Morpholino Monolayers: Preparation and Label-free DNA Analysis by Surface Hybridization

Abstract

Surface hybridization, a reaction in which nucleic acid molecules in solution react with nucleic acid partners immobilized on a surface, is widely practiced in life science research. In these applications the immobilized partner, or "probe", is typically single-stranded DNA. Because DNA is strongly charged, high salt conditions are required to enable binding between analyte nucleic acids ("targets") in solution and the DNA probes. High salt, however, compromises prospects for label-free monitoring or control of the hybridization reaction through surface electric fields, as well as stabilizes secondary structure in target species that can interfere with probe-target recognition. In this work, initial steps toward addressing these challenges are taken by introducing Morpholinos, a class of uncharged DNA analogues, for surface-hybridization applications. Monolayers of Morpholino probes on gold supports can be fabricated with methods similar to those employed with DNA, and are shown to hybridize efficiently and sequence-specifically with target strands. Hybridization-induced changes in the interfacial charge organization are analyzed with electrochemical methods and compared for Morpholino and DNA probe monolayers. Molecular mechanisms connecting surface hybridization state to the interfacial capacitance are identified and interpreted through comparison to numerical Poisson Boltzmann calculations. Interestingly, positive as well as negative capacitive responses (contrast inversion) to hybridization are possible, depending on surface populations of mobile ions as controlled by the applied potential. Quantitative comparison of surface capacitance with target coverage (targets/area) reveals a near-linear relationship, and demonstrates sensitivities (limits of quantification) in the pg mm −2 range.

Electrochemical Studies of Morpholino-DNA Surface Hybridization

“…Experimental data were taken from a prior study (17) in which 18mer single-stranded DNA targets of the sequence 5′ ATC ACT TGC AGA ATT TAA 3′ were hybridized to end-immobilized 20mer MO or DNA probes with the base composition 5′ TTT TAA ATT CTG CAA GTG AT 3′. A 0.2 mol L −1 pH 7.0 phosphate buffer served as electrolyte and hybridization medium, with no other salts added.…”

“…2A green regions) exist in a desolvated, precipitated state on the solid support. (17, 25) Ions can enter the desolvated layer under the influence of an electric field applied through the working electrode. Because probe coverage S 0 was in a range ( S 0 < ~ 5 × 10 12 cm −2 ) where consumption of probes by hybridization is expected to render the layer discontinuous,(18) patches of unhybridized probes coexist laterally with solvent-filled voids.…”

Impedimetric measurement of DNA–DNA hybridisation using microelectrodes with different radii for detection of methicillin resistant Staphylococcus aureus (MRSA)

“…In nucleic acid detection, a common approach is to use the negatively charged redox couple potassium ferri-ferrocyanide for the measurement of R CT upon DNA recognition 10 . EIS has been used to detect DNA-DNA recognition at an electrode surface and numerous biosensors have been developed using this measurement approach in concert with a wide range of electrode types [11][12][13][14] . Specifically, for the detection of nucleic acids, studies have demonstrated the successful fabrication of microelectode devices using photolithographic methods and the development of nucleic acid sensors which use EIS as the measurement technique.…”

Nucleotides and nucleic acids; oligo- and polynucleotides

“…36 Morpholino oligonucleotides have also been attached to gold surfaces for hybridisation studies; normal oligonucleotides require relatively high salt concentrations for hybridisation studies because of the negatively charged backbone, but lower salt concentrations may be used with morpholino oligonucleotides because it is a neutral modification. 37 A cationic morpholino nucleoside analogue (7) has been introduced into DNA where it was found that it was slightly destabilising at the 3'-end of a duplex, but more destabilising within the core of the duplex. 38 Incorporation of a 5'-5' inversion site within a G-quadruplex enhanced the thermal stability compared to the unmodified structure.…”

“…158 Cytidine photodimers are additionally susceptible to deamination, which leads to transition mutations, and the rate of deamination is decreased if the cytidine is methylated and increased if a flanking nucleotide is guanosine. 159 The C4'-oxidised abasic site will undergo elimination to give strand cleavage and an unsaturated aldehyde that forms adducts with cytidine on the opposite strand of a duplex (37). 160 Two of the adducts formed by the action of equine estrogen on cytosine residues have been synthesised and incorporated into DNA for NMR studies (see section 4.2).…”