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Young Scientist Journal

Young Scientist Journal

Using Fluorescence Resonance Energy-Transfer to Observe DNA Hybridization

ABSTRACT

This experiment utilized fluorescence resonance energy transfer (FRET) to investigate DNA hybridization. A 10-nucleotide DNA strand was labeled with a Cy3 donor fluorophore, and a 17-nucleotide DNA strand was labeled with a Cy5 acceptor fluorophore. The 10-nucleotide strand was hybridized to the 17-nucleotide strand, and FRET efficiency was calculated for the donor-only sample and samples containing an increasing amount of acceptor-labeled 17-nucleotide DNA sample. Specifically, we tested 0.5-, 1-, and 2-fold excess of the acceptor-labeled DNA relative to the donor strand. We observed that as the concentration of Cy5-labeled 17-nucleotide DNA increased, the FRET efficiency increased correspondingly. This result indicates that increasing the amount of acceptor-labeled DNA enhances hybridization efficiency, leading to higher FRET signals.

INTRODUCTION.

The proposed experiment uses fluorescence resonance energy transfer (FRET) to explore the hybridization of deoxyribonucleic acid (DNA). DNA consists of two complementary polynucleotide chains held together by hydrogen bonds between base pairs. The DNA chain, or strand, comprises four types of nucleotide subunits. Each subunit is made up of a deoxyribose sugar, a single phosphate group, and a nitrogen-containing base. The base can either be adenine (A), cytosine (C), guanine (G), or thymine (T). The sugars and phosphates are covalently bound in a chain to make a “backbone,” while only the base differs. Due to the way the nucleotide subunits attach, the ends of the chains are easily identifiable. One is the 5’ end (the 5’ phosphate), and the other is the 3’ end (the 3’ hydroxyl), giving the DNA polarity. When the two DNA strands are bound together by hydrogen bonds, it forms a three-dimensional double helix structure. All the bases are on the interior, while the sugar-phosphate backbones are on the exterior of the double helix. The two DNA strands must have complementary bases: A pairs with T and G pairs with C. Also, the strands must be antiparallel, meaning the polarity of the strands must have opposite orientations [1]. Several techniques are available for observing DNA hybridization, including Southern blotting, Northern blotting, colony and dot blotting, native polyacrylamide gel electrophoresis (PAGE), and fluorescence in situ hybridization [2,3,4]. In this study, we utilized FRET to monitor DNA hybridization (Figure 1). The DNA must first be labeled with donor and acceptor fluorescence probes to perform FRET experiments. Several FRET pairs can be used for this task; however, we used Cyanine 3 (Cy3) and Cyanine 5 (Cy5) fluorophores in this study [5]. FRET is a photophysical process in which energy is transferred non-radiatively from an excited donor fluorophore to a nearby acceptor fluorophore through dipole-dipole coupling (Figure 2A). The excited fluorophore acts as an oscillating dipole that can donate its energy to a second dipole with a similar resonance frequency. Therefore, this technique is often used to determine the proximity of labeled molecules [6]. The efficiency of energy transfer depends on the distance between the donor and acceptor fluorophores, typically ranging from 1 to 10 nanometers (nm), making FRET an efficient molecular ruler and a valuable tool for studying molecular interactions (Figure 2B) [7]. The donor fluorophore is typically excited by light at a specific wavelength, causing it to emit fluorescence at a characteristic emission wavelength. The acceptor fluorophore becomes excited and emits fluorescence at a longer wavelength by donor emission when the donor emission and acceptor absorption spectra overlap (Figure 2C) [8]. Fluorescence Spectrophotometers are typically used to measure emission intensities (a.u.) at specific wavelengths to calculate FRET efficiency [9].

Figure 1. Schematic illustration of FRET resulting from DNA hybridization. Left: Before hybridization, DNA strands remain unpaired, resulting in no FRET. Right: Upon hybridization, DNA strands of 10- and 17-nucleotides form a duplex, bringing the fluorophores into close proximity and enabling efficient FRET from the donor to the acceptor. [10]
Figure 2. Jablonski Diagram and FRET Illustration. (A) The Jablonski diagram shows the electronic transitions that occur in fluorescence. (B) A fluorescence intensity spectrum of Cy3 and Cy5 fluorophores. The blue lines represent the excitation spectra, and the red lines represent the emission spectra. The green-shaded area indicates the spectral overlap between the Cy3 emission and Cy5 excitation. (C) A curve representing R0, the Förster distance, is shown by a line where the energy transfer efficiency is 50% [9].
The purpose of this experiment is to test whether FRET can be used as a tool to monitor DNA hybridization efficiency. We hypothesize that an increase in FRET values will correspond to increasing hybridization efficiency of donor- and acceptor-labeled DNA strands, such that higher levels of strand hybridization led to greater energy transfer efficiency. Based on this hypothesis, DNA samples with a 2-fold excess of acceptor-labeled DNA are expected to show higher FRET values than those with a 0.5- or 1-fold excess, as increased acceptor availability enhances hybridization efficiency and brings more donor–acceptor pairs into proximity. In this way, the FRET technique can serve as a sensitive readout to monitor the extent of DNA hybridization. Reliable methods of detecting DNA hybridization for various research applications are necessary. Without appropriate hybridization, experimental results will be unreliable, leading to inaccurate conclusions.

MATERIALS AND METHODS.

Resuspending DNA.

DNA strands were ordered from Integrated DNA Technologies (IDT) with 5’ amino modifications for the fluorophore labeling as listed in Table 1. Those DNA strands were resuspended in the recommended volume of double-distilled water. To dissolve the DNA strands, the sample was vortexed multiple times and spun down using a benchtop centrifuge. After the resuspension, the concentration of each DNA sample was approximately 10 µM. These containers were stored at -20 °C.

Table 1. DNA sequences used in this study.
DNA Samples Sequence (5’ to 3’)
10 nt Cy3-CATTACAATG
17 nt Cy5-CATTGTAATGCAAAAGT

 Dissolving Fluorophores.

Cy3 NHS Ester (Cat# FP-1301-1) and Cy5 NHS Ester (Cat# FP-1321-1) fluorophores were purchased from Vector Labs. 1 mg of each fluorophore was dissolved in 50 µL Dimethyl Sulfoxide (DMSO) (Fisher Scientific, Cat# BP231-100), as the fluorophores are insoluble in water. We aliquoted 5 µL of each dye into 10 separate tubes, wrapped them in aluminum foil to protect from light, placed them in a desiccator to absorb moisture, and stored them in a -80 °C freezer [11].

Labeling of DNA with fluorescence probes.

First, we performed ethanol precipitation to remove impurities, including high-salt contaminants, from the DNA synthesis that may interfere with fluorophore labeling [12]. We started with 250 nmol of each DNA sample in 100 μL of distilled water. We then added 1/10th the volume of 3 M Sodium Acetate (Fisher Scientific, Cat# BP334500) and 2.5 times the volume of 100% Ethanol (Fisher Scientific, Cat# A412SK4) to the tube. We then mixed the solution using a pipette and kept it in the -20 °C freezer for an hour. Next, we centrifuged at 9,800 xg for 30 minutes at 4 °C. Afterward, we removed the supernatant and stored it in a separate tube. We immediately added 150 μL of 70% ice-cold Ethanol to the tube. We then centrifuged again at 9,800 xg for 30 minutes at 4 °C. This Ethanol was repeated once more. Finally, the supernatant was removed, and the tube was placed in the Vacufuge (Eppendorf, Vacufuge Plus) until it was dry. We then began labeling the DNA strands following a standard protocol [13]. The dried DNA pellet in the tube obtained from ethanol precipitation was resuspended in 75 μL of 100 mM Sodium Tetraborate Buffer, pH 8.5 (Fisher Scientific, Cat# S248-500). We then added 20 μL of double-distilled water and 5 μL of the previously prepared NHS Ester dye. The tubes were wrapped with aluminum foil to protect the dye from light. Finally, the reaction mixture was incubated overnight at room temperature on a shaking block at 300 rpm. After the labeling reaction was complete, the ethanol precipitation was performed to remove excess unreacted dye and to purify the labeled DNA samples. The final DNA samples were resuspended in 50 µL of double-distilled water, and DNA concentrations were measured using a NanoDrop spectrophotometer. Labeling efficiencies were calculated from absorbance measurements.

DNA Hybridization.

We hybridized the 10-nucleotide and 17-nucleotide DNA strands at different ratios: 0.5-, 1-, and 2-fold excess of the acceptor-labeled DNA strand. For each hybridization, the 10-nucleotide strand was added at a final concentration of 1 µM in a total volume of 50 µL, along with the calculated amount of acceptor-labeled strand in 20 mM HEPES (pH 7.5) and 100 mM NaCl buffer. A beaker of water was heated to a boil on a hot plate, and the tubes were submerged in the boiling water bath for approximately 2 minutes. After heating, the tubes were allowed to cool gradually to room temperature. The hybridized DNA samples were then stored at −20 °C until fluorescence emission measurements were performed [14].

FRET Measurements.

Fluorescence emission was measured for individual samples using a Cary Eclipse Fluorescence Spectrophotometer. In detail, 100 pM of hybridized DNA in buffer (20 mM HEPES, pH 7.5, and 100 mM NaCl) was placed in a quartz cuvette and excited at 550 nm with a 10-nm bandwidth. The emission spectrum was recorded with a 5 nm bandwidth. The maximum emission from both donor (Cy3) and acceptor (Cy5) fluorophores were used to calculate apparent FRET efficiencies as (IA)/(IA + ID), where ID and IA are the donor and acceptor fluorescence intensities, respectively.

\[FRET=\frac{I_A}{I_A+I_D}\tag{1}\]

RESULTS.

DNA strands of varying lengths with 5′ amino modifications were purchased from Integrated DNA Technologies (IDT) (Table 1). Among these, the 10-nucleotide strand is complementary to regions of the 17-nucleotide strand. Cy3 and Cy5 fluorophores are used as fluorescent probes that allow for the transfer of energy from the donor (Cy3) to the acceptor (Cy5). The amino-modified DNA strands were chemically labeled with these Ester dyes (Figure 1). The 10-nucleotide strand was labeled with the donor, while the 17-nucleotide strand was labeled with the acceptor, following standard labeling protocols [13]. To confirm that the DNA was efficiently labeled, we measured the absorbance spectra of the labeled DNA using a NanoDrop (Table 2). The resulting UV-Vis spectrum showed distinct peaks corresponding to the DNA and the attached dyes (Figure 3, top and middle panels).

Table 2. Properties of Cy3 and Cy5 fluorophores used in this study [7].
Fluorophores Excitation λmax
(nm)		 Emission λmax
(nm)		 Extinction Coefficient
(ε) (cm-1 M-1)		 Quantum Yield
Cy3 550 565 150,000 >0.15
Cy5 649 665 250,000 >0.28
Figure 3. Representative UV-Vis spectra of fluorophore-labeled DNA samples. Top: Donor (Cy3) labeled 10-nucleotide single-stranded DNA. Middle: Acceptor (Cy5) labeled 17-nucleotide single-stranded DNA. Bottom: Hybridized 10- and 17-nucleotide DNA strands at a 1:1 donor-to-acceptor ratio. Absorbance values on the y-axis were normalized to the highest DNA absorbance under each condition.

The 10-nucleotide DNA strand was hybridized with the 17-nucleotide DNA strand at various ratios, as described in the methods section. In detail, the sample was first heated in a boiling water bath for approximately 2 minutes to denature any secondary structure and then allowed to cool gradually to room temperature to facilitate hybridization. The hybridized DNA was then analyzed using a NanoDrop Spectrophotometer, and the Beer-Lambert Law was applied to determine the solution concentration by measuring its absorbance [15]. Distinct absorbance peaks were observed at 260 nm, 550 nm, and 650 nm, indicating the presence of DNA, Cy3, and Cy5, respectively (Figure 3, bottom panel). The calculated labeling efficiencies for each fluorophore exceeded 100%, which may be due to incomplete removal of free dye molecules and some nonspecific labeling. This indicates that ethanol precipitation alone was not sufficient to completely remove free dyes from the reaction mixture. These results indicate that both the fluorophores and DNA are present in the same double-stranded DNA sample. However, the data does not provide direct evidence that the DNA strands were successfully hybridized.

To determine whether energy transfer occurred between the donor and acceptor fluorophores, we used a Cary Eclipse Fluorescence Spectrophotometer to assess FRET efficiency. The presence of FRET would indicate successful hybridization of the DNA strands, as energy transfer only occurs when the donor and acceptor are in close proximity. We prepared a 150 pM, 120 µL single strand and hybridized DNA sample in buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl, and measured fluorescence emissions from both the donor and acceptor fluorophores following excitation at 550 nm. A fluorescence emission spectrum of the 10-nucleotide DNA strand labeled with the Cy3 fluorophore showed a single peak corresponding to donor emission at 565 nm (Figure 4, black). When the experiment was repeated using a 1:0.5-fold hybridized mixture of the 10-nucleotide (donor-labeled) and 17-nucleotide (acceptor-labeled) DNA strands, the spectrum displayed both donor and acceptor emissions at 565 and 665 nm wavelengths. However, the donor emission remained higher than the acceptor emission at 665 nm, indicating partial, rather than complete, energy transfer via FRET. Using this data, the apparent FRET efficiency was then calculated, where IA is the intensity of the acceptor and ID is the intensity of the donor (Figure 4, orange). The calculated FRET efficiency for the 0.5-fold Cy5 DNA was 0.14. This value increased to 0.25 when the experiment was repeated using a 1-fold Cy5 DNA (Figure 4, blue), suggesting enhanced hybridization and energy transfer under these conditions. Finally, when the experiment was repeated with 2-fold Cy5 DNA, the FRET value increased to 0.49 (Figure 4, red), providing further evidence supporting our hypothesis (Table 3).

Figure 4. Fluorescence emission spectra of the hybridized DNA samples, with emission values normalized to the sum of all intensity values.

 

Table 3. FRET values calculated from the data presented in Figure 4.
Samples Apparent FRET
Donor Only NA
0.5-fold Cy5 DNA 0.14
1-fold Cy5 DNA 0.25
2-fold Cy5 DNA 0.49

DISCUSSION.

The peak at 260 nm in the UV-Vis spectra confirms the presence of DNA, while the peaks at 550 nm and 650 nm indicate the presence of the Cy3 and Cy5 dyes, respectively (Figure 3). This demonstrates that the fluorophores are attached to the DNA strands, as further verified by calculating the labeling efficiency. According to our calculations, we still have residual free dye in the solution, which resulted in artificially high labeling efficiencies and could lead to inaccurate hybridization measurements. The best way to obtain only labeled samples would be to use High-Performance Liquid Chromatography (HPLC) purification, which removes free dye and helps separate the labeled DNA from the unlabeled fraction, allowing for more accurate hybridization of the samples [16]. The objective of this experiment was to use FRET to monitor the hybridization of the labeled DNA strands. The 10-nucleotide donor-only DNA showed no FRET signal, as expected, because there was no nearby acceptor fluorophore to facilitate energy transfer (Figure 2). The observation of a FRET efficiency of approximately 0.14 in the presence of a 0.5-fold excess of acceptor-labeled DNA indicates a significant level of energy transfer from the donor to the acceptor, confirming DNA hybridization. The increase in FRET in the presence of the 17-nucleotide DNA demonstrates that the Cy3 and Cy5 fluorophores were brought into proximity through hybridization. Additionally, we verified that the sample with a 2-fold excess of Cy5-labeled DNA (1:2 donor: acceptor) condition resulted in an increased high-FRET population, consistent with more hybridization compared to the 0.5- and 1-fold (1:1) ratios. This supports our hypothesis that an increase in concentration of acceptor-labeled DNA will increase the FRET efficiency. This means, in our experiment, the optimal energy transfer occurs with a 2-fold excess of Cy5-labeled DNA. These measurements were performed using a Cary Eclipse Fluorescence Spectrophotometer, which detects fluorescence from all molecules in bulk. As a result, the measurements include contributions from labeled and unlabeled molecules, as well as any residual free dye. In the future, single-molecule FRET experiments could be performed to detect only molecules that are successfully labeled and hybridized, thereby providing more accurate measurements, free of ensemble averaging. This experiment helps verify how concentration and the hybridization state affect FRET, providing insight into how energy transfer scales with hybridization efficiency. With this understanding, we used FRET as a molecular ruler to monitor conformational changes in DNA. This process also confirms hybridization, as evidenced by the transfer of energy from the donor to the acceptor fluorophore. It is crucial to ensure that the hybridization step is successful, as incorrect hybridization of the DNA can cause subsequent steps to fail.

ACKNOWLEDGMENTS.

Thank you to the Lamichhane Lab and Dr. Rajan Lamichhane for the opportunity to conduct my research. Additionally, I would like to thank the Webb School of Knoxville and Dr. Jason Abercrombie for the opportunity to participate in the Research Scholars Program.

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Posted by on Thursday, May 14, 2026 in May 2026.

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