Abstract
Diagnostic applications of morpholinos take advantage of their unique properties including backbone charge neutrality, a weak impact of ionic strength on their hybridization behavior, and their resistance to enzymatic degradation. This chapter overviews how these properties have advanced transduction and other capabilities useful for the analysis of nucleic acids. In many cases, the benefits stem from electrostatic mechanisms; for example, use of low ionic strengths improves sensitivity of detection while decreasing background signals because only the nucleic acid analyte is charged. While most literature reports focus on in vitro assays in buffer, morpholinos have been also used for biodistribution measurements of species such as fungal rRNA and miRNA. After reviewing the diagnostic applications of morpholinos, the chapter describes preparation of morpholino monolayers on metal supports for electrochemical diagnostics and the procedure for performing label-free detection of DNA from changes in surface capacitance.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Micklefield J (2001) Backbone modification of nucleic acids: synthesis, structure and therapeutic applications. Curr Med Chem 8:1157–1179
Summerton JE (2004) Morpholinos and PNAs compared. Lett Pept Sci 10:215–236
Wages JM, Wages GM, Matthews P, Weller D, Summerton J (1997) Affinity purification of RNA: sequence-specific capture by nonionic morpholino probes. Biotechniques 23:1116–1120
Qiao W, Kalachikov S, Liu Y, Levicky R (2013) Charge-neutral morpholino microarrays for nucleic acid analysis. Anal Biochem 434:207–214
Zu Y, Ting AL, Yi G, Gao Z (2011) Sequence-selective recognition of nucleic acids under extremely low salt conditions by using nanoparticle probes. Anal Chem 83:4090–4094
Zu Y, Ting AL, Gao Z (2011) Visualizing low-level point mutations: enzyme-like selectivity offered by nanoparticle probes. Small 7:306–310
Zu Y, Tan M-H, Chowbay B, Lee SC, Yap H, Lee MTM, Lu L-S, Chang C-P, Ying JY (2014) Nanoprobe-based genetic testing. Nano Today 9:166–171
Tercero N, Wang K, Gong P, Levicky R (2009) Morpholino monolayers: preparation and label-free DNA analysis by surface hybridization. J Am Chem Soc 131:4953–4961
Tercero N, Wang K, Levicky R (2010) Capacitive monitoring of morpholino-DNA surface hybridization: experimental and theoretical analysis. Langmuir 26:14351–14358
Gao Z, Deng H, Shen W, Ren Y (2013) A label-free biosensor for electrochemical detection of femtomolar microRNAs. Anal Chem 85:1624–1630
Gao Z, Ting BP (2009) A DNA biosensor based on a morpholino oligomer coated indium-tin oxide electrode and a cationic redox polymer. Analyst 134:952–957
Hu W, Hu Q, Li L, Kong J, Zhang X (2015) Detection of sequence-specific DNA with a morpholino-functionalized silicon chip. Anal Methods 7:2406–2412
Hu W, Ning Y, Li L, Kong J, Zhang X (2015) Highly sensitive detection of sequence-specific DNA with morpholino-functionalized magnetic microspheres. Anal Methods 7:6712–6717
Wang X, Smirnov S (2009) Label-free DNA sensor based on surface charge modulated ionic conductance. ACS Nano 3:1004–1010
Li SJ, Li J, Wang K, Wang C, Xu JJ, Chen HY, Xia XH, Huo Q (2010) A nanochannel array-based electrochemical device for quantitative label-free DNA analysis. ACS Nano 4:6417–6424
Gao H-L, Wang M, Wu Z-Q, Wang C, Wang K, Xia X-H (2015) Morpholino-functionalized nanochannel array for label-free single nucleotide polymorphisms detection. Anal Chem 87:3936–3941
Zhang GJ, Luo ZH, Huang MJ, Tay GK, Lim EJ (2010) Morpholino-functionalized silicon nanowire biosensor for sequence-specific label-free detection of DNA. Biosens Bioelectron 25:2447–2453
Martins D, Levicky R, Song Y-A (2015) Enhancing the speed of morpholino-DNA biosensor by electrokinetic concentration of DNA in a microfluidic chip. Biosens Bioelectron 72:87–94
Zhao Y, Cao L, Ouyang J, Wang M, Wang K, Xia XH (2013) Reversible plasmonic probe sensitive for pH in micro/nanospaces based on i-motif-modulated morpholino-gold nanoparticle assembly. Anal Chem 85:1053–1057
Liong M, Tassa C, Shaw SY, Lee H, Weissleder R (2011) Multiplexed magnetic labeling amplification using oligonucleotide hybridization. Adv Mater 23:H254–H257
Cao L, Zhao Y, Ji LN, Zhang J, Wang K, Xia XH (2013) The enhanced enzymolysis resistance of surface-immobilized DNA caused by hybridizing with morpholino. Electroanalysis 25:1074–1079
Burki U, Keane J, Blain A, O'Donovan L, Gait MJ, Laval SH, Straub V (2015) Development and application of an ultrasensitive hybridization-based ELISA method for the determination of peptide-conjugated phosphorodiamidate morpholino oligonucleotides. Nucleic Acid Ther 25:275–284
Cheng D, Wang Y, Liu X, Pretorius PH, Liang M, Rusckowski M, Hnatowich DJ (2010) A comparison of 18F PET and 99mTc SPECT imaging in phantoms and in tumored mice. Bioconjug Chem 21:1565–1570
Wang Y, Chen L, Liu X, Cheng D, Liu G, Liu Y, Dou S, Hnatowich DJ, Rusckowski M (2013) Detection of Aspergillus fumigatus pulmonary fungal infections in mice with 99mTc-labeled MORF oligomers targeting ribosomal RNA. Nucl Med Biol 40:89–96
Li H, Wu C, Aramayo R, Sachs MS, Harlow ML (2015) Synaptic vesicles contain small ribonucleic acids (sRNAs) including transfer RNA fragments (trfRNA) and microRNAs (miRNA). Sci Rep 5:14918
Lagendijk AK, Moulton JD, Bakkers J (2012) Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues. Biol Open 1:566–569
Schnell FJ, Crumley SL, Mourich DV, Iversen PL (2013) Development of novel bioanalytical methods to determine the effective concentrations of phosphorodiamidate morpholino oligomers in tissues and cells. Biores Open Access 2:61–66
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Levicky, R., Koniges, U., Tercero, N. (2017). Diagnostic Applications of Morpholinos and Label-Free Electrochemical Detection of Nucleic Acids. In: Moulton, H., Moulton, J. (eds) Morpholino Oligomers. Methods in Molecular Biology, vol 1565. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6817-6_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6817-6_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6815-2
Online ISBN: 978-1-4939-6817-6
eBook Packages: Springer Protocols