Hybridization Kinetics and Thermodynamics of Molecular Beacons

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2003 Feb 13

PMID 12582252

Citations 82

Authors

Andrew Tsourkas

Mark A Behlke

Scott D Rose

Gang Bao

Affiliations

Soon will be listed here.

Abstract

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.

Citing Articles

Challenges in observing transcription-translation for bottom-up synthetic biology.

Bogatyr V, Wuite G QRB Discov. 2025; 6:e5.

PMID: 39944880 PMC: 11811876. DOI: 10.1017/qrd.2024.27.

DNA Hybridization Kinetics Observed at the Single-Molecule Level Using Graphene Near-Field Effects.

Soares M, Freitas J, Queiros T, Purwidyantri A, Alpuim P, Nieder J J Phys Chem A. 2024; 128(49):10689-10696.

PMID: 39622497 PMC: 11647878. DOI: 10.1021/acs.jpca.4c05740.

Quantum-Enhanced Detection of Viral cDNA via Luminescence Resonance Energy Transfer Using Upconversion and Gold Nanoparticles.

Esmaeili S, Rajil N, Hazrathosseini A, Neuman B, Alkahtani M, Sen D ArXiv. 2024; .

PMID: 39483352 PMC: 11527101.

A method to measure molecular hybridization.

Rosencrantz S, Matyash V, Rosencrantz R, Fedorych O PLoS One. 2024; 19(8):e0308084.

PMID: 39150912 PMC: 11329138. DOI: 10.1371/journal.pone.0308084.

Sequestration within peptide coacervates improves the fluorescence intensity, kinetics, and limits of detection of dye-based DNA biosensors.

Green C, Sementa D, Mathur D, Melinger J, Deshpande P, Elbaum-Garfinkle S Commun Chem. 2024; 7(1):49.

PMID: 38424154 PMC: 10904739. DOI: 10.1038/s42004-024-01124-3.

References

Tsourkas A, Behlke M, Bao G . Structure-function relationships of shared-stem and conventional molecular beacons. Nucleic Acids Res. 2002; 30(19):4208-15. PMC: 140536. DOI: 10.1093/nar/gkf536. View

Kuhn H, Demidov V, Coull J, Fiandaca M, Gildea B, Frank-Kamenetskii M . Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. J Am Chem Soc. 2002; 124(6):1097-103. DOI: 10.1021/ja0041324. View

Roberts R, Crothers D . Specificity and stringency in DNA triplex formation. Proc Natl Acad Sci U S A. 1991; 88(21):9397-401. PMC: 52724. DOI: 10.1073/pnas.88.21.9397. View

Parkhurst K, Parkhurst L . Kinetic studies by fluorescence resonance energy transfer employing a double-labeled oligonucleotide: hybridization to the oligonucleotide complement and to single-stranded DNA. Biochemistry. 1995; 34(1):285-92. DOI: 10.1021/bi00001a035. View

Livak K, Flood S, Marmaro J, Giusti W, Deetz K . Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 1995; 4(6):357-62. DOI: 10.1101/gr.4.6.357. View

Allawi H, SantaLucia Jr J . Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry. 1997; 36(34):10581-94. DOI: 10.1021/bi962590c. View

Tyagi S, Bratu D, Kramer F . Multicolor molecular beacons for allele discrimination. Nat Biotechnol. 1998; 16(1):49-53. DOI: 10.1038/nbt0198-49. View

SantaLucia Jr J . A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 1998; 95(4):1460-5. PMC: 19045. DOI: 10.1073/pnas.95.4.1460. View

Allawi H, SantaLucia Jr J . Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA. Biochemistry. 1998; 37(8):2170-9. DOI: 10.1021/bi9724873. View

10.

Kostrikis L, Tyagi S, Mhlanga M, Ho D, Kramer F . Spectral genotyping of human alleles. Science. 1998; 279(5354):1228-9. DOI: 10.1126/science.279.5354.1228. View

11.

Mazumder A, Majlessi M, Becker M . A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics. Nucleic Acids Res. 1998; 26(8):1996-2000. PMC: 147504. DOI: 10.1093/nar/26.8.1996. View

12.

Matsuo T . In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim Biophys Acta. 1998; 1379(2):178-84. DOI: 10.1016/s0304-4165(97)00090-1. View

13.

Leone G, van Schijndel H, VAN Gemen B, Kramer F, Schoen C . Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res. 1998; 26(9):2150-5. PMC: 147533. DOI: 10.1093/nar/26.9.2150. View

14.

Piatek A, Tyagi S, Pol A, Telenti A, MILLER L, Kramer F . Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol. 1998; 16(4):359-63. DOI: 10.1038/nbt0498-359. View

15.

Allawi H, SantaLucia Jr J . Thermodynamics of internal C.T mismatches in DNA. Nucleic Acids Res. 1998; 26(11):2694-701. PMC: 147606. DOI: 10.1093/nar/26.11.2694. View

16.

Tyagi S, Kramer F . Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996; 14(3):303-8. DOI: 10.1038/nbt0396-303. View

17.

Allawi H, SantaLucia Jr J . Nearest-neighbor thermodynamics of internal A.C mismatches in DNA: sequence dependence and pH effects. Biochemistry. 1998; 37(26):9435-44. DOI: 10.1021/bi9803729. View

18.

Bonnet G, Krichevsky O, Libchaber A . Kinetics of conformational fluctuations in DNA hairpin-loops. Proc Natl Acad Sci U S A. 1998; 95(15):8602-6. PMC: 21122. DOI: 10.1073/pnas.95.15.8602. View

19.

Ratilainen T, Holmen A, Tuite E, Haaima G, Christensen L, Nielsen P . Hybridization of peptide nucleic acid. Biochemistry. 1998; 37(35):12331-42. DOI: 10.1021/bi9808722. View

20.

Sokol D, Zhang X, Lu P, Gewirtz A . Real time detection of DNA.RNA hybridization in living cells. Proc Natl Acad Sci U S A. 1998; 95(20):11538-43. PMC: 21676. DOI: 10.1073/pnas.95.20.11538. View