Skip to main content
SpringerLink
Log in

Thermodynamic and Kinetic Properties of Molecular Beacons

  • Chapter
  • First Online:
Molecular Beacons

Abstract

Molecular beacons are widely used for detection of nucleic acids both in vitro and in vivo. Compared with linear probes, molecular beacons have shown enhanced sensitivity and specificity primarily due to their stem–loop hairpin structures. The hairpin structures bring new considerations on thermodynamics and kinetics for designing of nucleic acid probes. This chapter has been designed to provide a better understanding of structure–performance relationship of molecular beacons based on analysis of their thermodynamic and kinetic properties. The conformational fluctuations of molecular beacons are discussed concerning the stability and kinetics of the hairpin-coil transformation. In the presence of target nucleic acids, molecular beacons hybridize with targets to form duplex complexes. We analyzed the theoretical models and the relevant parameters used to describe the hybridization reactions. Furthermore, studies on strategies for optimization of molecular beacon performance are summarized. The systematic analysis of studies about thermodynamic and kinetic properties of molecular beacons allows for sophisticated design of better molecular beacons for specific purposes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308

    Article  CAS  Google Scholar 

  2. Tsourkas A, Behlke MA, Rose SD, Bao G (2003) Hybridization kinetics and thermodynamics of molecular beacons. Nucleic Acids Res 31:1319–1330

    Article  CAS  Google Scholar 

  3. Bonnet G, Krichevsky O, Libchaber A (1998) Kinetics of conformational fluctuations in DNA hairpin-loops. Proc Natl Acad Sci U S A 95:8602–8606

    Article  CAS  Google Scholar 

  4. Vallone PM, Benight AS (1999) Melting studies of short DNA hairpins containing the universal base 5-nitroindole. Nucleic Acids Res 27:3589–3596

    Article  CAS  Google Scholar 

  5. Antao VP, Tinoco I (1992) Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops. Nucleic Acids Res 20:819–824

    Article  CAS  Google Scholar 

  6. Vallone PM, Paner TM, Hilario J, Lane MJ, Faldasz BD, Benight AS (1999) Melting studies of short DNA hairpins: influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability. Biopolymers 50:425–442

    Article  CAS  Google Scholar 

  7. Rentzeperis D, Shikiya R, Maiti S, Ho J, Marky LA (2002) Folding of intramolecular DNA hairpin loops: enthalpy-entropy compensations and hydration contributions. J Phys Chem B 106:9945–9950

    Article  CAS  Google Scholar 

  8. Breslauer KJ, Frank R, Blöcker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83:3746–3750

    Article  CAS  Google Scholar 

  9. SantaLucia J (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95:1460–1465

    Article  CAS  Google Scholar 

  10. Owczarzy R, Vallone PM, Gallo FJ, Paner TM, Lane MJ, Benight AS (1997) Predicting sequence‐dependent melting stability of short duplex DNA oligomers. Biopolymers 44:217–239

    Article  CAS  Google Scholar 

  11. Jaeger JA, Turner DH, Zuker M (1989) Improved predictions of secondary structures for RNA. Proc Natl Acad Sci U S A 86:7706–7710

    Article  CAS  Google Scholar 

  12. Haasnoot C, De Bruin S, Berendsen R, Janssen H, Binnendijk T, Hilbers C, Van der Marel G, Van Boom J (1983) Structure, kinetics and thermodynamics of DNA hairpin fragments in solution. J Biomol Struct Dyn 1:115–129

    Article  CAS  Google Scholar 

  13. Haasnoot CA, Hilbers CW, van der Marel GA, van Boom JH, Singh UC, Pattabiraman N, Kollman PA (1986) On loop folding in nucleic acid hairpin-type structures. J Biomol Struct Dyn 3:843–857

    Article  CAS  Google Scholar 

  14. Tinoco I, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J (1973) Improved estimation of secondary structure in ribonucleic acids. Nature 246:40–41

    Article  CAS  Google Scholar 

  15. Senior MM, Jones RA, Breslauer KJ (1988) Influence of loop residues on the relative stabilities of DNA hairpin structures. Proc Natl Acad Sci U S A 85:6242–6246

    Article  CAS  Google Scholar 

  16. Schildkraut C, Lifson S (1965) Dependence of the melting temperature of DNA on salt concentration. Biopolymers 3:195–208

    Article  CAS  Google Scholar 

  17. Owczarzy R, You Y, Moreira BG, Manthey JA, Huang L, Behlke MA, Walder JA (2004) Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures. Biochemistry (NY) 43:3537–3554

    Article  CAS  Google Scholar 

  18. Tan ZJ, Chen SJ (2006) Nucleic acid helix stability: effects of salt concentration, cation valence and size, and chain length. Biophys J 90:1175–1190

    Article  CAS  Google Scholar 

  19. Tan ZJ, Chen SJ (2007) RNA helix stability in mixed Na/Mg2 solution. Biophys J 92:3615–3632

    Article  CAS  Google Scholar 

  20. Tan ZJ, Chen SJ (2008) Salt dependence of nucleic acid hairpin stability. Biophys J 95:738–752

    Article  CAS  Google Scholar 

  21. Turner D, Sugimoto N, Freier S (1990) Thermodynamics and kinetics of base-pairing and of DNA and RNA self-assembly and helix coil transition. Nucleic Acids 1:201–227

    Google Scholar 

  22. Bloomfield VA, Crothers DM, Tinoco I (2000) Nucleic acids: structures, properties, and functions. University Science Books, Sausalito

    Google Scholar 

  23. Ansari A, Kuznetsov SV, Shen Y (2001) Configurational diffusion down a folding funnel describes the dynamics of DNA hairpins. Proc Natl Acad Sci U S A 98:7771–7776

    Article  CAS  Google Scholar 

  24. Shen Y, Kuznetsov SV, Ansari A (2001) Loop dependence of the dynamics of DNA hairpins. J Phys Chem B 105:12202–12211

    Article  CAS  Google Scholar 

  25. Kuznetsov SV, Shen Y, Benight AS, Ansari A (2001) A semiflexible polymer model applied to loop formation in DNA hairpins. Biophys J 81:2864–2875

    Article  CAS  Google Scholar 

  26. Bevilacqua PC, Blose JM (2008) Structures, kinetics, thermodynamics, and biological functions of RNA hairpins. Annu Rev Phys Chem 59:79–103

    Article  CAS  Google Scholar 

  27. Wallace MI, Ying L, Balasubramanian S, Klenerman D (2001) Non-Arrhenius kinetics for the loop closure of a DNA hairpin. Proc Natl Acad Sci U S A 98:5584–5589

    Article  CAS  Google Scholar 

  28. Wallace MI, Ying L, Balasubramanian S, Klenerman D (2000) FRET fluctuation spectroscopy: exploring the conformational dynamics of a DNA hairpin loop. J Phys Chem B 104:11551–11555

    Article  CAS  Google Scholar 

  29. Wemmer DE, Chou SH, Hare DR, Reid BR (1985) Duplex-hairpin transitions in DNA: NMR studies on CGCGTATACGCG. Nucleic Acids Res 13:3755–3772

    Article  CAS  Google Scholar 

  30. Youil R, Kemper BW, Cotton R (1995) Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc Natl Acad Sci U S A 92:87–91

    Article  CAS  Google Scholar 

  31. Nelson NC, Hammond PW, Matsuda E, Goud AA, Becker MM (1996) Detection of all single-base mismatches in solution by chemiluminescence. Nucleic Acids Res 24:4998–5003

    Article  CAS  Google Scholar 

  32. Dubertret B, Calame M, Libchaber AJ (2001) Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat Biotechnol 19:365–370

    Article  CAS  Google Scholar 

  33. Kostrikis LG, Tyagi S, Mhlanga MM, Ho DD, Kramer FR (1998) Spectral genotyping of human alleles. Science 279:1228–1229

    Article  CAS  Google Scholar 

  34. Marras SAE, Russell Kramer F, Tyagi S (1999) Multiplex detection of single-nucleotide variations using molecular beacons. Genet Anal Biomol Eng 14:151–156

    Article  CAS  Google Scholar 

  35. Meroueh M, Chow CS (1999) Thermodynamics of RNA hairpins containing single internal mismatches. Nucleic Acids Res 27:1118–1125

    Article  CAS  Google Scholar 

  36. Bonnet G, Tyagi S, Libchaber A, Kramer FR (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci U S A 96:6171–6176

    Article  CAS  Google Scholar 

  37. Tsourkas A, Behlke MA, Bao G (2002) Structure–function relationships of shared‐stem and conventional molecular beacons. Nucleic Acids Res 30:4208–4215

    Article  CAS  Google Scholar 

  38. Cantor CR (1980) Biophysical chemistry: Part III: The behavior of biological macromolecules. WH Freeman & Co, San Francisco

    Google Scholar 

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

    Article  Google Scholar 

  40. Demidov VV, Frank-Kamenetskii MD (2004) Two sides of the coin: affinity and specificity of nucleic acid interactions. Trends Biochem Sci 29:62–71

    Article  CAS  Google Scholar 

  41. Lomakin A, Frank-Kamenetskii MD (1998) A theoretical analysis of specificity of nucleic acid interactions with oligonucleotides and peptide nucleic acids (PNAs). J Mol Biol 276:57–70

    Article  CAS  Google Scholar 

  42. Kuhn H, Demidov VV, Coull JM, Fiandaca MJ, Gildea BD, Frank-Kamenetskii M (2002) Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. J Am Chem Soc 124:1097–1103

    Article  CAS  Google Scholar 

  43. Tan L, Li Y, Drake TJ, Moroz L, Wang K, Li J, Munteanu A, Yang CJ, Martinez K, Tan W (2005) Molecular beacons for bioanalytical applications. Analyst 130:1002–1005

    Article  CAS  Google Scholar 

  44. Goel G, Kumar A, Puniya A, Chen W, Singh K (2005) Molecular beacon: a multitask probe. J Appl Microbiol 99:435–442

    Article  CAS  Google Scholar 

  45. Bonnet G, Libchaber A (1999) Optimal sensitivity in molecular recognition. Phys A 263:68–77

    Article  CAS  Google Scholar 

  46. Wang K, Tang Z, Yang CJ, Kim Y, Fang X, Li W, Wu Y, Medley CD, Cao Z, Li J (2009) Molecular engineering of DNA: molecular beacons. Angew Chem Int Ed 48:856–870

    Article  CAS  Google Scholar 

  47. Bratu DP, Cha BJ, Mhlanga MM, Kramer FR, Tyagi S (2003) Visualizing the distribution and transport of mRNAs in living cells. Proc Natl Acad Sci U S A 100:13308–13313

    Article  CAS  Google Scholar 

  48. Beisel CL, Bayer TS, Hoff KG, Smolke CD (2008) Model-guided design of ligand-regulated RNAi for programmable control of gene expression. Mol Syst Biol 4:224

    Article  Google Scholar 

  49. Kim JR, Ostermeier M (2006) Modulation of effector affinity by hinge region mutations also modulates switching activity in an engineered allosteric TEM1 [beta]-lactamase switch. Arch Biochem Biophys 446:44–51

    Article  CAS  Google Scholar 

  50. Vallée-Bélisle A, Ricci F, Plaxco KW (2009) Thermodynamic basis for the optimization of binding-induced biomolecular switches and structure-switching biosensors. Proc Natl Acad Sci U S A 106:13802–13807

    Article  Google Scholar 

  51. Tsourkas A, Behlke MA, Bao G (2002) Hybridization of 2′‐O‐methyl and 2′‐deoxy molecular beacons to RNA and DNA targets. Nucleic Acids Res 30:5168–5174

    Article  CAS  Google Scholar 

  52. Yao G, Fang X, Yokota H, Yanagida T, Tan W (2003) Monitoring molecular beacon DNA probe hybridization at the single‐molecule level. Chem Eur J 9:5686–5692

    Article  CAS  Google Scholar 

  53. Nakano S, Kirihata T, Fujii S, Sakai H, Kuwahara M, Sawai H, Sugimoto N (2007) Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides. Nucleic Acids Res 35:486–494

    Article  CAS  Google Scholar 

  54. Chen C, Wang W, Wang Z, Wei F, Zhao XS (2007) Influence of secondary structure on kinetics and reaction mechanism of DNA hybridization. Nucleic Acids Res 35:2875–2884

    Article  CAS  Google Scholar 

  55. Porschke D, Eigen M (1971) Co-operative non-enzymatic base recognition III. Kinetics of the helix–coil transition of the oligoribouridylic · oligoriboadenylic acid system and of oligoriboadenylic acid alone at acidic pH. J Mol Biol 62:361–364

    Article  CAS  Google Scholar 

  56. Pörschke D, Uhlenbeck O, Martin F (1973) Thermodynamics and kinetics of the helix‐coil transition of oligomers containing GC base pairs. Biopolymers 12:1313–1335

    Article  Google Scholar 

  57. McConaughy BL, Laird CD, McCarthy BJ (1969) Nucleic acid reassociation in formamide. Biochemistry (NY) 8:3289–3295

    Article  CAS  Google Scholar 

  58. Kohne DE, Levison SA, Byers MJ (1977) Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique. Biochemistry (NY) 16:5329–5341

    Article  CAS  Google Scholar 

  59. Yao G, Tan W (2004) Molecular-beacon-based array for sensitive DNA analysis. Anal Biochem 331:216–223

    Article  CAS  Google Scholar 

  60. Tsourkas A, Bao G (eds) (2001) Detecting mRNA transcripts using FRET-enhanced molecular beacons. ASME BED

    Google Scholar 

  61. Vijayanathan V, Thomas T, Sigal LH, Thomas T (2002) Direct measurement of the association constant of HER2/neu antisense oligonucleotide to its target RNA sequence using a molecular beacon. Antisense Nucleic Acid Drug Dev 12:225–233

    Article  CAS  Google Scholar 

  62. Mhlanga MM, Vargas DY, Fung CW, Kramer FR, Tyagi S (2005) tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells. Nucleic Acids Res 33:1902–1912

    Article  CAS  Google Scholar 

  63. Santangelo PJ, Bao G (2007) Dynamics of filamentous viral RNPs prior to egress. Nucleic Acids Res 35:3602–3611

    Article  CAS  Google Scholar 

  64. Martinez K, Estevez M, Wu Y, Phillips JA, Medley CD, Tan W (2009) Locked nucleic acid based beacons for surface interaction studies and biosensor development. Anal Chem 81:3448–3454

    Article  CAS  Google Scholar 

  65. Wu Y, Yang CJ, Moroz LL, Tan W (2008) Nucleic acid beacons for long-term real-time intracellular monitoring. Anal Chem 80:3025–3028

    Article  CAS  Google Scholar 

  66. Vester B, Wengel J (2004) LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry (NY) 43:13233–13241

    Article  CAS  Google Scholar 

  67. Kurreck J, Wyszko E, Gillen C, Erdmann VA (2002) Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res 30:1911–1918

    Article  CAS  Google Scholar 

  68. Aartsma-Rus A, Kaman W, Bremmer-Bout M, Janson A, Den Dunnen J, van Ommen GJB, van Deutekom J (2004) Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells. Gene Ther 11:1391–1398

    Article  CAS  Google Scholar 

  69. Romani A (2007) Regulation of magnesium homeostasis and transport in mammalian cells. Arch Biochem Biophys 458:90–102

    Article  CAS  Google Scholar 

  70. Santangelo PJ (2010) Molecular beacons and related probes for intracellular RNA imaging. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2:11–19

    Article  CAS  Google Scholar 

  71. Koshkin AA, Nielsen P, Meldgaard M, Rajwanshi VK, Singh SK, Wengel J (1998) LNA (locked nucleic acid): an RNA mimic forming exceedingly stable LNA: LNA duplexes. J Am Chem Soc 120:13252–13253

    Article  CAS  Google Scholar 

  72. Koshkin AA, Singh SK, Nielsen P, Rajwanshi VK, Kumar R, Meldgaard M, Olsen CE, Wengel J (1998) LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54:3607–3630

    Article  CAS  Google Scholar 

  73. Wang L, Yang CJ, Medley CD, Benner SA, Tan W (2005) Locked nucleic acid molecular beacons. J Am Chem Soc 127:15664–15665

    Article  CAS  Google Scholar 

  74. Yoo SM, Keum KC, Yoo SY, Choi JY, Chang KH, Yoo NC, Yoo WM, Kim JM, Lee D, Lee SY (2004) Development of DNA microarray for pathogen detection. Biotechnol Bioprocess Eng 9:93–99

    Article  CAS  Google Scholar 

  75. Gracey AY, Cossins AR (2003) Application of microarray technology in environmental and comparative physiology. Annu Rev Physiol 65:231–259

    Article  CAS  Google Scholar 

  76. Fan J, Yang X, Wang W, Wood WH, Becker KG, Gorospe M (2002) Global analysis of stress-regulated mRNA turnover by using cDNA arrays. Proc Natl Acad Sci U S A 99:10611–10616

    Article  CAS  Google Scholar 

  77. Fang Y, Frutos AG, Lahiri J (2003) Ganglioside microarrays for toxin detection. Langmuir 19:1500–1505

    Article  CAS  Google Scholar 

  78. Sekar M, Bloch W, St John PM (2005) Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres. Nucleic Acids Res 33:366–375

    Article  CAS  Google Scholar 

  79. Gao Y, Wolf LK, Georgiadis RM (2006) Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison. Nucleic Acids Res 34:3370–3377

    Article  CAS  Google Scholar 

  80. Peterson AW, Heaton RJ, Georgiadis RM (2001) The effect of surface probe density on DNA hybridization. Nucleic Acids Res 29:5163–5168

    Article  CAS  Google Scholar 

  81. Vainrub A, Montgomery Pettitt B (2003) Surface electrostatic effects in oligonucleotide microarrays: control and optimization of binding thermodynamics. Biopolymers 68:265–270

    Article  CAS  Google Scholar 

  82. Halperin A, Buhot A, Zhulina E (2005) Brush effects on DNA chips: thermodynamics, kinetics, and design guidelines. Biophys J 89:796–811

    Article  CAS  Google Scholar 

  83. Tawa K, Knoll W (2004) Mismatching base‐pair dependence of the kinetics of DNA–DNA hybridization studied by surface plasmon fluorescence spectroscopy. Nucleic Acids Res 32:2372–2377

    Article  CAS  Google Scholar 

  84. Stillman BA, Tonkinson JL (2001) Expression microarray hybridization kinetics depend on length of the immobilized DNA but are independent of immobilization substrate. Anal Biochem 295:149–157

    Article  CAS  Google Scholar 

  85. Glazer MI, Fidanza JA, McGall GH, Trulson MO, Forman JE, Frank CW (2007) Kinetics of oligonucleotide hybridization to DNA probe arrays on high-capacity porous silica substrates. Biophys J 93:1661–1676

    Article  CAS  Google Scholar 

  86. Liu X, Tan W (1999) A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons. Anal Chem 71:5054–5059

    Article  CAS  Google Scholar 

  87. Wang H, Li J, Liu H, Liu Q, Mei Q, Wang Y, Zhu J, He N, Lu Z (2002) Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film. Nucleic Acids Res 30:e61

    Article  Google Scholar 

  88. Du H, Strohsahl CM, Camera J, Miller BL, Krauss TD (2005) Sensitivity and specificity of metal surface-immobilized molecular beacon biosensors. J Am Chem Soc 127:7932–7940

    Article  CAS  Google Scholar 

  89. Du H, Disney MD, Miller BL, Krauss TD (2003) Hybridization-based unquenching of DNA hairpins on Au surfaces: prototypical “molecular beacon” biosensors. J Am Chem Soc 125:4012–4013

    Article  CAS  Google Scholar 

  90. Sauthier ML, Carroll RL, Gorman CB, Franzen S (2002) Nanoparticle layers assembled through DNA hybridization: characterization and optimization. Langmuir 18:1825–1830

    Article  CAS  Google Scholar 

  91. Dave N, Liu J (2010) Fast molecular beacon hybridization fast molecular bea-con hybridization. J Phys Chem B 114:15694–15699

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA

    Lu Peng

  2. Department of Biomedical Engineering and Department of Chemistry, Hunan University, Changsha, P.R. China

    Weihong Tan

  3. Department of Chemistry, University of Florida, Gainesville, FL, USA

    Weihong Tan

Authors
  1. Lu Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Weihong Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lu Peng .

Editor information

Editors and Affiliations

  1. Department of Chemical Biology College of Chemistry and Chemical Xiamen University, Xiamen, Fujian, China, People's Republic

    Chaoyong James Yang

  2. Department of Biomedical Engineering and Department of Chemistry Hunan University, Changsha, China, People's Republic

    Weihong Tan

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer-Verlag Berlin Heidelberg

About this chapter

Cite this chapter

Peng, L., Tan, W. (2013). Thermodynamic and Kinetic Properties of Molecular Beacons. In: Yang, C., Tan, W. (eds) Molecular Beacons. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-39109-5_2

Download citation

Publish with us

Policies and ethics

Search

Navigation