,
Hamamatsu) under the following conditions: EX: 340/15 nm DM: 380 nm. The
exposure time for obtaining images was 1 sec and that for obtaining spectra was 5 sec.

24

Chapter 1

Results and Discussion
1.3.1. Probe preparation
RNA specificprobescontaining

twoconsecutive

pyrene-conjugatednucleotides (OMUpy2) were synthesized according to previous reports [18]. The
structure of the core region in OMUpy2 and typical fluorescence spectra of bispyrene
and monopyrene are shown in Figure 1. The target site for OMUpy2 must satisfy thefollowing three points. The first is the sequences of target RNA need to contain ApA
sequence, where UpyUpy in OMUpy2 can hybridize. Second, the base followed by
UpyUpy must be a pyrimidine base as to form an environment in which pyrene emits
excimer, namely the base followed by ApA mast be a purine base. Third, the target sites
have high potential to form a single-stranded region.
The nucleotide positions chosen as the target site for OMUpy2 were based on
the results of Loop Probability value (LP value) (Figure 2.) taking into account theconditions described above. Loop Probability represents the accessibility of probe toeach base. Each base belongs to a part of the loop structure or stem structure in thefolding RNA. Its belonging changes depending on the state of the folding RNA. For
each base, the probability to form a loop structure was calculated from secondary RNA
structure based on the software, “RNA Structure 4.6”, Mathews et al. [30, 31]
developed by the thermodynamic methods which are based on nearest neighbor rulesthat predict the stability of a structure quantified by folding free energy change [30, 32,33]. In brief, Loop Probability value (LP value) of the base which can always form aloop structure shows LP value=1. LP value of the base which always forms a stem
structure shows LP value=0. LP value is an average calculated based on twenty RNA
secondary structures which were predicted in ascending order of energy [34]. The entire
sequences were scanned sequentially for secondary structure formation in contiguous
frames of 100 bases. the frames for the simulation of secondary structures were
staggered down by 30 bases, resulting in an overlap of 70 bases on the 5’ side of thesecond set of fram (100 nt frame-30 nt shift). This procedure is repeated many times sothat any given sequences are scanned for its potential secondary structure in other25

Chapter 1

different frames. LP value was calculated from three types of RNA secondary structures,
100 nt frame-30 nt shift, 300 nt frame-100 nt shift, and 700nt frame (Figure 2a-g.).
Details of RNA probes and ORNs used in the present study are shown in Table 1.
As a backbone of OMUpy2, 2’-O-methyl RNA was employed because they
have high stability in terms of its enzyme resistance and the affinity with ORN.
2’-O-methyl RNA also permits selective binding at 37 °C [35-40].

26

Chapter 1

(1)

(2)

Fluorescence intensity

5’
800
600
400
200
0
350

400

450

500

550

600

Wavelength (nm)

3’
(3)
Py

Py

OMUpy2C A U U U G A U C G UPyPy

C A U U U G A U C G U

G U A A A C U A G C A U U CG U A A A C U A G C A U U Ctarget mRNAOMUpy2 hybridized to target mRNA.

py :Unexcited Pyrene
py py :Pyrene Excimer

Figure 1. (1) The structure of the core region in OMUpy2. (2) Fluorescence spectraof bispyrene and monopyrene. (3) Scheme of OMUpy2.
(2) light blue: bispyrene, dark blue: monopyrene. λex = 342 nm. (3) OMUpy2 emits
fluorescence at around 480 nm only when it binds to complementary RNA.

27

Chapter 1

5’

mRNA

3’

1) 100 nt frame-30 nt shift
100 nt30 nt

2) 300 nt frame-100 nt shift
300 nt
100 nt3) 700 nt frame

Figure 2a. A method of predicting RNA secondary structure to calculate the LP
value.

28

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Figure 2b. Loop probability values of bicoid mRNA.
red: 100 nt frame 30nt shift, blue: 300 nt frame 100 nt shift, yellow: 700 nt frame. The
target sites are painted in blue. The details of sequences are presented in Table 1.

29

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Figure 2c. Loop probability values of even-skipped mRNA.
red: 100 nt frame 30nt shift, blue: 300 nt frame 100 nt shift, yellow: 700 nt frame. The
target sites are painted in blue. The details of sequences are presented in Table 1.

30

Loop probability

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Figure 2d. Loop probability values of fushi-tarazu mRNA.
red: 100 nt frame 30nt shift, blue: 300 nt frame 100 nt shift, yellow: 700 nt frame. The
target sites are painted in blue. The details of sequences are presented in Table 1.

31

Loop probability

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Figure 2e. Loop probability values of giant mRNA.
red: 100 nt frame 30nt shift, blue: 300 nt frame 100 nt shift, yellow: 700 nt frame. The
target sites are painted in blue. The details of sequences are presented in Table 1.

32

Loop probability

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Figure 2f. Loop probability values of Krüppel mRNA.
red: 100 nt frame 30nt shift, blue: 300 nt frame 100 nt shift, yellow: 700 nt frame. The
target sites are painted in blue. The details of sequences are presented in Table 1.

33

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Figure 2g. Loop probability values of nanos mRNA.
red: 100 nt frame 30nt shift, blue: 300 nt frame 100 nt shift, yellow: 700 nt frame. The
target sites are painted in blue. The details of sequences are presented in Table 1.

34

Chapter 1

Table 1. Sequences used in the present study.
RNA target
bicoidmRNA

namebcd1-ORN
bcd1-OMUpy2
bcd2-ORN
bcd2-OMUpy2

even-skippedmRNA

eve1-ORN
eve1-OMUpy2
eve2-ORN
eve2-OMUpy2

fushi-tarazu
mRNA

ftz1-ORN
ftz1-OMUpy2
ftz2-ORN
ftz2-OMUpy2

giant
mRNA

gt1-ORN
gt1-OMUpy2
gt2-ORN
gt2-OMUpy2

KrüppelmRNA

Kr-ORN

28S rRNAhsa-mir-21
hsa-mir-24-2

GAU CAA AAC UUU UAC

GGG UAU GAA ACA GAGGAU ACC GAA CCU ACA

513-527

182-19645549169

CCA AAU GAA UGC CUA

307-321

UAG GCA UpyUpyC AUU UGG
GUU AGA GAA GAA AUC

38-52

19549961

GAU UUC UpyUpyC UCU AAC
CUA UCA GAA CAC CUC

190-204

GAG GUG UpyUpyC UGA UAG
UAC ACC GAA CAC CAU

99-11324639344

AUG GUG UpyUpyC GGU GUACGU AUC AAA CAA GCU

461-475

AGC UUG UpyUpyU GAU ACG
AGC ACA AAA UUA GGA

F-UCC UAA UUU UGU GCU

Kr-OMUnonpy

UCC UAA UUU UGU GCU

Control-OMUpy2

AGU CAC UpyUpyU UUC GUU
AAG UAC AAA AGU GUG

Kr2-OMUpy2

CAC ACU UpyUpyU GUA GUU

nos1-ORNACC ACA AAA UUC CUG

304-31822024037

157-17118-32

24648141

CAG GAA UpyUpyU UGU GGU

nos2-ORNUUG UGG AAA GUA GAG

nos2-OMUpy2

CUC UAC UpyUpyU CCA CAACGCUCAUCAGACCCCAGAAAAG

28S-OMUpy2

CUU UUC UGG GGUpy CpyUG AUG AGC G

miR21-ORN

UAG CUU AUC AGA CUG AUG UUG A

miR21-OMUpy2

UCA ACA UCA GUpyCpy UGA UAA GCU A

miR24-ORNUGG CUC AGU UCA GCA GGA ACA G

miR24-OMUpy27646

UGU AGG UpyUpyC GGU AUC

Kr-F

28S-ORN

19-33

CUC UGU UpyUpyC AUA CCC

UCC UAA UpyUpyU UGU GCU

nos1-OMUpy2

positions

GenBank ID/
miRBaseAccession

Nucleotide

GUA AAA GUpyUpy UUG AUC

Kr-OMUpy2

Kr2-ORN

nanos
mRNA

5’ SEQUENCES 3’
upper: target RNA / lower: probe

210-224

1903-1924

3373818-29MI0000077

50-71

MI0000081

CUG UUCpy CpyUG CUG AAC UGA GCC A

Italic letters: 2’-OMe nucleotides, capital letters: ribonucleotides.
Upy: 2’-O-pyrenylmethyluridine, Cpy: 2’-O-pyrenylmethylcytidine
Kr-F: 5’-fluorescein-labeled probe with same sequence as Kr-OMUpy2.
Kr-OMUnonpy: non-pyrene-labeled probe with the same sequence as Kr-OMUpy2.
Control-OMUpy2: scrambled sequence with the same base composition as Kr-OMUpy2.
28S-ORN: the sequence complementaery to nucleotide positions (1903-1924) in Human
28S ribosomal RNA gene.

35

Chapter 1

1.3.2. Fluorescence spectra of Kr-OMUpy2 and Control-OMUpy2 in the presence
and absence of Kr-ORN
Fluorescence spectra of Kr-OMUpy2 in the presence and absence of thecomplementary ORN (Kr-ORN) and oligo DNA (Kr-ODN) are shown in Figure 3a.
While the equimolar solution of Kr-OMUpy2 and Kr-ORN emitted the fluorescencepeaked at around 480 nm, the fluorescence of the equimolar solution of Kr-OMUpy2
and Kr-ODN was hardly observed. The spectrum showed the typical fluorescence of
bispyrene and these results were in accord with a previous report. Little to no
fluorescence around 480 nm from Kr-OMUpy2 itself, Control-OMUpy2 itself, and theequimolar solution of Control-OMUpy2 and Kr-ORN were observed (Figure 3b.). In
addition, we also observed little emission around 480 nm from the equimolar solution of
Kr-OMUpy2

andnoncomplementary

ORNs(28S-ORN,

miR24-ORN,

andmiR21-ORN) (Figure 3c.). These results indicate that 15-nt Kr-OMUpy2 has sufficientsequence specificity to identify complementary sequences because it did not
fluorescence almost around 480 nm unless in the presence of its complementary RNA,
Kr-ORN. Based on a previous report [18], we attributed the fluorescence around 480
nm to a pyrene excimer formed by a hybrid formation between OMUpy2 and its
complementary RNA.

36

Chapter 1

Fluorescence intensity

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0
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400

450
500
550
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600

650

Figure 3a. Fluorescence spectra of Kr-OMUpy2 in the presence of Kr-ORN and
Kr-ODN.
red line: Kr-OMUpy2/Kr-ORN, blue line: Kr-OMUpy2, green line:
Kr-OMUpy2/Kr-ODN, [Kr-OMUpy2] = [Kr-ORN] = [Kr-ODN] = 0.75 µM, 10 mM
phosphate buffer (pH 7.0) containing 100 mM NaCl, 11 °C, λex = 342 nm.

37

Chapter 1

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0
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400

450
500
550
Wavelength (nm)

600

650

Figure 3b. Fluorescence spectra of Kr-OMUpy2 and Control-OMUpy2 in thepresence and absence of Kr-ORN.
red line: Kr-OMUpy2/Kr-ORN, blue line: Control-OMUpy2/Kr-ORN, green line:
Control-OMUpy2, [Kr-OMUpy2] = [Control-OMUpy2] = [Kr-ORN] = 0.75 µM, 10mM phosphate buffer (pH 7.0) containing 100 mM NaCl, 11 °C, λex = 342 nm.

38

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Figure3c.

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400

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450
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of

600

650

Kr-OMUpy2

inthepresenceofnoncomplementary ORN
red line: Kr-OMUpy2/Kr-ORN, blue line: Kr-OMUpy2/28S-ORN, yellow line:
Kr-OMUpy2/miR21-ORN, green line: Kr-OMUpy2/miR24-ORN, [Kr-OMUpy2] =[Kr-ORN] = [28S-ORN] = [miR21-ORN] = [miR24-ORN] = 0.75 µM, 10 mM
phosphate buffer (pH 7.0) containing 100 mM NaCl, 11 °C, λex = 342 nm.

39

Chapter 1

1.3.3. The linear relationship between fluorescence intensity of OMUpy2 and theconcentration of ORN
In order to evaluate the capability of OMUpy2 in terms of the quantitative
detection of RNA, the fluorescence intensity of OMUpy2 in the presence of thecomplementary ORN (cORN) was measured in the range of 10 nM to 1000 nM cORN
concentration. The fluorescence intensity at 480 nm of OMUpy2-cORN hybrids versus
the concentration of cORN had a linear relationship at the range of 10 nM to 1000mM
as shown in Figure 4. This result shows that OMUpy2 is able not only to detect the
RNA target at the range of 10 nM to 1000mM, but also to evaluate the amount of the
RNA target from the fluorescence intensity.

30
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5
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Figure 4. The linear relationship between fluorescence intensity of OMUpy2 andthe concentration of ORN.
[Kr-OMUpy2] = 1000 nM, [Kr-ORN] = 10 nM-1000 nM, 10 mM phosphate buffer (pH7.0) containing 100 mM NaCl, λex = 340 nm, λem = 480 nm.

40Chapter 1

1.3.4. The detection of target RNA in total RNA extracted from Drosophila
embryos using OMUpy2
Although Mahara et al. [18] revealed that the 15-nt OMUpy2 bound to 120-nt
E. coli 5S-rRNA, this does not necessarily mean that the OMUpy2 can recognize thecomplementary sequence in the longer target mRNA such as 2500-nt Kr mRNA under
the presence of many other RNAs. We therefore measured emission spectra of the
Kr-OMUpy2 solution and the Control-OMUpy2 solution, respectively, both of whichwere added to the extracted mRNA purified through total RNA from Drosophila
embryo by the oligo dT cellulose column. Total mRNA (3-5-hr mRNA) was extracted
from 3-5-hr embryos expressing Kr mRNA. Kr-OMUpy2/3-5-hr mRNA solution andControl-OMUpy2/3-5-hr mRNA solution were concentrated into two microliters andtheir fluorescence characteristics were measured with a microvolume capillary adaptor
cell. As a result, Kr-OMUpy2/3-5-hr mRNA solution showed an emission at around 480
nm. However, Control-OMUpy2/3-5-hr mRNA solution did not emit signals (Figure5a.). The complementary sequence to the core region of Control-OMUpy2,
CUpyUpyUU, is included in eight places of Kr mRNA and in five places of them, six
consecutive bases are corresponding to a part of Kr mRNA. This means that OMUpy2
has the high sequence selectivity and was more likly to bind to the target part of KrmRNA.
To further confirm that the emission from Kr-OMUpy2 probe/3-5-hr mRNA
solution was attributed from the specific binding of Kr-OMUpy2 to Kr mRNA, thespectrum of the solution of Kr-OMUpy2/0-2-hr mRNA extracted from embryos before
Kr mRNA colud be expressed abundantly was measured. The fluorescence intensity at
480 nm of Kr-OMUpy2/0-2-hr mRNA solution decreased to 25 % compared to that of
the Kr-OMUpy2/3-5-hr mRNA solution (Figure 5a.). These results indicate that
Kr-OMUpy2 is capable of selectively detecting Kr mRNA in the solution containing
various RNAs, RNA pool.
To verify whether the emission in Kr-OMUpy2/0-2-hr mRNA solution was
attributed to sequence specific hybridization of Kr-OMUpy2 with the targeted sequence
in Kr mRNA, we performed a competition assay using Kr-OMUnonpy whose sequence41

Chapter 1

was the same as that of Kr-OMUpy2 except for the absence of two consecutive pyrenes.
Tm values of Kr-OMUpy2 and cORN duplex and Kr-OMUnonpy and cORN duplex are
60 ˚C and 65 ˚C, respectively. The addition of Kr-OMUnonpy to the solution of
Kr-OMUpy2/0-2-hr mRNA caused the large reduction of the fluorescence intensity ataround 480 nm (Figure 5b.). It suggests that Kr-OMUpy2 recognized Kr mRNA in asequence-specific manner and succeeded in detecting Kr mRNA.
To investigate relative RNA expression levels, bcd1-OMUpy2, bcd2-OMUpy2,
nos1-OMUpy2,

nos2-OMUpy2,

gt1-OMUpy2,

gt2-OMUpy2,

Kr-OMUpy2,

Kr2-OMUpy2, ftz1-OMUpy2, ftz2-OMUpy2, eve1-OMUpy2, and eve2-OMUpy2 wererespectively added to total RNA extracted from 0-9-hr Drosophila embryos after
fertilization. For each RNA target, two kinds of OMUpy2s were used. Theirfluorescence spectra were measured with a microspectroscope. They showed a typical
bis-pyrene fluorescence spectra having a maximum around 480 nm. The relative
expression levels between the six kinds of RNA targets were estimated from thefluorescence intensity at 480 nm (Figure 5c. (1)). There were a little differences in the
fluorescence intensities of the two kinds of OMUpy2 for the same target RNA,
respectively, but the tendency of the relative RNA expression levels in the six kinds oftarget RNA are similar between the two kinds of OMUpy2 for the same RNA. The
average of the fluorescence intensities by the two kinds of OMUpy2 for the same target
mRNA was shown in Figure 5c. (2). The relative expression levels of the six kinds of
RNA targets were similar to the tendency of total amount of 0-10-hr RNA expression
levels shown in the database (FlyBase) [41] (Figure 5c. (2) right). The expression levelof eve was the lowest and the expression level of Kr was the highest in the six kinds ofRNAs in both of database and experimental data. Relative values on the basis of theintensity of eve were shown above the bar graphs. The relative values of nos, gt, and ftz
in experimental data were according to the relative values in database. These results
indicate that it is possible to compare approximate relative RNA expression levels with
OMUpy2.

42

Chapter 1

700

Fluorescence intensity

600
500
400
300
200
100
0
360

400

440

480

520

560

600

Wavelength (nm)Figure 5a. Fluorescence spectra of Kr-OMUpy2 and Control-OMUpy2 in thepresence or the absence of mRNA extracted from Drosophila embryos expressed
Kr mRNA.
red line: Kr-OMUpy2/3-5-hr mRNA, blue line: Kr-OMUpy2/0-2-hr mRNA, green line:
Control-OMUpy2/3-5-hr mRNA, [Kr-OMUpy2] = [Control-OMUpy2] = 30 μM[mRNA] = 65 mg/ml, 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl.

43

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Fluorescence intensity

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560

600

Figure 5b. Competition assay by Kr-OMUnonpy.
blue line: Kr-OMUpy2/0-2-hr mRNA, orange line: Kr-OMUnonpy/Kr-OMUpy2/ 0-2-hr
mRNA, [Kr-OMUpy2] = 30 μM, [Kr-OMUnonpy] = 15 μM, [mRNA] = 65 mg/ml, 10mM phosphate buffer (pH 7.0) containing 100 mM NaCl.

44

Chapter 1(1)
Fluorescence intensity at 480 nm

40000

30000

20000

10000

0
bcd1 bcd2 nos1 nos2 gt1

gt2

Kr1

Kr2

ftz1 ftz2 eve1 eve2

(2)

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Average

Relative expression

35000

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1

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0

eve

bcd

bcd

eve

nos

nos

gt

gt

Kr

Kr

ftz

ftz

eve

eve

Figure 5c. Fluorescence intensity at 480 nm for the six types of RNA targets andrelative RNA expression levels.
(1) [OMUpy2] = 8 μM, [total RNA] = 70 mg/ml, 10 mM phosphate buffer (pH 7.0)
containing 100 mM NaCl, total RNA was extracted from 0-9hr embryos. (2) left: Theaverage of fluorescence intensity of the two kinds of OMUpy2 for the same target
mRNA. right: Relative RNA expression levels in 0-10hr embryos shown in the database(FlyBase: http://flybase.org/) The numbers above the bar graphs show a relative valueon the basis of the intensity of eve.

45

Chapter 1

1.3.5. The detection of target RNA in the fixed Drosophila embryo
The detection of Kr mRNA in the fixed embryos was carried out using bothstage-1 and stage-5 embryos at the beginning of formation of the cellular blastoderm.
Using a Kr-F whose 5’ end was modified with fluorescein isothiocyannate (FITC), we
first examined the distribution of probes in the fixed embryos at stage-5. Figure 6a.-A
shows differential interference contrast (DIC) images of the embryo at the stage-5, andFigure 6a.-E shows the fluorescence image after careful washing of Kr-F with PBT once.
The fluorescent signal of Kr-F was detected in the entire region of the embryo,indicating that probes were almost evenly distributed in the embryo.
Next OMUpy2 was applied to the detection of target RNA in fixed Drosophila
embryos with just one washing step by PBT. Figures 6a.-B and-C show the DIC imagesof the embryos at stage-5 after treatment with Kr-OMUpy2 and Control-OMUpy2,
respectively. Figure 6a.-D shows the result with an early embryo (stage-1) prior to
nuclei migration toward the cortex. In the embryo at stage-1, Kr mRNA is not expressed
yet. In stage-5 embryo treated with Kr-OMUpy2, a fluorescent signal was apparently
detected in the center region and around the posterior region of the embryo (Figure
6a.-F, Figure 6c.-E-H.). The localization of the signal in the center region of the embryo
corresponds to the reported Kr mRNA expression pattern in early embryos [9, 24-26].
The signal obtained in the posterior region of the embryo was similar to the observation
reported by Hoch et al. [42]. In the case of stages-5 embryo treated with
Control-OMUpy2, no fluorescence was observed (Figure 6a. G). Similarly, in thestages-1 embryo treated with Kr-OMUpy2, no fluorescence was observed (Figure
6a.-H). To confirm whether the signal in the stage-5 embryo treated with the
Kr-OMUpy2 (Figure 6a.-F) was derived from the bis-pyrene moiety, the fluorescence
spectra were measured with a microspectroscope (Figure 6b.). The spectrum of thestage-5 embryo treated with Kr-OMUpy2 peaked at around 480 nm (Figure 6a.-F and6b), and it was a waveform similar to the fluorescence spectrum of the
Kr-OMUpy2/Kr-ORN solution (Figure 3a.). In the spectrum of the stage-5 embryo
untreated with probes, small fluorescence considered to be the autofluorescence of anembryo was observed. The fluorescence observed in the spectra of the stage-5 embryo
46

Chapter 1

treated with Control-OMUpy2 and the stage-1 embryo treated with Kr-OMUpy2 wereno significant difference compared with the autofluorescence of an embryo (Figure 6b.).
These results also support that Kr-OMUpy2 detected Kr mRNA sequence specifically.
The embryos treated with the other OMUpy2 probes for bcd, nos, gt, ftz, and eve were
not observed fluorescence well. It is thought that this result relates to that Kr mRNA
was the most amount of expression of the six kinds of RNA. The detection of target
RNA by WISH is much more difficult than the detection of target RNA in the total RNA
extract because the success of the detection of target RNA in the fixed embryo largely
depends on the states of the specimen. It is not easy to solve this problem. But it isessential to improve various points including OMUpy2 for more successful detection
RNA in Drosophila embryo.
OMUpy2 has improved the protocol of whole-mount in situ hybridization in
the washing step. OMUpy2 enabled to the observation after a single wash because it
does not emit unless bound to the target RNA. In the commonly used in situ RNA
hybridization methods with Drosophila embryos, the steps from the embryo fixation toobservation take at least 3 days [10, 29]. In that protocol, cDNA probes are prepared byreverse transcription reaction on mRNA, and their lengths are usually more than 100-nt.
Using these cDNA probes, it can take a long time, sometimes one day, to attain
optimum hybridization with the target mRNA in the fixed embryos. And the repeats ofwashing steps are inevitably necessary to remove unbound signals. In contrast, using
Kr-OMUpy2, several washing steps could be omitted and the hybridization time wasshortened to a few hours. This was attributed to the unique characteristics of
Kr-OMUpy2 which consists of a short oligonucleotide (15-nt) and emits signals only
when excited in the presence of its complementary RNA. Therefore, unbound
Kr-OMUpy2 can be ignored in imaging protocols.
The development of RNA research has inaugurated a new era of life science,and the ripple effects extend to many different fields of life science. This study focuseson the technology aiming at RNA based diagnosis, which could be a key to research in
the future life sciences including medical science. From the information obtained by
DNA diagnosis, we can understand the presence of genetic alteration in specimens such
47

Chapter 1

as cells, tissue, and organs. For example, the finding of a single base alteration in DNA
suggests the possibility that the cells may develop into cancerous cells. However, thisalteration does not always mean the onset of cancer. In medical diagnosis, it is quite
important to find the signs of transformation in cells prior to their development into
malignancy. RNA could be a suitable target for detecting signs of cancerous change incells [5, 6]. In this study, we demonstrated that RNA specific probe, OMUpy2, wasapplicable to the estimation of relative RNA expression level in the total RNA extract.
We also showed the possibility of OMUpy2 as a probe to detect RNA target in
Drosophila embryo with relatively quick and easy protocol. The promising results ofthis manuscript show the great potential of RNA-based detection for both biologicalresearch and diagnostic medicine.

48

Chapter 1

Figure 6a. Differential Interference Contrast (DIC) and fluorescent images of fixed
Drosophila embryos.
Anterior is to the left. (A-D) DIC (differential interference contrast) images. (E-H)
Fluorescence images. (I-L) merge images. [OMUpy2] = [Kr-F] = 30 μM, OMUpy2:(Ex: 340/15 nm, Em: 480/30 nm), Kr-F: (Ex: 470/20 nm, Em: 520 nm (BA)) Objective:
Plan Fluor 20x, NA: 0.45, The merge images were converted to pseudo-color (I, yellow;
J, blue)49

Chapter 1

1200

Fluorescence intensity

1000
800
600
400
200
0
350

400

450

500

550

600

650

Wavelength (nm)Figure 6b. Microscopic fluorescence spectra of the embryos after whole-mount insitu hybridization.
red line: stage-5 embryo treated with Kr-OMUpy2, green line: stage-5 embryo treatedwith Control-OMUpy2, blue line: stage-5 embryo untreated with probes, yellow line:
stage-1 embryo treated with Kr-OMUpy2. Ex: 340/15 nm, DM: 380 nm, exposure time:
5 s.

50

Chapter 1

Figure 6c. Differential Interference Contrast (DIC) and fluorescent images of fixed
Drosophila embryos treated with Kr-OMUpy2.
Anterior is to the left. (A-D) DIC (differential interference contrast) images. (E-H)
Fluorescence images. (I-L) merge images. [OMUpy2] = 30 μM, OMUpy2: (Ex: 340/15
nm, Em: 480/30 nm), Objective: Plan Fluor 20x, NA: 0.45, The merge images wereconverted to pseudo-color (blue).

51

Chapter 1

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54

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Chapter 2
The quantitative analysis of RNA expression with RNA specific probes

2.1 Introduction
Many kinds of miRNAs have been found out one after another since the first
discovery in 1983 [1-4]. It has been revealed that miRNA is a molecule which
suppresses post-transcriptional level of mRNA translation in general [5-7]. Especially,
miRNA is involved in development and homeostasis, which means that the collapse ofmiRNA regulation leads to various diseases. In recent years, it has been revealed thatthe aberrant expression of miRNA is related to diseases such as cancer [2, 3, 8-10],cardiac disease [11, 12], rheumatism [13], and Alzheimer disease [14, 15]. miRNA canbe used as biomarkers of diagnosis, progression, and prognosis because many ofmiRNAs involved in certain diseases show characteristic expressions [16]. Though theexpressions of many miRNAs related to the diseases have been investigated by
microarray, probing, PCR assay, and northern blotting, most of them were theevaluation of relative expression levels or the existences of miRNAs [17-19]. In most ofother RNAs as well as miRNAs, average molecular copy number per cell is not
estimated though the relative expression levels are investigated. Therefore to calculate
the average molecule number of expressed RNA in a cell is a significant challenge even
if it is evaluated approximately.
With the aim of RNA diagnosis which can evaluate expression level in total
RNA extracts, we examined the abundance of target RNA in total RNA extracted fromHeLa cells using RNA specific probe, OMUpy2. Two characteristics of OMUpy2 makeit possible to decide the expression level of target RNA in the total RNA extract with therelations between fluorescence intensity and RNA concentration shown from OMUpy2
and its complementary RNA solution. The first is that OMUpy2 binds to target RNA ina one-to-one emission. Second is that OMUpy2 emits fluorescence at around 480 nm
55Chapter 2

only when it bound to target RNA [20, 21].

56

Chapter 2

2.2 Materials and Methods
2.2.1. Cell line
Human cervical carcinoma cell line, HeLa, was grown in Dulbecco’s modified
Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum, 25 units/mL
penicillin and 25 mg/mL streptomycin at 37 °C in a humidified chamber with 5 % CO2.

2.2.2. Extraction of total RNA from HeLa cells
Total RNA was extracted from 1×107 HeLa cells with 4 mL of TRIzol®
Reagent (Invitrogen). After stirring, 800 µL chloroform/isoamyl alcohol (49/1, v/v)
were added, and followed by vigorous shaking. After incubation on ice for 5 min, thesuspension was centrifuged at 10,000 rpm at 4 °C for 15 min. RNA in the aqueous
phase was transferred to a sterilized tube. After addition of 2 mL of isopropanol to thetube, the solution was stored on ice for 5 min followed by centrifugation at 10,000 rpm
at 4 °C for 10 min. Isopropanol was exchanged with 75 % aq. ethanol and the ethanol
solution was centrifuged at 7,500 rpm at 4 °C for 3 min three times. Decant thesupernatant, removing as much as possible without disturbing the pellet. Air-dry RNApellet.

2.2.3. Fluorescence measurements of OMUpy2 hybridized with complementaryoligo RNA (ORN) and OMUpy2 hybridized with target RNA in total RNA extract
Fluorescence measurements of OMUpy2 hybridized with complementary oligo
RNA (ORN) and OMUpy2 hybridized with total RNA extracted from Drosophila
embryos were measured with a spectrofluorophotometer (RF-5300PC, Shimadzu). The
concentration of ORN on creating calibration plots was changed in the range of 10 nM
to 1000 nM. The concentration of total RNA used for measurements was 1400 µg/mL
and that of OMUpy2s were each 1 µM in 10 mM phosphate buffer containing 100 mM
NaCl (pH 7.0). The excitation wavelength of fluorophore (OMUpy2) was 342 nm.

57

Chapter 2

2.3 Results and Discussion
2.3.1. Probe preparation
As target RNAs, 28S rRNA, hsa-mir-21, hsa-mir-24-2, hsa-mir-29a, and U6
snRNA were chosen. miR-21 is one of the most commonly and highly upregulated
miRNA in many kinds of cancer [22, 23]. miR-21, miR-24, and miR-29 are highly
transcribed in a HeLa cell [24]. The relative expression levels of them are high in orderof miR-21, miR-24, and miR-29 [24]. The expression level of U6 sn RNA is also highin a HeLa cell [25].
OMUpy2 were synthesized according to previous reports and purified byreverse phase HPLC [21]. The details of the sequences were shown in Table 1. The
fluorescence spectra of duplex between OMUpy2 and their ORN solution were peakedat around 480 nm.

Table 1. Sequences used in the present study.
target RNA
28S rRNAhsa-mir-21
hsa-mir-24-2

name
28S-ORNU6 snRNA

Nucleotide
positions

GenBank ID/
miRBaseAccession

CGC UCA UCA GAC CCC AGA AAA G

1903-1924

3373818-29MI0000077

50-71

MI0000081

42-63

MI0000087

16-36

17494328S-OMUpy2

CUU UUC UGG GGUpy CpyUG AUG AGC G

miR21-ORN

UAG CUU AUC AGA CUG AUG UUG A

miR21-OMUpy2

UCA ACA UCA GUpyCpy UGA UAA GCU A

miR24-ORNUGG CUC AGU UCA GCA GGA ACA G

miR24-OMUpy2

hsa-mir-29a

5’ SEQUENCES 3’
upper: target RNA / lower: probe

CUG UUCpy CpyUG CUG AAC UGA GCC A

miR29-ORN

UAG CAC CAU CUG AAA UCG GUU A

miR29-OMUpy2

UAA CCG AUpyUpy UCA GAU GGU GCU A

U6 snRNAACG CAA AUU CGU GAA GCG UU

U6-OMUpy2

AAC GCU UCA CGA AUpyUpy UGC GU

Italic letters: 2’-OMe nucleotides, capital letters: ribonucleotides.

58

Chapter 2

2.3.2. The calibration plots of the fluorescence intensity at 480 nm corresponding to
ORN concentration
In order to create the calibration plots of the fluorescence intensity at 480 nm
corresponding to ORN concentration, the fluorescence intensity at 480 nm of OMUpy2
in the presence of the complementary ORN (cORN) was measured in the range of 10
nM to 1000 nM cORN concentration. The fluorescence intensity of OMUpy2-cORN
hybrids versus the concentration of cORN had a linear relationship as shown in Figure
1.

59Chapter 2

(2)

250

30
20
10
0
0 20 40 60 80100

200
150

Fluorescence intensity at 480 nm

Fluorescence intensity at 480 nm

(1)

100
50
0
0

200

400
600
800
[ORN] (nM)

100
50
0
0

1000

(3)

200

125

20
15
10
5
0

100
75

0 20 40 60 80 100

50
25
0
0

200

400
600
800
[ORN] (nM)

1000

(5)

1000

125 2015
100 105
075
0 20 40 60 80 100
50
25
0
0

200

400
600
800
[ORN] (nM)

1000

Figure 1. The calibration plots of thefluorescence

Fluorescence intensity at 480 nm

400
600
800
[ORN] (nM)(4)

Fluorescence intensity at 480 nm

Fluorescence intensity at 480 nm

250 3020200 100150
0 20 40 60 80100

50 10
8
6
40 4
2
0
30
0 20 40 60 80 100

10
0
200

400
600
800
[ORN] (nM)

at

480

nm

corresponding to ORN concentration

20

0intensity

1000(1) 28S-OMUpy2/ORN,(2) miR21-OMUpy2/ORN,(3) miR24-OMUpy2/ORN,(4) miR29-OMUpy2/ ORN,
(5) U6-OMUpy2/ORN,[28S-OMUpy2] = [miR21-OMUpy2] =[miR24-OMUpy2] = [miR29-OMUpy2] =[U6-OMUpy2] = 1000 nM, [ORN] = 10
nM-1000 nM in 10 mM phosphate buffer(pH 7.0) containing 100 mM NaCl,
20 ˚C, λex=342 nm, λem = 480 nm.
60Chapter 2

2.3.3. The quantitative analysis of RNA expression levels in a HeLa cell caluculated
from fluorescence intensity of OMUpy2
To quantify the amount of RNA expression in a cell, the fluorescence spectra of
OMUpy2 and total RNA solution extracted from 1×107 HeLa cells were measured(Figure 2.). To determine the molecular abundance of target RNA in a cell, thefluorescence signals of the hybridization between OMUpy2 and target RNA in the total
RNA were compared with the calibration plots (Figure 3.). The number of mir-21, 104
molecules per cell, corresponded to a previous report by Lee et al. [26]. The number ofU6 snRNA was one order of magnitude smaller than previously reported by Lee et al.
[26]. Though the relative expression of miR-29 compared to miR-21 is reported to beabout one tenth by Schmittgen et al. [17], our result was larger than that. miRNAs aretranscribed as long primary transcripts (pri-miRNAs) whose maturation occurs throughfollowing processing; the nuclear processing of the pri-miRNAs into stem-loop
precursors, 70 nucleotides (pre-miRNAs), and the cytoplasmic processing of
pre-miRNAs into mature miRNAs [27]. OMUpy2 cannot distinguish that the detected
miRNAs are at any of these stages. But it is a great advantage to be able to detect target
RNA without the amplification of total RNA extract because an error based on theamplification efficiency does not occur. In order to adequately characterize theexpression of miRNA, it may be necessary to quantify both of mature and precursor
miRNA. Inferring the exact RNA expression level is difficult, but even approximationof RNA expression level would give us useful information to assist the judgment of
diagnosis, progress, and prognosis of various diseases.

61

Chapter 2

(1)

60

Fluorescence intensity

50
40
30
20
10
0
350

400

450

500

550

600

650

Wavelength (nm)(2)

Fluorescence intensity at 480 nm

60
50
40
30
20
10
0
28SrRNA miR21

miR24

miR29

U6

Figure 2. (1) Fluorescence spectra of OMUpy2 in the presence of total RNA. (2)Fluorescence intensity at 480 nm.
blue line: 28S-OMUpy2/total RNA, red line: miR21-OMUpy2/total RNA, green line:
miR24-OMUpy2/total RNA, yellow line: miR29-OMUpy2/total RNA, pink line:
U6-OMUpy2/total RNA, Conditions: [28S-OMUpy2] = [miR21-OMUpy2] =[miR24-OMUpy2] = [miR29-OMUpy2] = [U6-OMUpy2] = 1 µM, [total RNA] = 1400
µg/mL in 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl, 20 °C, λex =342 nm.
62

Chapter 2

5

Copy number of transcripts / cell

3x10

2.5x1052x105
1.5x105
1x105
5x104
0
28SrRNA miR21

miR24

miR29

U6

Figure 3. The quantitative analysis of RNA expression in a cell.
Molecular abundance of 28S rRNA, miRNAs, and U6 snRNA. Copy numbers of thetranscript per cell were calculated from fluorescence intensity (Figure1. and Figure 2.).

63

Chapter 2

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Ueda, T., Kobori, A., Yamayoshi, A., Yoshida, H., Yamaguchi, M., Murakami,
A. (2012) RNA-based diagnosis in a multicellular specimen by whole mount insitu hybridization using an RNA-specific probe. Bioorg. Med. Chem., 20,6034-6039.
Mahara, A., Iwase, R., Sakamoto, T., Yamana, K., Yamaoka, T., Murakami, A.
(2002) Bispyrene-conjugated 2’-O-methyloligonucleotide as a highly specificRNA-recognition probe. Angew. Chem. Int. Ed. Engl., 41, 19, 3648-3650.
Sayed, D., Rane, S., Lypowy, J., He, M., Chen, I.Y., Vashistha, H., Yan, L.,
Malhotra, A., Vatner, D., Abdellatif, M. (2008) MicroRNA-21 targets Sprouty2
and promotes cellular outgrowths. Mol. Biol. Cell., 19, 8, 3272-3282.
Volinia, S., Calin, G.A., Liu, C.G., Ambs, S., Cimmino, A., Petrocca, F., Visone,R., Iorio, M., Roldo, C., Ferracin, M., Prueitt, R.L., Yanaihara, N., Lanza, G.,
Scarpa, A., Vecchione, A., Negrini, M., Harris, C.C., Croce, C.M. (2006) A
microRNA expression signature of human solid tumors defines cancer genetargets. Proc. Natl. Acad. Sci. U. S. A., 103, 7, 2257-2261.
Lau, N.C., Lim, L.P., Weinstein, E.G., Bartel, D.P. (2001) An abundant class oftiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science,
294, 5543, 858-862.
Maroney, P.A., Chamnongpol, S., Souret, F., Nilsen, T.W. (2007) A rapid,
quantitative assay for direct detection of microRNAs and other small RNAs
using splinted ligation. RNA, 13, 6, 930-936.
Lim, L.P., Lau, N.C., Weinstein, E.G., Abdelhakim, A., Yekta, S., Rhoades,
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27.

M.W., Burge, C.B., Bartel, D.P. (2003) The microRNAs of Caenorhabditis
elegans. Genes. Dev., 17, 991-1008.
Lee, Y., Jeon, K., Lee, J.T., Kim, S., Kim, V.N. (2002) MicroRNA maturation:
stepwise processing and subcellular localization. EMBO J., 21, 4663-4670.

66

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Chapter 3
The development of multi-colored FRET RNA probes

3.1 Introduction
Fluorescent microscopy techniques with dye-conjugated biomolecules havebeen used to elucidate the expressions, localizations, and movements of RNA [1, 2].
The multi-colored fluorescent probes enabled to simultaneously detect multiple RNAtargets [3], transcription sites [4] and chromosomes [5] and have been applied todetermine the spatial organization of two or even more types of biomolecules [6, 7].
Multiple separate images are taken with different excitation emission filter setsaccording to the fluorescent dyes. Therefore it is very difficult to monitor the expressionof multiple RNA targets that are occuring at the same time using multi-excitation
wavelength systems.
Kool [8, 9] reported excimer- and exciplex-based multicolor imaging systems.
They synthesized oligodeoxyfluorosides with pyrene, benzopyrene, perylene, andstilbene derivatives at the 1’-position of nucleosides and took multicolor images with asingle-excitation wavelength. Tong [10, 11] and Ohya [12] also reported combinatorial
Förster resonance energy transfer (FRET) systems that exhibited different fluorescentemission patterns with a single-excitation wavelength and those were used in themultiplex detection of single nucleotide polymorphisms (SNPs). Based on these reports,
multi-color fluorescent probes with a single-excitation wavelength are highly useful forbiological assays and the development of new fluorescent probes having multi-color
fluorescent dyes with a single-excitation wavelength is a challenging task.
To simultaneously visualize multiple RNA targets in a cell, it is necessary todevelop probes which emit fluorescence with different colors when excited at asingle-wavelength. Here we synthesized OMUpy2 with a cyanine dye conjugated attheir 5’ end respectively. Cyanine dyes, Cy3 and Cy5, emit fluorescence around 570 nm
or 670 nm when excited at 550 nm or 650 nm respectively (Figure 1.). Cy3 and Cy5
67Chapter 3

emit no fluorescence by the 342 nm excitation which is the excitation wavelength ofpyrene. Cyanine dyes conjugated probes, Cy3-OMUpy2 and Cy5-OMUpy2, caused
large Stokes shifts by FRET in the presence of the complementary RNA when excited at
342 nm. OMUpy2, Cy3-OMUpy2, and Cy5-OMUpy2 emitted blue, yellow and pink
fluorescence respectively with single-wavelength excitation. It means that three RNAtargets can be detected in a cell at the same time.

68Chapter 3

(1)

Cy3

py

hν

hν

Cy3

py
py

FRET

FRET
ex: 342 nm

ex: 342 nm

Cy5

py

hν
ex: 342 nm

hν
FRET

Cy5

py
py

ex: 342 nm

FRETAbsorbance / Fluorescence

(2)

300

400

500

600

700

Wavelength (nm)Figure 1. (1) Scheme of OMUpy and OMUpy2 modified with Cy3 or Cy5. (2)Absorption and fluorescence spectra of Cy3 and Cy5.

blue line: absorption spectrum of Cy3, yellow line: fluorescence spectrum of Cy3, green
line: absorption spectrum of Cy5, red line: fluorescence spectrum of Cy5.

69

Chapter 3

3.2 Materials and Methods
3.2.1. Probe preparation
Single-stranded region in the secondary RNA structure for Krüppel mRNA wastargeted.

2’OMe

RNA

oligonucleotides

weresynthesizedusing

standard

phosphoramidite chemistry and purified by reverse phase HPLC. 2’OMe RNA probes
were synthesized using standard 2’OMe phosphoramidite monomers. Fluorescent dyes
were linked to the 5’-end via a Cy5™ Phosphoramidite and Cy3™ Phosphoramidite(Glen Research) respectively [13]. Ion-molecular weights of probes were determined bymass spectrometry using ESI-MASS.

3.2.2. Fluorescence measurements of the hybridization solutions with cyanine dye
conjugated RNA probes
Probes and the complementary oligoribonucleotides (ORN) were solved in 10
mM phosphate buffer (100mM NaCl, pH=7.0) (final concentration, 750 nM).
Fluorescence emission was measured with a spectrofluorometer (RF-5300PC,
Shimadzu). The excitation wavelength of donor fluorophore (pyrene) was 342 nm.

3.2.3. Tm values of probes with the complementary oligoribonucleotides
Melting curves (absorbance versus temperature curves) were measured at 260
nm for the equimolar mixture of the probe and the complementary oligoribonucleotides(ORN) in 10 mM sodium phosphate (pH=7.0) containing 100 mM NaCl. (final
concentration, 750 nM).

3.2.4. Visualization of fluorescent dye-conjugated oligoribonucleotides in thepresence of ORN
The solutions of hybrids between Cyanine dye-conjugated oligoribonucleotides
and the complementary ORN were visualized by UV irradiation at 365 nm using a UVtransilluminator (excitation wavelength = 365 nm) with 8 s exposure. Conditions:[OMUpy2] = [ORN] = 3 µM, [Cy3-OMUpy2] = [ORN] = 750 nM, [Cy5-OMUpy2] =70

Chapter 3[ORN] = 750 nM in phosphate buffer (10 mM sodium phosphate (pH=7.0) containing
100 mM NaCl).

71

Chapter 3

3.3 Results and Discussion
3.3.1. Probe preparation
Cyanine dye conjugated OMUpy probes used in the presence of study are
shown in table. 1. Using our procedures for synthesizing Upy and conventional
phosphoramidite methods [13], oligoribonucleotides with cyanine dyes at the 5’-end
were synthesized and purified by reversed-phase HPLC.
The obtained oligoribonucleotides were characterized using ESI-MASS.
([Cy3-OMUpy1-5H]-5; calcd. 1112.2, found 1112.6, [Cy3-OMUpy2-6H]-6; calcd. 960.0,
found 960.0, [Cy5-OMUpy1-5H]-5; calcd. 1117.4, found 1117.8, [Cy5-OMUpy2-5H]-5;
calcd. 1447.0, found 1447.1).
Melting curves (absorbance versus temperature curves) were measured at 260
nm for the equimolar mixture of the probe and the complementary oligoribonucleotides(ORN) in 10 mM sodium phosphate (pH=7.0) containing 100 mM NaCl (table.2). The
Tm value of OMUnonpy which has no fluorophore modification and ORN was 65 °C.
Compared with it, the Tm values of pyrene and cyanine dyes modified probes and ORN
were a little lower but they neatly formed duplexes. From these results it is found that
Upy, Cy3, and Cy5 hardly affected the stability of the duplex formed by the probe and
ORN.

72

Chapter 3

Table 1. Sequences used in the present study.
name5’ SEQUENCES 3’

Cy3-OMUpy1 (*)
Cy3-OMUpy2
Cy5-OMUpy1
Cy5-OMUpy2
Cy3-T
Cy5-T
OMUpy2
OMUnonpy
ORN

Cy3-UCC UAA UUpyU UGU GCU
Cy3-UCC UAA UpyUpyU UGU GCU
Cy5-UCC UAA UUpyU UGU GCU
Cy5-UCC UAA UpyUpyU UGU GCU
Cy3-T
Cy5-T
UCC UAA UpyUpyU UGU GCU
UCC UAA UUU UGU GCU
AGC ACA AAA UUA GGA

Italic letters: 2’-OMe nucleotides, capital letters: ribonucleotides. Cy5 and Cy3 are
conjugated at the 5’ ends of the hydroxyl groups of the nucleotides. (*) The sequence iscomplementary to the 304-318 nucleotide position of the Kr mRNA (GenBank ID:
22024037).

Table 2. Tm values of probes with the complementary oligonucleotides (ORN).
Tm value [ ̊C]

Probe / ORN
OMUnonpy/ ORN
OMUpy1/ ORN
OMUpy2/ ORN
Cy3-OMUpy1/ ORN
Cy3-OMUpy2/ ORN
Cy5-OMUpy1/ ORN
Cy5-OMUpy2/ ORN

65
61
60
63
59
62
60

Melting temperatures of the equimolar mixture of the probe and ORN in 10 mM sodium
phosphate (pH=7.0) containing 100 mM NaCl.

73

Chapter 3

3.3.2.

Absorption

spectraandfluorescence

spectraofCy3-conjugatedoligoribonucleotides and Cy5-conjugated oligoribonucleotides
The fluorescent properties of Cy3-conjugated oligonucleotides were examined(Figure 2a. (2)). Cy3-conjugated oligonucleotides were excited at 342 nm.
Cy3-OMUpy2 had faint fluorescence around 480 nm derived from pyrene excimer andstrong fluorescence around 570 nm derived from Cy3. The Stokes shift of
Cy3-OMUpy2 was nearly 230 nm. Cy3-OMUpy1 also had strong fluorescence around
570 nm with small fluorescence peaking at 378 nm and 398 nm derived from pyrene
monomer. In contrast to Cy3-OMUpy1 and Cy3-OMUpy2, Cy3-conjugated thymidine(Cy3-T) had nearly no fluorescence around 570 nm with excitation at 342 nm. These
results suggested that Cy3 chromophore conjugated with OMUpy were not directly
excited by UV irradiation at 342 nm, but that Upy in Cy3-OMUpy1 and Cy3-OMUpy2strongly enhanced Cy3 emission. Yamana reported that the pyrene group incorporated
into OMUpy1 is intercalated between adjacent nucleobases, and that the fluorescenceintensity of OMUpy1 derived from the pyrene monomer around 370 nm is largely
quenched due to the formation of exciplexes between pyrene and the nucleobases [14].
In the case of OMUpy2, the fluorescence derived from pyrene monomer was quenched,
whereas large fluorescence around 480 nm derived from pyrene excimer was observedin the presence of the complementary RNA [15]. Kawai reported that largely quenchedpyrene attached to the DNA strand could serve as a donor for Förster resonance energy
transfer (FRET) [16]. Therefore, Cy3 chromophore conjugated to OMUpy2 andOMUpy1 might be excited by FRET from pyrene chromophore under the originallyquenched environment.
Next, fluorescence spectra of cyanine dye-conjugated oligoribonucleotides in
the presence of the complementary RNA (ORN) were examined. Cy3-OMUpy2/ORN
and Cy3-OMUpy1/ORN had strong emission around 570 nm with excitation at 342 nm(Figure 2b. (2)), as in the case of Cy3-conjugated oligoribonucleotides (Figure 2a. (2)).
The fluorescence intensity at 570 nm of Cy3-OMUpy2/ORN was four-fold stronger
than that of Cy3-OMUpy1/ORN.
We

also

examinedthefluorescent74

propertiesofCy5-conjugatedChapter 3

oligoribonucleotides, (Figure 2c. (2), 2d. (2)). Cy5-OMUpy1 and Cy5-OMUpy2 hadstrong fluorescence at 670 nm with an extremely large Stokes shift of 330 nm.
Compared to Cy3-OMUpy2, Cy5-OMUpy2 has stronger fluorescence around 480 nm.
This result suggests that the efficiency of energy transfer might be relatively low.
Cy5-OMUpy2/ORN and Cy5-OMUpy1/ORN also had fluorescence around 670 nmwith excitation at 342 nm (Figure 2d. (2)). The fluorescence intensity at 670 nm of
Cy5-OMUpy2/ORN was a little stronger than that of Cy5-OMUpy1/ORN. It isconsidered that this result is attributed to the number of pyrene in the OMUpy like acase of Cy3-conjugated oligoribonucleotides.
We showed that cyanine-conjugated OMUpy in the presence of the ORN havestrong fluorescence emission derived from cyanine chromophores excited by theemission of pyrene and cyanine-conjugated OMUpy2 are more suitable for the
fluorescence detection of the complementary RNA than that of OMUpy1.

75

Chapter 3

(1)

0.2

Absorbance

0.15

0.1

0.05

0
250 300 350 400 450 500 550 600 650
Wavelength (nm)

(2)

500

Fluorescence intensity

400
300
200
100
0
350 400

450 500 550 600 650 700 750
Wavelength (nm)Figure 2a. (1) Absorption spectra and (2) Fluorescence spectra of Cy3-conjugatednucleotides.
[Cy3-OMUpy2] = [Cy3-OMUpy1] = [Cy3-dT] = 750 nM, λex=342 nm, room
temperature, green line: Cy3-OMUpy2, blue line: Cy3-OMUpy1, red line: Cy3-dT.

76

Chapter 3

(1)

0.3
0.25

Absorbance

0.2
0.15
0.1
0.05
0
250 300 350 400 450 500 550 600 650
Wavelength (nm)(2)

Fluorescence intensity

200

150

100

50

0
350 400

450 500 550 600 650 700 750
Wavelength (nm)Figure 2b. (1) Absorption spectra and (2) Fluorescence spectra of Cy3-conjugatednucleotides in the presence of ORN.
[Cy3-OMUpy2] = [Cy3-OMUpy1] = [ORN] = 750 nM, λex=342 nm, room temperature,green line: Cy3-OMUpy2/ORN, blue line: Cy3-OMUpy1/ORN.

77

Chapter 3

(1)

0.2

Absorbance

0.15

0.1

0.05

0
250

350

450
550
Wavelength (nm)

650

750

(2)

120

Fluorescence intensity

100
80
60
40
20
0
350 400

450 500 550 600 650 700 750
Wavelength (nm)Figure 2c. (1) Absorption spectra and (2) Fluorescence spectra of Cy5-conjugatednucleotides.
[Cy5-OMUpy2] = [Cy5-OMUpy1] = [Cy5-dT] = 750 nM, λex=342 nm, room
temperature, green line: Cy5-OMUpy2, blue line: Cy5-OMUpy1, red line: Cy5-dT.

78

Chapter 3

(1)

0.3
0.25

Absorbance

0.2
0.15
0.1
0.05
0
250

350

450
550
Wavelength (nm)

650

750

(2)

300

Fluorescence intensity

250
200
150
100
50
0
350 400

450 500 550 600 650 700 750
Wavelength (nm)Figure 2d. (1) Absorption spectra and (2) Fluorescence spectra of Cy5-conjugatednucleotides in the presence of ORN.
[Cy5-OMUpy2] = [Cy5-OMUpy1] = [ORN] = 750 nM, λex=342 nm, room temperature,green line: Cy5-OMUpy2/ORN, blue line: Cy5-OMUpy1/ORN.

79

Chapter 3

3.3.3. Visualization of fluorescent dye-conjugated oligoribonucleotides in thepresence of ORN
OMUpy2, Cy3-OMUpy2 and Cy5-OMUpy2 in the presence of ORN werevisualized by UV irradiation at 365 nm using a UV transilluminator (Figure. 3.).
OMUpy2 has blue fluorescence in the presence of ORN. Cy3-OMUpy2 at aconcentration of 750 nM emitted bright yellow fluorescent light. Cy5-OMUpy2 gave
pink light under the same conditions. These images indicated that Cy3-OMUpy2 andCy5-OMUpy2 will be feasible to simultaneously visualize multiple RNA targets in acell by single-excitaion wavelength.

(a)

(b)

(c)

Figure 3. Visualization of fluorescent dye-conjugated oligoribonucleotides in thepresesnce of ORN.
(a) [OMUpy2] = [ORN] = 3 µM, (b) [Cy3-OMUpy2] = [ORN] = 750 nM, (c)[Cy5-OMUpy2] = [ORN] = 750 nM in 10 mM sodium phosphate (pH=7.0) containing
100 mM NaCl. The oligoribonucleotides solutions were excited by a UVtransilluminator (excitation wavelength = 365 nm) with 8 s exposure.

80

Chapter 3

References
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Weil, T.T., Parton, R.M., Davis, I. (2010) Making the message clear:visualizing mRNA localization. Trends Cell Biol., 20, 380–390.
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Kosman, D., Mizutani, C.M., Lemons, D., Cox, W.G., McGinnis, W., Bier, E.
(2004) Multiplex detection of RNA expression in Drosophila embryos. Science,
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Levsky, J.M., Shenoy, S.M., Pezo, R.C., Singer, R.H. (2002) Single-cell gene
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Macville, M., Veldman, T., Padilla-Nash, H., Wangsa, D., O’Brien, P., Schröck,
E., Ried, T. (1997) Spectral karyotyping, a 24-colour FISH technique for theidentification of chromosomal rearrangements. Histochem. Cell Biol., 108,299-305.
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Teo, Y.N., Kool, E.T. (2009) Polyfluorophore excimers and exciplexes as FRET
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Tong, A.K., Li, Z., Jones, G.S., Russo, J.J., Ju, J. (2001) Combinatorial
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Tong, A.K., Ju, J. (2002) Single nucleotide polymorphism detection by
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Ohya,Y., Yabuki, K., Hashimoto, M., Nakajima, A., Ouchi, T. (2003) Multistep
fluorescence resonance energy transfer in sequential chromophore array
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deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids
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Nakamura, M., Fukunaga, Y., Sasa, K., Ohtoshi, Y., Kanaori, K., Hayashi, H.,
Nakano, H., Yamana, K. (2005) Pyrene is highly emissive when attached to the
RNA duplex but not to the DNA duplex: the structural basis of this difference.
Nucleic Acids Res., 18, 5887-5895.
Mahara, A., Iwase, R., Sakamoto, T., Yamana, K., Yamaoka, T., Murakami, A.
(2002) Bispyrene-conjugated 2’-O-methyloligonucleotide as a highly specific81

Chapter 3

16.

RNA-recognition probe. Angew. Chem. Int. Ed., 41, 3648-3650.
Kawai, K., Kawabata, K., Tojo, S., Majima, T. (2002) Synthesis of ODNs
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82

Summary
Chapter 1.
The detection of mRNA expressed in Drosophila embryo using OMUpy2 wasdescribed. The performance of OMUpy2 for RNA detection was estimated using oligo
RNA solution, total RNA extracted from Drosophila embryo, and Drosophila embryo.
In this thesis, OMUpy2 was used as RNA-specific probe. OMUpy2 emitted
fluorescence at 480 nm only when it hybridized with its complementary RNA in
homogeneous media. OMUpy2 also emitted similar fluorescence around 480 nm in total
RNA extracted from Drosophila embryos containing a large amount of RNA other thantarget mRNA. These results suggest that target mRNA in total RNA extracts was
successfully detected by OMUpy2. Furthermore, from the fluorescence intensity of
OMUpy2, relative mRNA expression levels could be estimated. The detection of target
mRNA in Drosophila fixed embryo was carried out using Kr-OMUpy2, and theemission was observed in the center region of the fixed embryo. It was confirmed thatthe emission observed in the embryo treated with Kr-OMUpy2 was derived frombis-pyrene by the fluorescence spectrum measured with a microspectroscope under the
microscope.

Chapter 2.
The average molecule numbers of target RNA expressed in a HeLa cell were
estimated using OMUpy2. In general, inferring the exact RNA expression level isdifficult, but even approximation of RNA expression level would give us useful
information to assist the diagnosis and the judgment of progress, and prognosis forvarious diseases. We examined whether the abundance of target RNA in total RNA
extracted from HeLa cells could be estimated using OMUpy2. Two characteristics ofOMUpy2 make it possible to estimate the expression level of target RNA in total RNA
extract. The first is that OMUpy2 binds to target RNA in a one-to-one. Second is thatOMUpy2 emits fluorescence at around 480 nm only when it bound to target RNA. In
order to create the calibration plots of the fluorescence intensity at 480 nm
83corresponding to cORN concentration, the fluorescence intensity at 480 nm of OMUpy2
in the presence of cORN was measured in the range of 10 nM to 1000 nM cORN
concentration. The expression levels of 28S rRNA, mir-21, mir-24, mir-29, and U6
snRNA in a HeLa cell were calculated from the calibration plots of the fluorescenceintensity at 480 nm corresponding to ORN concentration, respectively.

Chapter 3.
The development of multi-colored RNA FRET probes to simultaneously
observe multiple target RNAs was described. The excitation and fluorescencewavelength of Cy3 are 550 nm and 570 nm, respectively. Those of Cy5 are 650 nm and
670 nm, respectively. Both Cy3 and Cy5 emitted no fluorescence with 342 nm
excitation. However OMUpy2 modified with Cy3, Cy3-OMUpy2, emitted fluorescenceat around 650 nm with 342 nm excitation. OMUpy2 modified with Cy5, Cy5-OMUpy2,
emitted fluorescence at around 670 nm with 342 nm excitation. FRET might beoccurred from pyrene in OMUpy2 to Cy3 or Cy5. Equimolar mixtures of OMUpy2 andoligo RNA, Cy3-OMUpy2 and oligo RNA, and Cy5-OMUpy2 and oligo RNA solutions
emitted blue, yellow, and pink fluorescence, respectively, by the excitation at 342 nm.
These results suggest that these probes are applicable to simultaneous detection ofmultiple target RNAs in a cell.

84

List of Publications
Chapter 1: Ueda, T., Kobori, A., Yamayoshi, A., Yoshida, H., Yamaguchi, M.,
Murakami, A.
RNA-based diagnosis in a multicellular specimen by whole mount in situ
hybridization using an RNA-specific probe
Bioorganic & Medicinal Chemistry, 20, 6034-6039. (2012)
doi: 10.1016/j.bmc.2012.08.028.

Chapter 2: Ueda, T., Murakami, A.
Quantitative analysis of RNA expression with RNA specific probes
In preparation

Chapter 3: Kobori, A., Ueda, T., Sanada, Y., Yamayoshi, A., Murakami, A.
Dual-Fluorescent RNA Probes with an Extremely Large Stokes Shift
Biosci. Biotechnol. Biochem., 77, 1117-1119. (2013)
doi:10.1271/bbb.121018

85

Acknowledgements
The present study in this doctoral dissertation was carried out under theguidance of Professor Akira Murakami at the Bio-regulation Science Laboratory of the
Department of Molecular Engineering, Kyoto Institute of Technology from 2006 to2013.
It would not have been possible to write this doctoral dissertation without thehelp and support of the kind people around me. I would like to express my sincere
gratitude to them here.
I am grateful to Professor Akira Murakami for giving me the opportunity to
study under his group and his guidance. I am also grateful to Professor Masamitsu
Yamaguchi for his collaboration, advice on writing papers, and direction of
microinjection technique. I would like to thank Associate professor Akio Kobori for his
suggestion and advice on writing papers. I appreciate Assistant professor Asako
Yamayoshi and Hideki Yoshida for their discussion and advices.
I also thank my friends and members of the Bio-regulation Science laboratory
and Chromosome technology laboratory for their help and support.
Last but not the least, I would like to extend my deepest gratitude to my family,
Makoto Ueda, Makiko Ueda, Tomoko Ueda, and Hiroko Ueda. Without theirencouragement, support, and patience over a long period of time, my doctoral
dissertation could not have been accomplished. I’m really grateful for all the family.

April 4, 2013

TAKAKO86

UEDA