Independent Research Proposal
A Novel Molecular Beacon with Long-wavelength Fluorescent Dyes for High Sensitivity DNA/Protein Analysis
Libo Cao
Department of Chemistry, Center for Intelligent Chemical Instrumentation, OhioUniversity, Athens, OH45701-2979
IDEA
DNA/RNA analysis is of great importance in molecular biology, genetics, and molecular medicine. Molecular beacons (MBs) are single-stranded oligonucleotide probes with a hairpin structure that can identify the mutations in the human genome caused by DNA hybridization.
A fluorophore and a quencher are linked to the two ends of the strand. The bases at both ends of the beacon are complementary to each other, forming the stem, which keeps the fluorophore and the quencher in proximity to each other. The fluorescence of the fluorophore, which are usually dyes, is thus quenched by the quencher through energy transfer. This proposal attempts to design a novel molecular beacon with long-wavelength fluorescent dyes for high sensitivity and efficiency DNA/Protein analysis. With the long-wavelength fluorescent dyes, which will have emission and excitation greater than 600 nm, the background noise will be attenuated. By using two fluorophores, as acceptor and donor, defined in FRET theory in stead of a fluorophore and a quencher, higher molecular beacon efficiency can be obtained. The cyanine dyes Cy3 and Cy5 are presented as the fluorophore donor and acceptor in MBs.
Another advantage of this method is that 630-650 nm laser diode can be used as the excitation source if using near infrared donor and acceptor pair to label the ends of MB. The traditional laser sources are He-Cd (325 nm and 441 nm) and Ar ion (488 nm and 512 nm), which excite auto-fluorescence. However the diode laser will circumvent this shortcoming.1-3Further more, diode lasers have several other advantageous characteristics: they are small, have a low flicker noise (<0.05%), and the available wavelengths are in the red (>630nm) to near-infrared region, where the light scattering is generally low.
BACKGROUND
1. Molecular beacon
DNA and RNA analysis is of great importance in molecular biology, genetics, and molecular medicine. Molecular beacons are a new class of DNA/RNA probes. The first molecular beacon, which is a single-stranded oligonucleotide probe with a hairpin structure, was developed in 1996.4A fluorophore and a quencher are linked to the two ends of the strand. The five to seven bases at both ends of the beacon are complementary to each other, forming the stem, which brings the fluorophore and quencher close enough to allow quenching to occur by fluorescence resonance energy transfer. Upon hybridization to a complementary DNA sequence, the hairpin loop is broken and the fluorophore and quencher are separated, resulting in the restoration of fluorescence.
1
Fig. 1. Principle of operation of molecular beacons.
Because molecular beacons can possess a wide variety of differently colored fluorophores, multiple targets can be distinguished in the same solution, using several different molecular beacons, each designed to detect a different target, and each labeled with a different fluorophore.
Single-nucleotide differences in a DNA sequence can thus be detected in homogeneous assays.5The region surrounding the site of a sequence variation is amplified in a polymerase chain reaction and the identity of the variant nucleotide is determined by observing which of four differently colored molecular beacons binds to the amplification product.
Anthony developed the method for detection of single-base mismatches using molecular beacon with high selectivity. The method is based on fluorescence resonance energy transfer (FRET) between a fluorophore attached to an immobilized DNA strand (“probe”) and a quencher-containing sequence that is complementary except for an artificial mismatch at the site of interrogation.6, 7 Also, since the signal transduction mechanism is built within the MB molecules, no DNA intercalation reagent or labeling of the target molecule is needed. Molecular beacons have shown many other advantages over other DNA probes. These include the excellent capability of studying biological process in real time and in vivo.
1
1
Fig. 2.Autofluorescence of Gray Snapper (L.griseus) oocyte8
1
For current molecular beacons, the fluorophore usually chosen is fluorescein (FAM),9which has emission at 520nm and excitation at 480nm. However, for some amino acids, such as Oocyte, Tryptophane, and Tyrosine, autofluorescence will occur at this region.8The figure 2 above shows the autofluorescence of a different Gray Snapper (L. griseus) oocyte excited at 490 nm and 685nm. The accompanying surface plot shown to the right of the corresponding image graphically illustrates the autofluorescent signal or background noise using 490 nm and the lack of autofluorescence using 685nm, the LaJolla Blue excitation wavelength. Thus using these molecular beacons to detect protein with these amino acids will reduce the signal-to-noise ratio and the sensitivity.
2. Fluorescence resonance energy transfer (FRET)
FRET involves non-radiative transfer of electronic excitation from an excited donor, D* to a ground state acceptor molecule A, and occur on time scales from femto seconds to milliseconds at distances ranging from 10 to approximately 100. Therefore, FRET suits for the detection of dynamic distance changes, for example the distance change during the process of open and close of the hairpin loop. The particular advantage of FRET is the dependence (and thus sensitivity) of the energy transfer efficiency (E) on the sixth power of the distance (R) between the chromophores.10The energy transfer yield is given by Eq. (1).11
1
（1）
1
wherecritical distance (R0) is a dye-pair-dependent parameter in the range up to 70 . R is the distance between the donor and acceptor in a biological environment. The transferred energy is depended on both the critical distance, which is characterized by the FRET pairs themselves and the distance between the donor and acceptor fluorophores.
An addition method that is utilized to calculate transfer efficiency, E, is from the fluorescence intensities (quantum yields) using Eq. (2): 12
（2）
Where Fda is the donor fluorescence intensity determined at a given wavelength in the presence of the acceptor and Fd is the corresponding quantity determined in the absence of the acceptor. This method was used to roughly calculate the transfer efficiency of the FRET fluorophore pairs.
3. Efficiency evaluation of the molecular beacon
In most of the applications, MBs use a fluorophore and a quencher attached to both ends of the stem. The sensitivity and dynamic range of MBs as probes are determined mainly by two parameters: the residual fluorescence intensity when the MBs are in the stem-closed form and the fluorescence intensity when they are in the stem-open form. Efficiency of the MBs can be evaluated as the ratio of the fluorescence intensity of the fluorophore at the stem-open form to the stem-close form, which can be written as Ifl,open/Ifl,close,however, in reality, the residual fluorescence varies greatly due to many factors, and usually cause incomplete quenching. Thus a new strategy to design MBs with two fluorophores instead of one fluorophore and one quencher was invented.13
1
Fig. 3. A schematic representation of the MB with two fluorophores.
1
In Figure 3, two different fluorophores (F1 and F2) are attached to the two ends of the stem. F1 and F2 are chosen such that FRET will occur within a certain distance. When the MB is in the stem-closed form and excited at the absorption band of F1, due to the energy transfer, the fluorescence of F1 is quenched by F2, and the fluorescence of F2 is increasing. When a hybrid is formed between the MB and target, because of the increased distance between two ends of the stem, FRET is reduced or eliminated and the fluorescence of F1 will rise while that of F2 will diminish or disappear. By using the two fluorophores, the ratio of the intensities of the fluorescence of F1 and F2 (IF1/IF2) can provide significant advantages over measurement of IF1 only. Because (IF1,open/IF2,open)/( IF1,close/IF2,close), which can be rewrite as (IF1,openIF2,close)/( IF1,closeIF2,open), will always be larger than Ifl,open/Ifl,close according to the mechanism, the fluorescence intensity of F1 will increase upon hybridization, while that for F2 will decrease. In this proposal, twofluorophores are chosen, as acceptor and donor, defined in FRET theory.
EXPERIMENTS AND METHODS
1. Choose of acceptor and donor dyes
The structure of a molecular beacon is given in Figure 4 below.
Fig. 4. The structure of a molecular beacon
The known helical structure of double-stranded DNA and RNA can be exploited to determine the global structure of nucleic acids and nucleoprotein complexes. Well-defined sequences of DNA and RNA oligomers are now routinely synthesized and fluorescently labeled at 5′the 3′or termini, as well as within the DNA sequence.The structure of the molecular beacon can be referred to as Donor-(N)n-Acceptor, where n indicates the base pair separation between the dyes. Many acceptor and donor pair can be chosen for this purpose to label to the end of DNA.End labeling has three main advantages (when the shortest possible DNA is used): the dyes can be spatially separated from the binding protein to avoid protein–dye interactions; the DNA end sequence can be specifically designed to provide an identical chemical environment and, thus, the fluorescence properties of the dyes in a series of molecules can be derived; and the exact location of Cy3 on the DNA is known and evidence is accumulated on the position of fluorescein and rhodamine. In most studies, fluorescein and rhodamine are attached to the 5′ends.12 Because of its improved absorption and fluorescence properties, Cy3 will largely replace rhodamine as the acceptor in FRET studies. Additionally, in contrast to rhodamine, Cy3 can be directly attached to the oligonucleotides during synthesis.14In this proposal, the cyanine dyes of Cy3 and Cy5, which showed in Figure 5, are used as donor and acceptor respectively because of their long-wavelength character (Cy3 fluorophores were excited by argon laser at 514.5 nm and Cy5 fluorophores were excited by a He-Ne laser, or Diode laser-induced fluorescence detector (DIO-LIF) at 632.8nm) that can minimize the background noise and other excellent parameters showed in applications on bimolecular analysis.
The FRET donor and acceptor are linked to the ends of DNA strand, Cy3 (donor) can be labeled at the 5’ end in this oligonucleotide model.7The position of Cy3 stacking on top of a C-G base pair (bp) at the ends of helical DNA is also known.14Dyes with group COOH- or SO3- can be labeled with the biomolecular as showed in Figure 6:6
Fig. 5. The molecular structures of Cy3 and Cy5
Fig. 6. Label dyes with the biomolecular
2. FRET resultsfor Cy3 and Cy5 pair
FRET used to bethe technique used to measure distances in protein structures and their assemblies in solution. This technique recently is applied to make more efficient molecular beacon.7, 15Protein can be labeled with dyes at defined sites, for example, cysteine residues.
The FRET efficiency of different donor-acceptor labeled model DNA system in aqueous solution from ensemble measurements and at the single molecule level are studied. A set of differently labeled FRET constructs with D/A base pair separation was synthesized to investigate and compare the distance dependence and the influence of the dye structure on the measured spectroscopic characteristics.
The radii, R0 of the D/A pair of Cy3/Cy5 is 55.8 . It is calculated from the spectral overlap of the separate absorption and emission spectra of the donor and acceptor only double-stranded oligonucleotides, and the fluorescence quantum yields of the donor only constructs, respectively. Cy3 and Cy5 were assumed as free rotors.
Figure 7 is the model of the D/A DNA constructs with varying distance: D-(N)5-A; D-(N)15-A;D-(N)25-A; and D-(N)35-A. the 40mer complementary oligonucleotideswere custom synthesized.
Fig. 7. Model of the D/A DNA constructs with varying distance.11
The donor dye Cy3 was coupled to the 5’ end of oligonucleotide, and acceptor Cy5 was also coupled to C6 amino-modified thymidine bases at four different positions in the complementary oligonucleotide, resulting in D/A distances of 5, 15, 25 and 35 base pairs, respectively.
From table 1 we can see that the donor dye Cy3 exhibit monoexponential fluorescence decay times attached at the double-stranded DNA. From the relatively high fluorescence quantum yields, the data imply that the donor dye is not quenched by DNA nucleotides. In other words, the donor only constructs exhibit relatively homogeneous spectroscopic characteristics, which is important for successful FRET experiments.
Lieberwirth also proved that the rhodamine derivative JA133 and most indocarbocyanine dyes such as Cy5 are not quenched by DNA nucleotides.16 Due to this observation, we can use FRET constructs where guanosine residues are so far apart from the donor dyes that any quenching influence of the DNA base guanine should be minimized.
Table 1. Ensemble spectroscopic characteristics of the different FRET constructs in aqueous buffer containing 1 M NaCl.11
-- relative fluorescence quantum yield; D – donor; A—acceptor; -- fluorescence lifetime; -- FRET efficiency from the donor average fluorescence lifetime.
Fig. 8. Fluorescence emission spectra of the different FRET constructs in aqueous buffer containing 1 M NaCl 25 oC, 10-6 M. Cy3/Cy5 excited at 520 nm. 11
Figure 8 shows the emission spectra of the FRET constructs with D/A distances of 15, 25, and 35 base pairs show the expected decrease in donor fluorescence and increase in acceptor fluorescence. Furthermore, the emission curves intersect in one point, which demonstrates a direct correlation between the decrease of donor fluorescence and increase of acceptor fluorescence. As we may also see, the energy is not completely transferred to the acceptor at even 35 bases distance level.
In single molecule measurements, the single pair FRET (spFRET) efficiencies, Esp were calculated from the background corrected fluorescence intensities of the donor and acceptor, as showed in equation (3)
1
(3)
1
The cross-talk, C between the donor and acceptor channel was calculated from ensemble emission spectra and the transmission of the filter set (8.1% for Cy3).
Fig. 9. FRET histograms extracted from single molecule data of 10-11 M solutions of the differently labeled D/A constructs and corresponding Gaussian fits.11
Figure 9 shows the spFRET efficiency histograms that were generated from the single molecule fluorescence intensity data of Cy3-(N)x-Cy5 constructs, and of only donor labeled oligonucleotides measured in aqueous buffer containing 1 M NaCl. Without acceptor, the donor only labeled oligonucleotides show only one peak with zero FRET efficiency. Two peaks can be seen for the 5-bp separation constructs, the donor at approximately zero efficiency and the acceptor at very high (>0.95) efficiency. As the increasing of the bp length, the acceptor peak clearly shifts to lower FRET efficiency, as expected for energy transfer.
3. Molecular beacondesign
FRET can occur between them within a certain distance. Several applications on protein and DNA analysis haven been done that relate to these two dyes.11, 15, 17 However, no application up to now has been carried out to apply this pair of dyes on molecular beacon. MB and target DNA sequence with 5-25 bp with this pair of fluorophores are evaluated. The Cy3 fluorophores were excited by argon laser at 514.5 nm and Cy5 fluorophores were excited by a He-Ne laser, or Diode laser-induced fluorescence detector (DIO-LIF) at 632.8nm.
Since the two fluorophore MB will provide higher efficiency than one fluorophore MB, this proposal applied this strategy by synthesizing a two fluorophore MB with Cy3 dye labeled on one end of the stem as a donor and Cy5 as an acceptor on the other. The sequences of the MB and target DNA used in this work is showed in table 2.
Table 2. MB and target DNA sequences.
______
MB: 5’-Cy3-GCTCGTCCATGCCCAGGAAGGAGGCAACGACACGAGC-Cy5-3’
Target: 5’-GTCGTTGCCTCCTTCCTGGGCATGG-3’
______
MB and target DNAs used in this work can be synthesized by Trilink Bio Technologies, Inc. (San Diego, CA). MB was synthesized using standard phosphoramidite coupling procedures. The sample was deprotected with concentrated ammonium hydroxide at room temperature for 30 hours, and then purified on a polyacrylamide gel. The labeled oligonucleotide was isolated on reversed-phase HPLC.13 In the absence of a target sequence MB adopts a hairpin structure containing a stem of 6 base pairs (the underlined segment) and a loop of 25 bases. The stem keeps the two fluorophores in proximity, which causes a quenching of the Cy5 and an emission of Cy3 as a result of energy transfer. A conformational change that opens the stem leads to the separation of the two fluorophores (35 bases distance), and therefore restores the fluorescence of Cy5 while decreasing that of Cy3, and fluorescence spectra can be read from figure 7. All fluorescence experiments were performed at room temperature. The fluorescence emission spectra of the different FRET are obtained in aqueous buffer containing 1 M NaCl 25 oC, 10-6 M. Cy3/Cy5 excited at 520 nm.
Figure 10 is the schematic diagram of the optical setup for detection. ‘For efficient excitation of the donor dye Cy3, a frequency-doubled Nd:YAG laser emitting at 532 nm. The fluorescence light was collected through the same objective and imaged onto a 100 m pinhole to reject out-of-focus light. The collimated laser beam was directed into an inverted microscope and coupled into the microscope objective with high numerical apertures (oil immersion, 100 x, NA 1.4) via a dichroic beam splitter. Within the microscope objective, the beam was focused into the sample to detect freely diffusing FRET constructs. The transmitted fluorescence light is then split by a dichroic mirror and focused onto the active areas of two avalanche photodiodes. To further isolate the donor and acceptor signal, additional band pass filters in front of the APDs can b e used. The signals of both APDs were coupled to a counting board and a personal computer. Sample solutions (10-11 M) were prepared from 10-6 M stock solutions by several dilution steps. For diffusion measurements, the average excitation power at the sample was adjusted to be 325 W. ’