Morpholinos in diagnostic applications
Morpholinos have been used as probes in many styles of diagnostic systems. Their complete resistance to nucleases and their absence of electrostatic charge at neutral pH allow development of novel diagnostic applications. Methods for measuring Morpholino concentration or structure also fall under diagnostics. Here are some citations from those projects.
Karasawa K, Duchoslav E, Burton L, Kawakami J, Baba T. Sequencing of Morpholino Antisense Oligonucleotides Using Electron Capture Dissociation Mass Spectrometry . Anal Chem. 2023 Oct 23. doi: 10.1021/acs.analchem.3c03621. Online ahead of print. Li K, Liang C, Pan Y, Mei L, Zhang GJ. Protocol for constructing GQD-PMO functionalized FET-biosensor for ultrasensitive exosomal miRNA detection. STAR Protoc. 2023 Aug 16;4(3):102516. doi: 10.1016/j.xpro.2023.102516. Online ahead of print. Oda H, Sato Y, Kawashima SA, Fujiwara Y, Pálfy M, Wu E, Vastenhouw NL, Kanai M, Kimura H. Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo. Biol Open. 2023 May 15;12(5):bio059783. doi: 10.1242/bio.059783. Epub 2023 May 17.
Fluorochrome-labeled Morpholino used as a diagnostic probe in a zebrafish to visualize transcription of the miRNA mir-430 Zhou Y, Liu B, Lei Y, Tang L, Li T, Yu S, Zhang GJ, Li YT. Acupuncture Needle-Based Transistor Neuroprobe for In Vivo Monitoring of Neurotransmitter. Small. 2022 Nov 7:e2204142. doi: 10.1002/smll.202204142. Online ahead of print. Becette OB, Marino JP, Brinson RG. Structural Fingerprinting of Antisense Oligonucleotide Therapeutics by Solution NMR Spectroscopy. Pharm Res. 2022 Oct 4. doi: 10.1007/s11095-022-03403-x. Online ahead of print. Li K, Tu J, Zhang Y, Jin D, Li T, Li J, Ni W, Xiao MM, Zhang ZY, Zhang GJ. Ultrasensitive detection of exosomal miRNA with PMO-graphene quantum dots-functionalized field-effect transistor biosensor. iScience. 2022 Jun 3;25(7):104522. doi: 10.1016/j.isci.2022.104522. eCollection 2022 Jul 15. Klaus A, Clapes T, Yvernogeau L, Basu S, Weijts B, Maas J, Smal I, Galjart N, Robin C. CLASP2 safeguards hematopoietic stem cell properties during mouse and fish development. Cell Rep. 2022 Jun 14;39(11):110957. doi: 10.1016/j.celrep.2022.110957. Li K, Tu J, Zhang Y, Jin D, Li T, Li J, Ni W, Xiao M-M, Zhang Z-Y, Zhang G-J. Ultrasensitive Detection of Exosomal miRNA with PMO- Graphene Quantum Dots Functionalized Field Effect Transistor Biosensor. iScience. 2022;[Epub] doi:10.1016/j.isci.2022.104522 Yang L, Ye X, Li X, Huang Z, Chen F, Yang W, Wang Z. Colorimetric aptasensor for sensitive detection of quinclorac based on exonuclease III-assisted cyclic release of phosphorodiamidate morpholino oligomer mimic enzyme strategy. Anal Chim Acta. 2022;1207:339815 doi:10.1016/j.aca.2022.339815 Mana T, Kundu J, Lahiri H, Bera S, Kolay J, Sinha S, Mukhopadhyay R. Molecularly resolved, label-free nucleic acid sensing at solid-liquid interface using non-ionic DNA analogues. RSC Adv. 2022;12:9263-74. doi:10.1039/D2RA00386D Yousuf S, Kim J, Orozaliev A, Dahlem MS, Song Y-A, Viegas J. Label-Free Detection of Morpholino-DNA Hybridization Using a Silicon Photonics Suspended Slab Micro-Ring Resonator. IEEE Photon J. 2021;14(4):6600809. doi:10.1109/JPHOT.2021.3098680 Hadzhiev Y, Wheatley L, Cooper L, Ansaloni F, Whalley C, Chen Z, Gustincich S, Sanges R, Burgess SM, Beggs AD, Mueller FD. The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation. bioRxiv. 2021;[preprint] doi:10.1101/2021.08.09.455629
Labeled Morpholinos as probes for miRNA transcription Li J, Wu D, Yu Y, Li T, Li K, Xiao M-M, Li Y, Zhang Z-Y, Zhang G-J. Rapid and Unamplified Identification of COVID-19 with Morpholino-Modified Graphene Field-Effect Transistor Nanosensor. Biosens Bioelectron. 2021;[Epub] doi:10.1016/j.bios.2021.113206 D'Orazio FM, Jasiulewicz A, Hadzhiev Y, Müller F. Visualization of Transcriptional Activity in Early Zebrafish Primordial Germ Cells. Methods Mol Biol. 2021;2218:185-194. doi: 10.1007/978-1-0716-0970-5_15. Victorio CB, Novera W, Tham JY, Watanabe S, Vasudevan SG, Chacko A-M. Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication. Int J Molec Sci. 2020;21(23):1422-0067 doi:10.3390/ijms21239260 Lorenson MY, Chen KE, Walker AM. Enzyme-linked oligonucleotide hybridization assay for direct oligo measurement in blood. Biol Methods Protoc. 2019 Jan 7;4(1):bpy014. doi: 10.1093/biomethods/bpy014. eCollection 2019. Riback JA, Zhu L, Ferrolino MC, Tolbert M, Mitrea DM, Sanders DW, Wei M-T, Kriwacki RW, Brangwynne CP. Composition-dependent thermodynamics of intracellular phase separation. Nature. 2020;[Epub]. doi:10.1038/s41586-020-2256-2
"To detect 16S rRNA localization, the 16S rRNA was mixed with an equimolar amount of a 13mer morpholino complementary to nts 110-122 (Gene Tools, LLC) labeled with Alexa 555 (Life Technologies) prior to NPM1 addition. The morpholino used was labeled via its 5’ amine using with an Alexa Flour succinimdyl ester dye conjugate according to the manufacturer’s protocol. Excess dye was removed thru dialysis. " D'Orazio FM, Balwierz P, Guo Y, Rodriguez B, Jasiulewicz A, Vaquerizas JM, Cairns B, Lenhard B, Mueller F. Germ plasm localisation dynamics mark distinct phases of transcriptional and post-transcriptional regulation control in primordial germ cells. bioRxiv. 2020;[preprint] doi:10.1101/2020.01.12.903336
Morpholinos as probes to reveal transcription Riback JA, Zhu L, Ferrolino MC, Tolbert M, Mitrea DM, Sanders DW, Wei M-T, Kriwacki RW, Brangwynne CP. Composition dependent phase separation underlies directional flux through the nucleolus. bioRxiv. 2019;[preprint] doi:10.1101/809210
Morpholinos used as ribosomal RNA probes Sato Y, Hilbert L, Oda H, Wan Y, Heddleston JM, Chew TL, Zaburdaev V, Keller P, Lionnet T, Vastenhouw N, Kimura H. Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis. Development. 2019 Sep 30;146(19). pii: dev179127. doi: 10.1242/dev.179127.
Morpholinos used as transcription probes. Sato Y, Hilbert L, Oda H, Wan Y, Heddleston JM, Chew T-L, Zaburdaev V, Keller P, Lionnet T, Vastenhouw N, Hiroshi Kimura H. Quantitative measurements of chromatin modification dynamics during zygotic genome activation. bioRxiv. 2019;[preprint] doi:10.1101/601393
Morpholino used as live-embryo ISH probe for miRNA dre-miR-430. Hadzhiev Y, Qureshi HK, Wheatley L, Cooper L, Jasiulewicz A, Van Nguyen H, Wragg JW, Poovathumkadavil D, Conic S, Bajan S, Sik A, Hutvàgner G, Tora L, Gambus A, Fossey JS, Müller F. A cell cycle-coordinated Polymerase II transcription compartment encompasses gene expression before global genome activation. Nat Commun. 2019 Feb 11;10(1):691. doi: 10.1038/s41467-019-08487-5. Li C, He L, Jiang S, Mo R, Yan X, Qian Z-J, Zhou C, Sun S, Hong P, Huang Y. Ultrasensitive detection of microRNA using an array of Au nanowires deposited within the channels of a porous anodized alumina membrane. Elecrochem Comm. 2019;[Epub ahead of print] doi:10.1016/j.elecom.2019.03.008 Kumari P, Adeloju SB. Fabrication of a novel DNA affinity biosensor based on hybridisation induced current by electrostatic repulsion of silicotungstic acid as a redox indicator. Talanta. 2019 Mar 1;194:127-133. doi: 10.1016/j.talanta.2018.09.074. Epub 2018 Sep 20. Langford G, Raeburn J, Ferrier DC, Hands PJW, Shaver MP. Morpholino Oligonucleotide Crosslinked Hydrogels as Portable Optical Oligonucleotide Biosensors. ACS Sens. 2018 Dec 28. doi: 10.1021/acssensors.8b01208. [Epub ahead of print] Bagi A, Soelberg SD, Furlong CE, Baussant T. Implementing Morpholino-Based Nucleic Acid Sensing on a Portable Surface Plasmon Resonance Instrument for Future Application in Environmental Monitoring. Sensors. 2018;18(10):3259. doi:10.3390/s18103259 Ouyang W, Li Z, Han J. Pressure-Modulated Selective Electrokinetic Trapping for Direct Enrichment, Purification, and Detection of Nucleic Acids in Human Serum. Anal Chem. 2018 Aug 29. doi: 10.1021/acs.analchem.8b02330. [Epub ahead of print] Hadzhiev Y, Qureshi H, Wheatley L, Cooper L, Jasiulewicz A, Nguyen HV, Wragg J, Poovathumkadavil D, Conic S, Bajan S, Sik A, Hutvagner G, Tora L, Gambus A, Fossey JS, Mueller F. A cell cycle-coordinated nuclear compartment for Polymerase II transcription encompasses the earliest gene expression before global genome activation. BioRxiv. 2018;[Epub] doi:doi.org/10.1101/366468
Morpholinos as probes to visualize transcription dynamics. Xiong Y, McQuistan TJ, Stanek JW, Summerton JE, Mata JE, Squier TC. Detection of unique Ebola virus oligonucleotides using fluorescently-labeled phosphorodiamidate morpholino oligonucleotide probe pairs. Anal Biochem. 2018 Jul 18;557:84-90. doi: 10.1016/j.ab.2018.07.006. [Epub ahead of print]
FRET Mei J, Li Y-T, Zhang H, Xiao M-M, Ning Y, Zhang Z-Y, Zhang G-J. Molybdenum Disulfide Field-Effect Transistor Biosensor for Ultrasensitive Detection of DNA by Employing Morpholino as Probe. Biosens Bioelectr. 2018;{Epub ahead of print] doi:10.1016/j.bios.2018.03.043 Rosenfeld T, Bercovici M. Amplification-free detection of DNA in a paper-based microfluidic device using electroosmotically balanced isotachophoresis. Lab Chip. 2018 Feb 20. doi: 10.1039/c7lc01250k. [Epub ahead of print] Zeidman Kalman T, Khalandovsky R, Tenenbaum Gonikman E, Bercovici M. Monitoring dissociation kinetics during electrophoretic focusing enables high-specificity nucleic acid detection. Angew Chem. 2018;[Epub ahead of print] doi:10.1002/ange.201711673 Cheung LS, Wei X, Martins D, Song Y-A. Rapid detection of exosomal microRNA biomarkers by electrokinetic concentration for liquid biopsy on chip. Biomicrofluidics. 2018;12:014104. doi:10.1063/1.5009719 Pavlova AS, Dyudeeva ES, Kupryushkin MS, Amirkhanov NV, Pyshnyi DV, Pyshnaya IA. SDS-PAGE Procedure: Application for Characterization of New Entirely Uncharged Nucleic Acids Analogues. Electrophoresis. 2017 Nov 7. doi: 10.1002/elps.201700415. [Epub ahead of print] Ruffin S, Hung IA, Koniges UM, Levicky R. Electrostatic Cycling of Hybridization Using Nonionic DNA Mimics. ACS Sens. 2017 Jul 28;2(7):892-896. doi: 10.1021/acssensors.7b00100. Epub 2017 Jun 20. Meng M, Zhang J, Liu A, Reuschel S, Sazani P, Wong M. Quantitative determination of AVI-7100 (Radavirsen), a phosphorodiamidate morpholino oligomer (PMOplus®), in human plasma using LC-MS/MS. Bioanalysis. 2017 May 18. doi: 10.4155/bio-2016-0289. [Epub ahead of print] Liao T, Li X, Tong Q, Zou K, Zhang H, Tang L, Sun Z, Zhang G-J. Ultrasensitive Detection of MicroRNAs with Morpholino-Functionalized Nanochannel Biosensor. Anal Chem. 2017;[Just accepted] doi:10.1021/acs.analchem.7b00487 Levicky R, Koniges U, Tercero N. Diagnostic Applications of Morpholinos and Label-Free Electrochemical Detection of Nucleic Acids. Methods Mol Biol. 2017;1565:181-190. doi: 10.1007/978-1-4939-6817-6_15. Burki U, Straub V. Ultrasensitive Hybridization-Based ELISA Method for the Determination of Phosphorodiamidate Morpholino Oligonucleotides in Biological samples. Methods Mol Biol. 2017;1565:265-277. doi: 10.1007/978-1-4939-6817-6_22. Boutilier J, Moulton HM. Surface Plasmon Resonance-Based Concentration Determination Assay: Label-Free and Antibody-Free Quantification of Morpholinos. Methods Mol Biol. 2017;1565:251-263. doi: 10.1007/978-1-4939-6817-6_21. Zu Y, Tan MH, Ren EC, Ying JY. Facile and phase-defined determination of HLA alleles with morpholino-functionalized nanoparticle probes. Nanomedicine. 2016 Oct 5. pii: S1549-9634(16)30161-7. doi: 10.1016/j.nano.2016.09.009. Wei X, Panindre P, Zhang Q, Song Y-A. Increasing the Detection Sensitivity for DNA-Morpholino Hybridization in Sub-Nanomolar Regime by Enhancing the Surface Ion Conductance of PEDOT:PSS Membrane in a Microchannel. ACS Sens. 2016;[Epub ahead of print] doi:10.1021/acssensors.6b00169 Hu W, Zhang J, Kong J. Fluorescence Detection of DNA Based on Non-covalent π-π Stacking Interaction between 1-Pyrenebutanoic Acid and Hypericin. Anal Sci. 2016;32(5):523-7. doi: 10.2116/analsci.32.523. Liu A, Zhang J, Zhao N, Burton S, Carver M, Liu S, Meng M, Reuschel S, Voelker T. Two Quantitative Assays for the Phosphorodiamidate Morpholino Oligomer (PMO) SRP-4045 in Mouse Plasma Using HPLC-MS/MS Coupled with Solid Phase Micro-Extraction. Presented at ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, Texas, June 5-9, 2016. He Y, Zhang J, Ruffin S, Ji L, Wang K, Levicky R, Xia X. An Electrochemical Study of the Surface Hybridization Process of Morpholino-DNA: Thermodynamics and Kinetics. Electroanalysis. 2016;[Epub ahead of print] doi:10.1002/elan.201501109 Martins D, Wei X, Levicky R, Song Y-AR. Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization. Anal Chem. 2016;[Epub ahead of print] doi:10.1021/acs.analchem.5b03875 Chen L, Cheng D, Liu G, Dou S, Wang Y, Liu X, Liu Y, Rusckowski M. Detection of Klebsiella pneumoniae Infection with an Antisense Oligomer Against its Ribosomal RNA. Mol Imaging Biol. 2016 Feb 1. [Epub ahead of print] Chen J, Wu J, Hong Y. Morpholino molecular beacon for specific RNA visualization in vivo. Chem Commun. 2016;[Epub ahead of print] doi:10.1039/C5CC07124K
microinjection into single-cell medaka zygotes Li H, Wu C, Aramayo R, Sachs MS, Harlow ML. Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA). Sci Rep. 2015 Oct 8;5:14918. doi: 10.1038/srep14918.
Morpholino used as an in situ probe. Qiao W, Chiang H-C, Xie H, Levicky R. Surface vs. solution hybridization: effects of salt, temperature, and probe type. Chem Commun. 2015;[Epub ahead of print] doi:10.1039/C5CC06674C Burki U, Keane J, Blain A, O'Donovan L, Gait MJ, Laval SH, Straub V. Development and Application of an Ultrasensitive Hybridization-Based ELISA Method for the Determination of Peptide-Conjugated Phosphorodiamidate Morpholino Oligonucleotides. Nucleic Acid Ther. 2015 Jul 15. [Epub ahead of print] Hu W, Ning Y, Li L, Kong J, Zhang X. Highly sensitive detection of sequence-specific DNA with morpholino-functionalized magnetic microspheres. Anal Methods. 2015;[Epub ahead of print] doi:10.1039/C5AY00628G Martins D, Levicky R, Songa Y-A. Enhancing the speed of morpholino-DNA biosensor by electrokinetic concentration of DNA in a microfluidic chip. Biosensors Bioelectronics. Gao HL, Wang M, Wu ZQ, Wang C, Wang K, Xia XH. A morpholino-functionalized nanochannel-array for label-free SNPs detection. Anal Chem. 2015 Apr 7;87(7):3936-41. doi: 10.1021/ac504830e. Epub 2015 Mar 11 Hu W, Hu Q, Li L, Kong J, Zhang X. Detection of sequence-specific DNA with morpholino-functionalized silicon chip. Anal Methods. 2015;[Epub ahead of print] doi:10.1039/C4AY02780A Zu Y, Tan M-H, Chowbay B, Lee SC, Yap H, Lee MT, Lu L-S, Chang C-P, Ying JY. Nanoprobe-based genetic testing. NanoToday. 2014;[Epub ahead of print] doi:10.1016/j.nantod.2014.04.003 Cao L, Zhao Y, Ji L-N, Zhang J, Wang K, Xia X-H. The Enhanced Enzymolysis Resistance of Surface-Immobilized DNA Caused by Hybridizing with Morpholino. Electroanalysis. 2013;[Epub ahead of print] doi:10.1002/elan.201200612 Chen L, Wang Y, Cheng D, Liu X, Dou S, Liu G, Hnatowich DJ, Rusckowski M. 99mTc-MORF oligomers specific for bacterial ribosomal RNA as potential specific infection imaging agents. Bioorg Med Chem. 2013 Aug 28. doi:pii: S0968-0896(13)00728-1. 10.1016/j.bmc.2013.08.034. [Epub ahead of print]. Schnell FJ, Crumley SL, Mourich DV, Iversen PL. Development of Novel Bioanalytical Methods to Determine the Effective Concentrations of Phosphorodiamidate Morpholino Oligomers in Tissues and Cells. BioResearch Open Access. 2013;[Epub ahead of print] doi:10.1089/biores.2012.0276.
In this paper, it is the Morpholino concentration being analyzed. Gao Z, Deng H, Shen W, Ren Y. A Label-Free Biosensor for Electrochemical Detection of Femtomolar MicroRNAs. Anal Chem. 2013 Jan 16. [Epub ahead of print] Qiao W, Kalachikov S, Liu Y, Levicky R. Charge-Neutral Morpholino Microarrays for Nucleic Acid Analysis. Anal Biochem. 2012;[Epub ahead of print] doi:10.1016/j.ab.2012.12.001 Zhao Y, Cao L, Ouyang J, Wang M, Wang K, Xia X. A Reversible Plasmonic Probe Sensitive for pH in Micro/nano-spaces Based on i-motif-modulated Morpholino-AuNPs Assembly. Anal Chem. 2012;[Epub ahead of print] doi:10.1021/ac302915a Wang Y, Chen L, Liu X, Cheng D, Liu G, Liu Y, Dou S, Hnatowich DJ, Rusckowski M. Detection of Aspergillus fumigatus pulmonary fungal infections in mice with 99mTc-labeled MORF oligomers targeting ribosomal RNA. Nuclear Med Biol. 2012;[epub ahead of print] doi:10.1016/j.nucmedbio.2012.10.001. Morpholinos as ISH probes for miRNA:
Lagendijk AK, Moulton JD, Bakkers J. Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues. Biology Open. 2012;[Advance Online Publication] doi:10.1242/bio.2012810 Liu Y, Irving D, Qiao W, Ge D, Levicky R. Kinetic Mechanisms in Morpholino-DNA Surface Hybridization. J. Am. Chem. Soc. 2011;[Epub ahead of print] DOI: 10.1021/ja202631b Zu Y, Ting AL, Yi G, Gao Z. Sequence-Selective Recognition of Nucleic Acids under Extremely Low Salt Conditions by Using Nanoparticle Probes. Anal Chem. 2011;[Epub ahead of print] DOI: 10.1021/ac2001516 Liong M, Tassa C, Shaw SY, Lee H, Weissleder R. Multiplexed magnetic labeling amplification using oligonucleotide hybridization. Adv Mater. 2011 Sep 22;23(36):H254-7. doi: 10.1002/adma.201101401. Epub 2011 Jul 22. No abstract available. O'Connor R, Tercero N, Qiao W, Levicky R. Electrochemical Studies of Morpholino-DNA Surface Hybridization. ECS Trans. 2011;(35)7:99-110. doi: 10.1149/1.3571981 Zu Y, Ting AL, Gao Z. Visualizing Low-Level Point Mutations: Enzyme-like Selectivity Offered by Nanoparticle Probes. Small. 2011 Feb 7;7(3):306-10. doi: 10.1002/smll.201001774. Epub 2010 Dec 22. No abstract available. Liu G, Dou S, Liu Y, Liang M, Chen L, Cheng D, Greiner D, Rusckowski M, Hnatowich DJ. Unexpected side products in the conjugation of an amine-derivatized morpholino oligomer with p-isothiocyanate benzyl DTPA and their removal. Nucl Med Biol. 2011 Feb;38(2):159-63. Epub 2010 Oct 27. Li SJ, Li J, Wang K, Wang C, Xu JJ, Chen HY, Xia XH, Huo Q. A Nanochannel Array-Based Electrochemical Device for Quantitative Label-free DNA Analysis. ACS Nano. 2010 Oct 19. [Epub ahead of print] Cheng D, Wang Y, Liu X, Pretorius PH, Liang M, Rusckowski M, Hnatowich DJ. Comparison of (18)F PET and (99m)Tc SPECT Imaging in Phantoms and in Tumored Mice. Bioconjug Chem. 2010 Aug 18;21(8):1565-70 Tercero N, Wang K, Levicky R. Capacitive Monitoring of Morpholino-DNA Surface Hybridization: Experimental and Theoretical Analysis. Langmuir. 2010 Sep 7;26(17):14351-8. Gong P, Wang K, Liu Y, Shepard K, Levicky R. Molecular Mechanisms in Morpholino-DNA Surface Hybridization. J Am Chem Soc. 2010 Jun 24. [Epub ahead of print] Zhang GJ, Luo ZH, Huang MJ, Tay GK, Lim EJ. Morpholino-functionalized silicon nanowire biosensor for sequence-specific label-free detection of DNA. Biosens Bioelectron. 2010 Apr 9. [Epub ahead of print] Tercero N, Wang K, Gong P, Levicky R. Morpholino Monolayers: Preparation and Label-free DNA Analysis by Surface Hybridization. J Am Chem Soc. 2009 Mar 18. [Epub ahead of print] Wang X, Smirnov S. Label-Free DNA Sensor Based on Surface Charge Modulated Ionic Conductance. ACS Nano. 2009 Mar 13. [Epub ahead of print] Gao Z, Boon PT. A DNA biosensor based on a morpholino oligomer coated indium-tin oxide electrode and a cationic redox polymer. Analyst. 2009; [Epub ahead of print] Detecting labeled Morpholinos in tissues with an anti-fluorescein antibody:
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