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Draft:Multifunctional Hybridization Probes

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  • Comment: This draft reads like an attempt at a review article, which is the equivalent of WP:ESSAY for scientific topics. I'm not totally opposed to an article on this subject, and there are secondary sources (reviews) available, but this is an encyclopedia; the article should be written for a general audience and provide some background such as what this technology is used for, why it is distinct from other methods, and how it works in the broadest terms. It should not be written for practitioners. The jargon should be kept to a minimum, and terms that are obvious to experts but not to a general reader (such as "signal") should be defined. WeirdNAnnoyed (talk) 11:45, 30 July 2025 (UTC)
  • Comment: There are several sections that are missing inline citations. It is also highly unusual for an article to describe "advantages and disadvantages". This is intended to be an encyclopedic article and not a review article; otherwise, you may be interested in Wikiversity. -- Reconrabbit 14:25, 29 July 2025 (UTC)


Multicomponent (Multifunctional) Hybridization Probes/Sensors (MHPs) are nucleic acid detection systems in which two or more oligonucleotide strands assemble on a target to produce a signal, unlike conventional single-stranded hybridization probes. In an MHP, each component carries part of the recognition, binding, reporting or other function to achieve high selectivity, tightly bind nucleic acid analytes or produce detectable signals.

History and examples

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Earlier examples of two-component hybridization probes use two fluorophore-labeled oligonucleotides that hybridize to the abutting positions of a nucleic acid analyte and produce Förster Resonance energy transfer (FRET) signal.[1] The idea was later commercialized as LightCycler technology.[2] This was followed by the introduction of a series of binary (split) probes for nucleic acid analysis.[3] In binary probes two components act in cooperation with each other to produce a detectable signal.[4]

FRET probes use two DNA stands to produce FRET signals.

Next step in conceptualization of MHPs was done in 2011 when in Molecular beacon (MB) probe-based MHP also known as X sensor named after DNA crossover structure was described.[5] In the X sensor, each strand (f and m) has two parts: one binds the MB probe, and the other binds the target nucleic acid (analyte). Strand f has a long analyte-binding arm that can unwind and strongly bind the analyte, while strand m has a short arm that forms a stable complex only with a perfectly matched target. The MB probe generates a fluorescent signal. Each component of the X sensor performs a distinct role but works cooperatively. This design enables the sensor to: (1) produce a fluorescent signal, (2) detect targets with high selectivity, and (3) tightly bind structured analytes like natural RNA.[6]

MB-based X probe.[7] The three strands of the MHP serve three different functions: (1) Strand F – tingly binds folded analyte; (2) stand m senses single nucleotide variation (SNV) at temperatures 5-40oC[8]; 3) MB probe produces fluorescent signal. Single nucleotide variation (SNV) site is shown in magenta.

The MHP concept evolved into "DNA machines" for nucleic acid analysis, where some probe components were integrated into a single DNA nanostructure.[9] For example, in a DNAzyme (Dz)-based machine, Arms 1, 3 and 4 were linked to a common platform to cooperatively bind viral RNA, while Arm 2 provided high target selectivity.[10] The Dz core produces a fluorescent signal. A tile-associated hook delivered the fluorogenic substrate, greatly enhancing sensor sensitivity. This design highlights the value of a shared platform that unifies multiple components for coordinated action.[11]

A DNAzyme (Dz)-based DNA machine tightly binds viral RNA via Arms 1, 3, and 4, which are linked to a common DNA platform. Dz 2 recognizes the RNA target with high selectivity and forms the active DNAzyme core only when fully complementary, leading to the cleavage of a fluorogenic F_sub strand labeled with a fluorophore and quencher. The hook strand (shown in red) captures F_sub in solution and is responsible for the increased cleavage rate when multiple F_sub molecules are present.

Other examples

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  • Split light-up aptamer sensors[12]
  • Three-way junction probe[13]
  • Split G-quadruplex probes[14]
  • 5-WJ junction probe[15]
  • DX-tile sensor[16]
  • OC sensor[17]
  • OWL sensor[18]

See also

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References

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  1. ^ Cardullo, R. A.; Agrawal, S.; Flores, C.; Zamecnik, P. C.; Wolf, D. E. (1988). "Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer". Proceedings of the National Academy of Sciences. 85 (23): 8790–8794. Bibcode:1988PNAS...85.8790C. doi:10.1073/pnas.85.23.8790. PMC 282592. PMID 3194390.
  2. ^ Lyon, E. (2001). "Mutation detection using fluorescent hybridization probes and melting curve analysis". Expert Review of Molecular Diagnostics. 1 (1): 92–101. doi:10.1586/14737159.1.1.92. PMID 11901805.
  3. ^ Kolpashchikov, D. M. (2010). "Binary probes for nucleic acid analysis". Chemical Reviews. 110 (8): 4709–4723. doi:10.1021/cr900323b. PMID 20583806.
  4. ^ Kolpashchikov, D. M. (2010). "Binary probes for nucleic acid analysis". Chemical Reviews. 110 (8): 4709–4723. doi:10.1021/cr900323b. PMID 20583806.
  5. ^ Nguyen, C.; Grimes, J.; Gerasimova, Y. V.; Kolpashchikov, D. M. (2011). "Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids". Chemistry (Weinheim an der Bergstrasse, Germany). 17 (46): 13052–13058. doi:10.1002/chem.201101987. PMC 3221966. PMID 21956816.
  6. ^ Nguyen, C.; Grimes, J.; Gerasimova, Y. V.; Kolpashchikov, D. M. (2011). "Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids". Chemistry (Weinheim an der Bergstrasse, Germany). 17 (46): 13052–13058. doi:10.1002/chem.201101987. PMC 3221966. PMID 21956816.
  7. ^ Nguyen, C.; Grimes, J.; Gerasimova, Y. V.; Kolpashchikov, D. M. (2011). "Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids". Chemistry (Weinheim an der Bergstrasse, Germany). 17 (46): 13052–13058. doi:10.1002/chem.201101987. PMC 3221966. PMID 21956816.
  8. ^ Stancescu, M.; Fedotova, T. A.; Hooyberghs, J.; Balaeff, A.; Kolpashchikov, D. M. (2016). "Nonequilibrium Hybridization Enables Discrimination of a Point Mutation within 5-40 °C". Journal of the American Chemical Society. 138 (41): 13465–13468. Bibcode:2016JAChS.13813465S. doi:10.1021/jacs.6b05628. PMC 5645261. PMID 27681667.
  9. ^ https://pubmed.ncbi.nlm.nih.gov/31243970/ https://pubmed.ncbi.nlm.nih.gov/27886299/
  10. ^ Hussein, Z.; Nour MAY; Kozlova, A. V.; Kolpashchikov, D. M.; Komissarov, A. B.; El-Deeb, A. A. (2023). "DNAzyme Nanomachine with Fluorogenic Substrate Delivery Function: Advancing Sensitivity in Nucleic Acid Detection". Analytical Chemistry. 95 (51): 18667–18672. doi:10.1021/acs.analchem.3c04420. PMID 38079240.
  11. ^ Cox, A. J.; Bengtson, H. N.; Rohde, K. H.; Kolpashchikov, D. M. (2016). "DNA nanotechnology for nucleic acid analysis: Multifunctional molecular DNA machine for RNA detection". Chemical Communications (Cambridge, England). 52 (99): 14318–14321. doi:10.1039/c6cc06889h. PMC 5645153. PMID 27886299.
  12. ^ Kolpashchikov, D. M.; Spelkov, A. A. (2021). "Binary (Split) Light-up Aptameric Sensors". Angewandte Chemie (International ed. In English). 60 (10): 4988–4999. Bibcode:2021ACIE...60.4988K. doi:10.1002/anie.201914919. PMID 32208549.
  13. ^ Nakayama, S.; Yan, L.; Sintim, H. O. (2008). "Junction probes - sequence specific detection of nucleic acids via template enhanced hybridization processes". Journal of the American Chemical Society. 130 (38): 12560–12561. doi:10.1021/ja803146f. PMID 18759403.
  14. ^ Kolpashchikov, D. M. (2008). "Split DNA enzyme for visual single nucleotide polymorphism typing". Journal of the American Chemical Society. 130 (10): 2934–2935. Bibcode:2008JAChS.130.2934K. doi:10.1021/ja711192e. PMC 2948481. PMID 18281995.
  15. ^ Foguel, M. V.; Zamora, V.; Ojeda, J.; Reed, M.; Bennett, A.; Calvo-Marzal, P.; Gerasimova, Y. V.; Kolpashchikov, D.; Chumbimuni-Torres, K. Y. (2024). "DNA nanotechnology for nucleic acid analysis: Sensing of nucleic acids with DNA junction-probes". The Analyst. 149 (3): 968–974. Bibcode:2024Ana...149..968F. doi:10.1039/d3an01707a. PMC 11439508. PMID 38197474.
  16. ^ Kolpashchikov, D. M.; Gerasimova, Y. V.; Khan, M. S. (2011). "DNA nanotechnology for nucleic acid analysis: DX motif-based sensor". ChemBioChem : A European Journal of Chemical Biology. 12 (17): 2564–2567. doi:10.1002/cbic.201100545. PMC 3221779. PMID 22006680.
  17. ^ Cornett, E. M.; O'Steen, M. R.; Kolpashchikov, D. M. (2013). "Operating Cooperatively (OC) sensor for highly specific recognition of nucleic acids". PLOS ONE. 8 (2): e55919. Bibcode:2013PLoSO...855919C. doi:10.1371/journal.pone.0055919. PMC 3575382. PMID 23441157.
  18. ^ Karadeema, R. J.; Stancescu, M.; Steidl, T. P.; Bertot, S. C.; Kolpashchikov, D. M. (2018). "The owl sensor: A 'fragile' DNA nanostructure for the analysis of single nucleotide variations". Nanoscale. 10 (21): 10116–10122. doi:10.1039/c8nr01107a. PMID 29781024.
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