Molecular Beacons Protocol

Recently, controlled-pore glass columns that introduces a dabcyl moiety at the 3' end of an oligonucleotide have become available, which enable the synthesis of molecular beacons completely on a DNA synthesizer. Alternatively, the starting material for the synthesis of molecular beacons is an oligonucleotide that contains a sulfhydryl group at its 5' end and a primary amino group at its 3' end. Dabcyl is coupled to the primary amino group utilizing an amine-reactive derivative of dabcyl. The oligonucleotides that are coupled to dabcyl are then purified. The protective trityl moiety is then removed from the 5'-sulfhydryl group and a fluorophore is introduced in its place, using an iodoacetamide derivative.

1.  Materials

Equipment

–  High-pressure liquid chromatograph
–  Spectrofluorometer
–  Spectrofluorometric thermal cycler with a capacity to monitor fluorescence in real time

Buffers

–  0.1 M sodium bicarbonate, pH 8.5
–  0.2 M sodium bicarbonate, pH 9.0
–  HPLC Buffer A:  0.1 M triethylammonium acetate, pH 6.5, filtered and degassed
–  HPLC Buffer B:  0.1 M triethylammonium acetate in 75% acetonitrile, pH 6.5, filtered and degassed
–  TE buffer:  1 mM EDTA, 10 mM Tris-HCl, pH 8.0
–  Molecular beacon buffer:  1 mM MgCl₂, 20 mM Tris-HCl, pH 8.0

2. Synthesis and purification

2.1. Coupling of dabcyl

Dissolve 50-250 nanomoles of dry oligonucleotide in 500 µl of 0.1 M sodium bicarbonate, pH 8.5. Dissolve about 20 mg of dabcyl (4-(4'-dimethylaminophenylazo)benzoic acid) succinimidyl ester (Molecular Probes) in 100 µl N,N-dimethylformamide and add to a stirring solution of the oligonucleotide in 10-µl aliquots at 20-minute intervals. Continue stirring for at least 12 hours.
Remove particulate material by spinning the mixture in a microcentrifuge for one minute at 10,000 rpm. In order to remove unreacted dabcyl, pass the supernatant through a gel-exclusion column. Equilibrate a Sephadex G-25 column (NAP-5, Pharmacia) with buffer A, load the supernatant and elute with 1 ml buffer A. Filter the eluate through a 0.2 µm Centrex MF-0.4 filter (Schleicher & Schuell).
Purify the oligonucleotides by high-pressure liquid chromatography (HPLC) on a C-18 reverse-phase column (Waters), utilizing a linear elution gradient of 20 to 70% buffer B in buffer A and run for 25 minutes at a flow rate of 1 ml/min. Monitor the absorption of the elution stream at 260 nm and 491 nm. A typical chromatogram is shown in Figure 1. Collect the peak that absorbs in both wavelengths, which contains oligonucleotides with a protected sulfhydryl group at their 5'-ends and dabcyl at their 3'-ends (peak D).
Precipitate the collected material with ethanol and salt, and spin in a centrifuge for 10 minutes at 10,000 rpm, discard the supernatant, dry the pellet and dissolve it in 250 µl buffer A.

Figure 1. Chromatographic separation of oligonucleotides coupled to dabcyl. The blue line represents absorption at 260 nm and the red line represents absorption at 491 nm. The oligonucleotides in peaks A and B do not contain trityl moieties, whereas the oligonucleotides in peaks C and D are protected by trityl moieties. The oligonucleotides in peaks B and D are coupled to dabcyl, whereas the oligonucleotides in peaks A and C are not coupled to dabcyl. Peak D should be collected.

2.2. Coupling of fluorophore

In order to remove the trityl moiety, add 10 µl of 0.15 M silver nitrate and incubate for 30 minutes. Add 15 µl of 0.15 M dithiothreitol to this mixture and shake for 5 minutes. Spin for 2 minutes at 10,000 rpm and transfer the supernatant to a new tube. Dissolve about 40 mg 5-iodoactamidofluorescein (Molecular Probes) in 250 µl of 0.2 M sodium bicarbonate, pH 9.0, and add it to the supernatant. Incubate the mixture for 90 minutes. Each of these solutions should be prepared just before use.
Remove excess fluorescein from the reaction mixture by gel exclusion chromatography and purify the oligonucleotides coupled to fluorescein by HPLC, following the instructions in steps 2 and 3 of the previous section. A sample chromatogram is shown in Figure 2. Collect the fractions corresponding to peak F, which absorb at wavelengths 260 nm and 491 nm and are fluorescent when observed with an ultraviolet lamp in a dark room. If a different fluorophore is coupled in place of fluorescein, its maximum absorption wavelength should be used instead of 491 nm.
Precipitate the collected material and dissolve the pellet in 100 µl TE buffer. Determine the absorbance at 260 nm and estimate the yield (1 OD₂₆₀= 33 µg/ml).

Figure 2. Chromatographic separation of oligonucleotides coupled to both dabcyl and fluorescein. The blue line represents absorption at 260 nm and the red line represents absorption at 491 nm. The oligonucleotides present in peak E are not coupled to fluorescein, whereas the oligonucleotides in peak F are coupled to fluorescein. Peak F should be collected.

2.3. Automated synthesis

Use a controlled-pore glass column to introduce dabcyl (Biosearch Technologies or Glen Research) at the 3' end of the oligonucleotide during automated synthesis. At the 5' end of the oligonucleotide, either a thiol or an amino modifier (Glen Research) can be introduced for a subsequent coupling to a fluorophore, or a fluorophore can directly be introduced during automated synthesis using a phosphoramidite. The 5' modifiers and fluorophores should remained protected with a trityl moiety during the synthesis. Perform post-synthetic steps as recommended by the manufacturer of the DNA synthesizer. Dissolve the oligonucleotide in 600 µl Buffer A.
When the fluorophore is to be introduced manually, purify the oligonucleotide protected with a trityl moiety. Remove the trityl moiety from the purified oligonucleotide and continue with the coupling of the fluorophore, as described above.
When a 5' fluorophore is introduced via automated synthesis, purify the oligonucleotide protected with the trityl moiety and then remove the trityl moiety from the purified oligonucleotide. Precipitate the molecular beacon with ethanol and salt and dissolve the pellet in 100 µl TE buffer. Determine the absorbance at 260 nm and estimate the yield.

3. Characterization of molecular beacons

3.1. Signal to background ratio

Determine the fluorescence of 200 µl of molecular beacon buffer solution (F_buffer), using 491 nm as the excitation wavelength and 515 as the emission wavelength. If the fluorophore is not fluorescein, choose wavelengths that are optimal for the fluorophore in the molecular beacon.
Add 10 µl of 1 µM molecular beacon to this solution and record the new level of fluorescence (F_closed).
Add a two-fold molar excess of a complementary oligonucleotide target and monitor the rise in fluorescence until it reaches a stable level (F_open).
Calculate the signal-to-background ratio as (F_open-F_buffer)/(F_closed-F_buffer).

3.2. Thermal denaturation profiles

Prepare two tubes containing 50 µl of 200 nM molecular beacon dissolved in 3.5 mM MgCl₂ and 10 mM Tris-HCl, pH 8.0 and add the oligonucleotide target to one of the tubes at a final concentration of 400 nM.
Determine the fluorescence of each solution as a function of temperature using a spectrofluorometric thermal cycler. Decrease the temperature of these tubes from 80 °C to 10 °C in 1 °C steps, with each hold lasting one minute, while monitoring the fluorescence during each hold.

3.3. Results

An example of a hybridization reaction performed for the determination of the signal-to-background ratio is shown in Figure 3. The signal-to-background ratio in this example was 190. Usually the ratio ranges from 30 to 200. An example of a thermal denaturation profile is shown in Figure 4. The probe-target hybrid denatures at 58 °C and the stem of the molecular beacon denatures at 64 °C. In the range of 10 °C to 50 °C, the free probe has very little fluorescence, whereas the target-bound form is fluorescent. The sequence of the molecular beacon used throughout this section is: fluorescein-5'- GCG AGC TAG GAA ACA CCA AAG ATG ATA TTT GCT CGC -3'-dabcyl, where underlines identify the arm sequences.

Figure 3. The spontaneous fluorogenic response of molecular beacons to the addition of target. The first segment of the data is due to the fluorescence of the buffer, the second segment is due to the fluorescence of the buffer containing the molecular beacons, and the third segment shows the increase in fluorescence that occurs upon the addition of target oligonucleotides.

Figure 4. Thermal denaturation profiles of a molecular beacon (red-dotted line) and the hybrid formed between the molecular beacons and its oligonucleotide target (blue-dashed line). The profiles indicate that this molecular beacon can be used below 55 °C.

4. Real-time monitoring of polymerase chain reactions

Utilize molecular beacons that are complementary to a sequence in the middle of the expected amplicon. The length of their arm sequences should be chosen so that a stem is formed at the annealing temperature of the polymerase chain reaction. The length of the loop sequence should be chosen so that the probe-target hybrid is stable at the annealing temperature. Whether a molecular beacon actually exhibits these design features is determined by obtaining thermal denaturation profiles, as detailed in the previous section. Molecular beacons with appropriate thermal denaturation characteristics are included in each reaction at a concentration similar to the concentration of the primers. During the denaturation step, the molecular beacons assume a random-coil configuration and fluoresce. As the temperature is lowered to allow annealing of the primers, stem hybrids form rapidly, preventing fluorescence. However, at the annealing temperature, molecular beacons also bind to the amplicons, undergo conformational reaorganization, and generate fluorescence. When the temperature is raised to allow primer extension, the molecular beacons dissociate from their targets and do not interfere with polymerization. A new hybridization takes place in the annealing step of every cycle, and the intensity of the resulting fluorescence indicates the amount of accumulated amplicon. In the procedure below, the synthesis of an 84-nucleotide-long amplicon is monitored with the same molecular beacon whose synthesis and characterization was described in previous section.

4.1. Procedure

Set up six 50 µl reactions so that each contains a different number of targets (1,000,000; 100,000; 10,000; 1,000; 100; 10; 1 copies), 0.34 µM molecular beacon, 1 µM of each primer, 2.5 units of Amplitaq Gold DNA polymerase (Perkin Elmer), 0.25 mM of each deoxyribonucleotide, 3.5 mM MgCl₂, 50 mM KCl, and 10 mM Tris-HCl, pH 8.0.
Program the spectrofluorometric thermal cycler to incubate the tubes at 95 °C for 10 min to activate the Amplitaq Gold DNA polymerase, followed by 40 cycles of 30 sec at 95 °C, 60 sec at 50 °C, and 30 sec at 72 °C. Monitor fluorescence during the 50 °C annealing steps.

4.2. Results

Figure 5 shows the level of fluorescence as a function of the number of temperature cycles completed. The level of fluorescence is proportional to the amount of amplicons present in each cycle. The reaction that did not contain any template, did not show any rise in fluorescence. The number of temperature cycles required before the fluorescence signal becomes detectable over the background is inversely proportional to the logarithm of the initial number of template molecules. This relationship is true over a wide range of template concentrations.

Figure 5. (A) The threshold cycle (Ct) is the cycle at which the fluorescence rises significantly above the background. The fluorescence increases as the molecular beacons bind to the amplification products that accumulate during each successive cycle. In the early cycles of amplification, the change in fluorescence is usually undetectable, but at some point during amplification, the accumulation of amplified DNA results in a detectable change in the fluorescence of the reaction mixture. The threshold cycle number decreases as the number of target molecules initially present in a reaction increases. (B) The standard curve can be used to determine the starting amount of an unknown template, based on its threshold cycle. Given known starting amounts of the target, a standard curve can be constructed by plotting the log of the starting amount versus the threshold cycle. The threshold cycle is inversely proportional to the logarithm of the number of target molecules initially present.

5. Notes and troubleshooting

Usually the 50 nmol, 200 nmol, and 1000 nmol scales of syntheses yield 10 nmol, 40 nmol, and 200 nmol molecular beacons, respectively.
Low yield in coupling reactions:

–  Check the pH of the buffers used in the coupling reactions and use fresh dyes. The reactive dyes should be stored at -20 °C in the presence of a desiccant.

–  Before performing the coupling reactions, prepare fresh solutions of both silver nitrate and dithiothreitol.

–  Refer to the data-sheets of the fluorophore derivatives for information on their solubility. In case a fluorophore derivative is not soluble in water, as is the case for most succinimidyl ester derivatives, dissolve it in a small amount of dimethylformamide and then add this solution to the reaction mixture in small aliquots.
In order to remove unincorporated fluorophore derivatives from the coupling reactions without using column chromatography, the reaction mixtures can be precipitated with salt and ethanol, as the fluorophores remain dissolved in ethanol.
Store stock solutions of molecular beacons at -20 °C or -70 °C in TE buffer (10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA) and prevent them from being exposed to light. For the longtime storage, store the molecular beacons as a dried pellet.
Low signal-to-background ratio:

–  The most likely reason is high background due to contamination by either free fluorophores or oligonucleotides that contain the fluorophore but not the quencher. Free fluorophores can be removed by passage through a Sephadex column. In order to ensure that every molecule contains a quencher, repeat the purification of oligonucleotides that are protected by a trityl moiety and labeled with dabcyl prior to coupling with the fluorophore.

–  The assay medium may contain insufficient salt and the stem opens up. There should be at least 1 mM MgCl₂ in the solution in order to ensure that stem hybrids form.

–  The molecular beacon may fold into an alternate conformation that results in a sub-population that is not quenched well. Change the stem sequence (and probe sequence, if necessary) to eliminate that possibility.
Incomplete restoration of fluorescence at low temperatures:

– If the stem of a molecular beacon is too strong, at low temperatures it may remain closed while the probe is bound to the target. This may happen inadvertently if the probe sequence can participate in the formation of a hairpin that results in a stem longer and stronger than originally designed. Change the sequence at the edges of the probe and the stem sequence to avoid this problem.
Although false amplicons and primer dimers are not detected by molecular beacons, when they do appear, the sensitivity of the PCR assay is reduced. Therefore, DNA polymerases that become active after a brief incubation at 95 °C are recommended, as they minimize false priming.
Low signals in real-time PCR:

– Try one of the following: Optimize the concentration of the molecular beacons, decrease the size of the amplicon, decrease the annealing temperature, and alter the relative concentrations of the two primers so that the PCR becomes asymmetric, favoring of the target strand.
Poor discrimination between the alleles:

–  Check if there is no bleed through of fluorescence from one optical color channel to the other.
–  If the instrument is able to distinguish between the two fluorophores perfectly, increase the annealing temperature of the PCR.
–  If poor discrimination is still observed, increase the length of the stems of the molecular beacons or decrease the length of the probe sequences.

6. Literature

Detailed descriptions on the design, synthesis and application of molecular beacons appeared in:

Tyagi S, Marras SAE, Vet JAM, and Kramer FR (2000) Molecular beacons: hybridization probes for detection of nucleic acids in homogeneous solutions. In Kessler, C (ed.), Nonradioactive analysis of biomolecules, Second edition. Springer Verlag, Berlin, Germany, pp. 606-616.
Marras SAE, Kramer FR, and Tyagi S (2003) Genotyping single nucleotide polymorphisms with molecular beacons. In Kwok, PY (ed.), Single nucleotide polymorphisms: Methods and Protocols. Humana Press, Totowa, NJ, Vol. 212, pp. 111-128.
Vet, J.A.M. and Marras, S.A.E. (2004) Design and optimization of molecular beacon real-time polymerase chain reaction assays. In Herdewijn, P. (ed.), Oligonucleotide synthesis: Methods and Applications. Humana Press, Totowa, NJ, Vol. 288, pp. 273-290.

If you have any further questions or remarks on the synthesis, purification and characterization of molecular beacons, please contact:

Salvatore A.E. Marras, Ph.D.
Public Health Research Institute
255 Warren Street, Newark, NJ 07103, USA
marrassa@umdnj.edu