Skip to main content

Advertisement

SpringerLink
Log in

Fluorescent hybridization probes for nucleic acid detection

  • Review
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Reference

  1. Tan W, Wang K, Drake TJ (2004) Molecular beacons. Curr Opin Chem Biol 8:547–553

    Article  CAS  Google Scholar 

  2. Ju J, Kim DH, Bi L, Meng Q, Bai X, Li Z, Li X, Marma MS, Shi S, Wu J, Edwards JR, Romu A, Turro NJ (2006) Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proc Natl Acad Sci USA 103:19635–19640

    Article  CAS  Google Scholar 

  3. Guo J, Xu N, Li Z, Zhang S, Wu J, Kim DH, Sano Marma M, Meng Q, Cao H, Li X, Shi S, Yu L, Kalachikov S, Russo JJ, Turro NJ, Ju J (2008) Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc Natl Acad Sci USA 105:9145–9150

    Google Scholar 

  4. Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402

    Article  CAS  Google Scholar 

  5. Guo J, Yu L, Turro NJ, Ju J (2010) An integrated system for DNA sequencing by synthesis using novel nucleotide analogues. Acc Chem Res 43:551–563

    Article  CAS  Google Scholar 

  6. Martí AA, Jockusch S, Stevens N, Ju J, Turro NJ (2007) Fluorescent hybridization probes for sensitive and selective DNA and RNA detection. Acc Chem Res 40:402–409

    Article  Google Scholar 

  7. Kolpashchikov DM (2010) Binary probes for nucleic acid analysis. Chem Rev 110:4709–4723

    Article  CAS  Google Scholar 

  8. Sokol DL, Zhang X, Lu P, Gewirtz AM (1998) Real time detection of DNA•RNA hybridization in living cells. Proc Natl Acad Sci USA 95:11538–11543

    Article  CAS  Google Scholar 

  9. 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 USA 100:13308–13313

    Article  CAS  Google Scholar 

  10. Liming SH, Bhagwat AA (2004) Application of a molecular beacon—real-time PCR technology to detect Salmonella species contaminating fruits and vegetables. Int J Food Microbiol 95:177–187

    Article  CAS  Google Scholar 

  11. Giesendorf BA, Vet JA, Tyagi S, Mensink EJ, Trijbels FJ, Blom HJ (1998) Molecular beacons: a new approach for semiautomated mutation analysis. Clin Chem 44:482–486

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Franzini RM, Kool ET (2009) Efficient nucleic acid detection by templated reductive quencher release. J Am Chem Soc 131:16021–16023

    Article  CAS  Google Scholar 

  14. Vet JA, Majithia AR, Marras SA, Tyagi S, Dube S, Poiesz BJ, Kramer FR (1999) Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci USA 96:6394–6399

    Article  CAS  Google Scholar 

  15. Silverman AP, Kool ET (2006) Detecting RNA and DNA with templated chemical reactions. Chem Rev 106:3775–3789

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Santangelo PJ, Nix B, Tsourkas A, Bao G (2004) Dual FRET molecular beacons for mRNA detection in living cells. Nucleic Acid Res 32:e57

    Article  Google Scholar 

  18. Tanke HJ, Dirks RW, Raap T (2005) FISH and immunocytochemistry: towards visualizing single target molecules in living cells. Curr Opin Biotechnol 16:49–54

    Article  CAS  Google Scholar 

  19. Jockusch S, Martí AA, Turro NJ, Li Z, Li X, Ju J, Stevens N, Akins DL (2006) Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences. Photochem Photobiol Sci 5:493–498

    Article  CAS  Google Scholar 

  20. Zhang P, Beck T, Tan W (2001) Design of a molecular beacon DNA probe with two fluorophores. Angew Chem Int Ed 40:402–405

    Article  CAS  Google Scholar 

  21. Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci USA 58:719–726

    Article  CAS  Google Scholar 

  22. Martí AA, Jockusch S, Li Z, Ju J, Turro NJ (2006) Molecular beacons with intrinsically fluorescent nucleotides. Nucleic Acid Res 34:e50

    Article  Google Scholar 

  23. Jean JM, Hall KB (2001) 2-Aminopurine fluorescence quenching and lifetimes: role of base stacking. Proc Natl Acad Sci USA 98:37–41

    Article  CAS  Google Scholar 

  24. Holzhauser C, Wagenknecht HA (2011) In-stem-labeled molecular beacons for distinct fluorescent color readout. Angew Chem Int Ed 50:7268–7272

    Article  CAS  Google Scholar 

  25. Guo J, Wang S, Dai N, Teo YN, Kool ET (2011) Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging. Proc Natl Acad Sci USA 108:3493–3498

    Article  CAS  Google Scholar 

  26. Tyagi S, Marras SA, Kramer FR (2000) Wavelength-shifting molecular beacons. Nat Biotechnol 18:1191–1196

    Article  CAS  Google Scholar 

  27. Li X, Li Z, Martí AA, Jockusch S, Stevens N, Akins DL, Turro NJ, Ju J (2006) Combinatorial fluorescence energy transfer molecular beacon for probing nucleic acid sequences. Photochem Photobiol Sci 5:896–902

    Article  CAS  Google Scholar 

  28. Tong AK, Li Z, Jones GS, Russo JJ, Ju J (2001) Combinatorial fluorescence energy transfer tags for multiplex biological assays. Nat Biotechnol 19:756–759

    Article  CAS  Google Scholar 

  29. Tong AK, Jockusch S, Li Z, Zhu HR, Akins DL, Turro NJ, Ju J (2001) Triple fluorescence energy transfer in covalently trichromophore-labeled DNA. J Am Chem Soc 123:12923–12924

    Article  CAS  Google Scholar 

  30. Snare MJ, Thistlethwaite PJ, Ghiggino KP (1983) Kinetic studies of intramolecular excimer formation in dipyrenylalkanes. J Am Chem Soc 105:3328–3332

    Article  CAS  Google Scholar 

  31. Fujimoto K, Shimizu H, Inouye M (2004) Unambiguous detection of target DNAs by excimer-monomer switching molecular beacons. J Org Chem 69:3271–3275

    Article  CAS  Google Scholar 

  32. Conlon P, Yang CJ, Wu Y, Chen Y, Martinez K, Kim Y, Stevens N, Marti AA, Jockusch S, Turro NJ, Tan W (2008) Pyrene excimer signaling molecular beacons for probing nucleic acids. J Am Chem Soc 130:336–342

    Article  CAS  Google Scholar 

  33. Häner R, Biner SM, Langenegger SM, Meng T, Malinovskii VL (2010) A highly sensitive, excimer-controlled molecular beacon. Angew Chem Int Ed 49:1227–1230

    Article  Google Scholar 

  34. Zhang W, Dichtel WR, Stieg AZ, Benítez D, Gimzewski JK, Heath JR, Stoddart JF (2008) Folding of a donor–acceptor polyrotaxane by using noncovalent bonding interactions. Proc Natl Acad Sci USA 105:6514–6519

    Google Scholar 

  35. Kashida H, Takatsu T, Fujii T, Sekiguchi K, Liang X, Niwa K, Takase T, Yoshida Y, Asanuma H (2009) In-stem molecular beacon containing a pseudo base pair of threoninol nucleotides for the removal of background emission. Angew Chem Int Ed 48:7044–7047

    Article  CAS  Google Scholar 

  36. Socher E, Bethge L, Knoll A, Jungnick N, Herrmann A, Seitz O (2008) Low-noise stemless PNA beacons for sensitive DNA and RNA detection. Angew Chem Int Ed 47:9555–9559

    Article  CAS  Google Scholar 

  37. Grossmann TN, Röglin L, Seitz O (2007) Triplex molecular beacons as modular probes for DNA detection. Angew Chem Int Ed 46:5223–5225

    Article  CAS  Google Scholar 

  38. Rich A (1993) DNA comes in many forms. Gene 135:99–109

    Article  CAS  Google Scholar 

  39. Bourdoncle A, Estévez Torres A, Gosse C, Lacroix L, Vekhoff P, Le Saux T, Jullien L, Mergny JL (2006) Quadruplex-based molecular beacons as tunable DNA probes. J Am Chem Soc 128:11094–11105

    Article  CAS  Google Scholar 

  40. Williamson JR (1994) G-quartet structures in telomeric DNA. Annu Rev Biophys Biomol Struct 23:703–730

    Article  CAS  Google Scholar 

  41. Sei-Iida Y, Koshimoto H, Kondo S, Tsuji A (2000) Real-time monitoring of in vitro transcriptional RNA synthesis using fluorescence resonance energy transfer. Nucleic Acids Res 28:e59

    Article  CAS  Google Scholar 

  42. Tsuji A, Koshimoto H, Sato Y, Hirano M, Sei-Iida Y, Kondo S, Ishibashi K (2000) Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope. Biophys J 78:3260–3274

    Article  CAS  Google Scholar 

  43. Martí AA, Puckett CA, Dyer J, Stevens N, Jockusch S, Ju J, Barton JK, Turro NJ (2007) Inorganic-organic hybrid luminescent binary probe for DNA detection based on spin-forbidden resonance energy transfer. J Am Chem Soc 129:8680–8681

    Article  Google Scholar 

  44. Martí AA, Li X, Jockusch S, Li Z, Raveendra B, Kalachikov S, Russo JJ, Morozova I, Puthanveettil SV, Ju J, Turro NJ (2006) Pyrene binary probes for unambiguous detection of mRNA using time-resolved fluorescence spectroscopy. Nucleic Acids Res 34:3161–3168

    Article  Google Scholar 

  45. Martí AA, Li X, Jockusch S, Stevens N, Li Z, Raveendra B, Kalachikov S, Morozova I, Russo JJ, Akins DL, Ju J, Turro NJ (2007) Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection. Tetrahedron 63:3591–3600

    Article  Google Scholar 

  46. Dirks RM, Pierce NA (2004) Triggered amplification by hybridization chain reaction. Proc Natl Acad Sci USA 101:15275–15278

    Article  CAS  Google Scholar 

  47. Huang J, Wu Y, Chen Y, Zhu Z, Yang X, Yang CJ, Wang K, Tan W (2011) Pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids. Angew Chem Int Ed 50:401–404

    Article  CAS  Google Scholar 

  48. Yang CJ, Martinez K, Lin H, Tan W (2006) Hybrid molecular probe for nucleic acid analysis in biological samples. J Am Chem Soc 128:9986–9987

    Article  CAS  Google Scholar 

  49. Rosmarin D, Pei Z, Blaser MJ, Tyagi S (2006) C-shaped probe. US Patent Appl US20060040275A1

  50. Audic S, Claverie JM (1997) The significance of digital gene expression profiles. Genome Res 7:986–995

    CAS  Google Scholar 

  51. Schork NJ, Fallin D, Lanchbury JS (2000) Single nucleotide polymorphisms and the future of genetic epidemiology. Clin Genet 58:250–264

    Article  CAS  Google Scholar 

  52. Larson DR, Zenklusen D, Wu B, Chao JA, Singer RH (2011) Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332:475–478

    Article  CAS  Google Scholar 

  53. Joo C, Balci H, Ishitsuka Y, Buranachai C, Ha T (2008) Advances in single-molecule fluorescence methods for molecular biology. Annu Rev Biochem 77:51–76

    Article  CAS  Google Scholar 

  54. Bastiaens PI, Squire A (1999) Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol 9:48–52

    Article  CAS  Google Scholar 

  55. Huang B, Bates M, Zhuang X (2009) Super-resolution fluorescence microscopy. Annu Rev Biochem 78:993–1016

    Article  CAS  Google Scholar 

  56. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248:73–76

    Article  CAS  Google Scholar 

  57. Alivisatos AP, Gu W, Larabell C (2005) Quantum dots as cellular probes. Annu Rev Biomed Eng 7:55–76

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the US National Institutes of Health grant R01NS060762 and the National Science Foundation grant NSF-CHE 07–17518.

Author information

Authors and Affiliations

  1. Department of Chemistry, Columbia University, New York, NY, 10027, USA

    Jia Guo & Nicholas J. Turro

  2. Department of Chemical Engineering, Columbia University, New York, NY, 10027, USA

    Jia Guo, Jingyue Ju & Nicholas J. Turro

  3. Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY, 10032, USA

    Jia Guo & Jingyue Ju

Authors
  1. Jia Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jingyue Ju
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicholas J. Turro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nicholas J. Turro.

Additional information

Published in the topical collection Biomimetic Recognition Elements for Sensing Applications with guest editor María Cruz Moreno-Bondi.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, J., Ju, J. & Turro, N.J. Fluorescent hybridization probes for nucleic acid detection. Anal Bioanal Chem 402, 3115–3125 (2012). https://doi.org/10.1007/s00216-011-5526-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5526-x

Keywords

Search

Navigation