Design rules for Molecular Beacons

Many innovative technologies and methods for sensitive and accurate genetic analysis have been developed during the last few years. The polymerase chain reaction (PCR) has made it possible to detect tiny amounts of DNA or RNA sequences in cells, tissue, or blood samples. The real-time polymerase chain reaction (RT-PCR) is the most commonly used method for this type of analysis. The implementation of fluorescent detection strategies in combination with sensitive instrumentation allows for the accurate quantification of nucleic acids.

Since their introduction in 1996 molecular beacons have become widely used in the biosciences. Specific hybridization of complementary sequences in DNA is the basic mechanism for the identification of target genes. Stem-loop oligonucleotide probes have been developed for specific and selective target detection and are the key for a well-designed molecular beacon based assay.

The principle of operation of molecular beacons is illustrated in figure 1.

Figure1: Principle of operation of molecular beacons. A molecular beacon contains a fluorophore-quencher pair, sometimes also called a donor-acceptor pair, a loop region, and a stem region. The stem region contains two complementary sequences. The sequence in the loop region is complementary to the target sequence. If the target sequence is not presence the complementary sequence regions in the stem hybridize and bring the fluorophore and quencher in close contact. In this conformation the fluorescence of the molecule is quenched. Hence no fluorescent signal is detected. The beacon is extended when the probe sequence binds to the target and increased fluorescence occurs.  

A molecular beacon is a fluorescent-labeled oligonucleotide, usually 25 to 35 nucleotides long. A typical molecular beacon can be divided into four parts:

1.  Loop

An 18 to 30 single-stranded sequence region complementary to the target sequence.

2.  Stem

Two short, 5 to 7 nucleotide residues long oligonucleotides complementary to each other. The stem is attached to both termini of the loop.

3.  5’-Fluorophore 

A fluorescent dye is covalently attached at the 5’-end.

4.  3’-Quencher 

A nonfluorescent quencher dye is covalently attached at the 3’- end.

To establish a molecular beacon RT-PCR assay the following steps are necessary:

1.  Target design.

2.   Primer design.

3.   Optimization of the amplification reaction conditions using SYBR Green.

4.   Molecular beacon design.

5.   Molecular beacon synthesis and characterization.

Molecular beacons hybridize at the annealing temperature with the target sequence, the amplification product in PCR. They do not interfere with with primer annealing and extension. Correctly designed molecular beacons allow the detection of different targets in the same assay tube. Molecular beacon based real-time PCR can be used for a variety of studies including the detection of genomic DNA sequences, single nucleotide polymorphisms (SNPs), messenger ribonucleic acids (mRNA) expression levels, as well as pathogens. In addition, molecular beacons can also be used as intracellular probes for the detection of DNA and mRNA.

Target Design

1.  The source and of the template and the sequence of the primers and template
     will affect the efficiency of the PCR.

2.  Design primer pairs that amplify a target region of 75 to 250 basepairs.

3.  Avoid selecting a molecular beacon target sequence that forms strong secondary
     structures.

4.  Analyze the selected sequences using a DNA folding program such as DNA mfold
     or UNAFold (http://www.bioinfo.rpi.edu/applications/mfold).

Primer Design

1.  Design primers with a 50 to 60 % GC content.

2.  Target a Tm between 50 to 65 °C.

3.  Avoid strong secondary structures. 

4.  Avoid repeats og Gs or Cs longer than three (3) bases.

5.  Check that the primers are not complementary to each other and avoid
     primer-dimers.

6.  Place Gs and Cs on the ends of the primers. 

7.  Avoid false priming by verifying the primers specificity. Us BLAST, the
     “Basic Local Alignment Search Tool” ,
     to do so.

8.  Test primer sets using PCR and SYBR Green as a reporter molecule.

 Figure 2: Structure of SYBR Green I.

SYBR Green I is a commonly used fluorescent DNA binding dye. The dye binds all double–stranded DNA and allows detection and monitoring by measuring the increase in fluorescence throughout the PCR cycle. The excitation and emission maxima of SYBR Green I are 494 nm and 521 nm, respectively.

Molecular Beacon Design

Molecular beacons should be designed such that they are able to hybridize to their targets at the annealing temperature of the PCR. However, the free molecular beacon should stay closed and be not fluorescent at this temperature. Proper design will achieve this. The Tm of the probe can be predicted with the help of the “percent-GC” rule. Several software packages are now available for this purpose.

Usually a probe length of 22 to 30 nucleotides is used but can be expanded from 18 to 30 bases. The Tm of the probe should be 7 to 10 °C above the annealing temperature of the PCR. To test the designed beacon, thermal denaturation profiles can be performed. Longer loop sequences make the probe-target hybrids containing mismatches more stable at the annealing temperature of the PCR.


1.  In the presence of a perfectly complementary target the molecular beacon
     must form a stable probe-target hybrid.

2.  In the presence of a mismatched target the molecular beacon must
     remain closed.

3.  Select a probe sequence that will dissociate from its target at temperatures 5 to
     8 °C higher than the annealing temperature of PCR.

4.  Determine the window of discrimination, the range of temperature in which
     perfectly complementary probe targets can form and in which mismatched
     probe-target hybrids cannot form, by measuring the fluorescence of solutions
     of molecular beacons in the presence of each kind of target as a function of
     temperature. 

STEM-LOOP FORMAT DESIGN

After selecting the sequence for the probe, two complementary arm sequences need to be added on either side of the probe sequence. The stem should be stable at the annealing temperature of the PCR. PREMIER Biosoft offers a free design tool http://www.premierbiosoft.com/qpcr/ for this. A list of design guidelines are listed below.


1.  The probe may contain some bases (usually someGs and Cs) to form the 
     beginnings of a stem.

2.  Divide the primer element from the stem by a C18 (also called HEG,
     hexethylene glycol) group.

3.  Use fluorophores that can be added by phosphoramidite chemistry for
     optimum yields, although other labels can be attached through post-
     synthesis labeling chemistries.

4.  Include a dark quencher adjacent to the spacer, at the end of the 5'-stem.

5.  Avoid placing aG next to the fluorophore as this leads to lower fluorescence.

6.  Design the stem to have aΔG₀ of about -1.5 to -2 kcal/mol. The stem strength
     will also depend upon the length of the intervening loop. Short loops give
     higherTms.

7.  Stems should be as short as possible, 5 or 6 bases are preferred (seven or
     more may lead to baseline drift).

8.  Stems will be mostly GC. A standard stem: CCGCGC-loop-GCGCGG may be
     used but the exactTm of this stem will vary depending upon the length of the
     probe element. 

9.  Model the structures of:

9.a. The unincorporated Scorpion to confirm theTm of the stem.

9.b. The extended Scorpion (essentially the amplicon, plus the probe element).
       Ensure the correct strand of the amplicon is used: if the probe selected goes
       on the reverse primer, use the reverse-complement of the sequence, plus the
       forward strand probe. The ΔG₀ of the second construct MUST be more negative
       than that of the stem although theTm may be higher for the unincorporated
       version than the extended product.

Fluorophore/quencher pairs

The classic molecular beacon first presented by Kramer and colleagues was designed with an EDANS/Dabcyl fluorophore/quencher pair. Molecular beacon based assays containing this type of fluorophore/quencher pairs rely upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact molecular beacon. Efficient fluorescence quenching is a result of favorable energetic overlap of the EDANS excited state and the absorption by DABCYL. The relatively long excited state lifetime of the EDANS fluorophore is also an additional key factor.

Figure 3: Structure of the original molecular beacon used by Tyagi and Kramer in 1996. The molecular beacon consists of a 15 nucleotide long probe sequence. 

This sequence is embedded between two complementary 5 nucleotide long arm or stem sequences. The fluorophore, EDANS (5-((2-Aminoethyl)-amino)-naphthalene-1-sulfonic acid) is conjugated to the 5’-terminal phosphate using a –(CH₂)6-S-CH₂-CO- linker. The quencher, DABSYL (4-(4-Dimethylaminophenylazo) benzenesulfonyl chloride, 4-(Dimethylamino) azobenzene-4′-sulfonyl chloride, DABS-Cl, Dabsyl chloride) is connected to the 39-terminal hydroxyl group using a –(CH₂)₇-NH- linker.

Many more fluorophore/quencher pairs are now commercial available for the synthesis of molecular beacons. Table 1 list the quenching efficiency for different fluorophore-quencher combinations.

Table 1:  Static quenching efficiency of different fluorophore-quencher combinations (Marras et al. 2002).

E_max the emission maximum of the fluorophore, A_max absorption maximum of the quencher

Reference

Basic Local Alignment Search Tool - BLAST:

Beacon Designer 7.9: http://beacon-designer.software.informer.com/7.9/

G. Goel, A. Kumar, A.K. Puniya, W. Chen and K. Singh; Molecular beacon: a multitask probe Journal of Applied Microbiology 2005, 99, 435–442.

Jacqueline A. M. Vet, Salvatore A. E. Marras; Design and Optimization of Molecular Beacon Real-Time Polymerase Chain Reaction Assays. Oligonucleotide Synthesis, Volume 288 of the series Methods in Molecular Biology pp 273-290.

Marras SA, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30(21):e122.

mfold or UNAFold: http://www.bioinfo.rpi.edu/applications/mfold

Molecular Beacons – Yang, C. J., and Tan, W.; Editors. Springer Heidelberg New York Dordrecht London. ISBN 978-642-39109-5.

Molecular Beacons: www.molecular-beacons.org

NUPACK – nucleic acid package: http://nupack.org/partition/new

OligoCalc – SimGene.com:  http://www.simgene.com/OligoCalc

PREMIER Biosoft offers a free design tool: http://www.premierbiosoft.com/qpcr/

Primer 3 – SimGene.com:  http://www.simgene.com/Primer3

POLAND - thermal denaturation profiles of double-stranded RNA, DNA or RNA/DNA-hybrids:

Sfold – Wadsworth Center NYS: http://sfold.wadsworth.org/cgi-bin/index.pl

Tyagi S, Kramer FR.; Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996 Mar;14(3):303-8.

Zipper H, Brunner H, Bernhagen J, Vitzthum F. Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications. Nucleic Acids Research. 2004;32(12):e103. doi:10.1093/nar/gnh101.