I would especially like to thank Anna Arnold and Mike Grodick for their work on the. Furthermore, I would like to thank Luis Guerra, who has been a dedicated SURF and a valuable addition to the hemoglobin project.
Multiplexed Electrochemistry of DNA-bound Metalloproteins 101 Abstract
NHS-ester modified Methylene Blue Synthesis 142 Introduction
Probe-oligonucleotide synthesis and purification 153 Introduction
Multiplexed Chip Fabrication 169 Introduction
Electrochemistry as a function of assembly conditions Effect of neighboring duplex integrity for MB-dT-C8-DNA Effect of neighboring duplex integrity for MB-dT-C12-DNA. EndoIII signal accumulation as a function of time and concentration Comparison of wild type and Y82A EndoIII.
LIST OF TABLES
DNA-mediated electrochemistry
INTRODUCTION
The choice of the redox-active reporter depends on the properties one wants to investigate. Ultimately, the successful electrochemical characterization of DNA CT by theses platforms depends on the extent and specificity of the electronic coupling of the reporter to the π-stack (25).
DNA Electrochemistry with Tethered Methylene Blue
ABSTRACT
Association with the base stack has been shown to be possible by a variety of mechanisms, including intercalation (18), end capping (20), and direct conjugation to the nucleic acid base (17). MB'-DNA, covalently bound by this flexible linker, binds to the core stack by intercalation.
MATERIALS AND METHODS
Concentrated hydrochloric acid was added dropwise to the aqueous layer to adjust the pH to 5.5, then the product was extracted with ether (3 x 100 mL), dried over MgSO 4 , and the solvent was removed under reduced pressure. This correction was made by adding the extinction coefficient MBꞌ at 260 nm (10,300 M-1cm-1) to the calculated extinction coefficient for single-stranded DNA.
RESULTS
Under these assembly conditions, the percentage of residual signal was essentially equivalent regardless of position in the duplex, due to the inclusion of the CA mismatch. The area of the reduction peaks was used to quantify the size of the reduction signal (black) and to determine the percentage of residual signal ([MM]/[WM] *100) (gray) from the inclusion of a single mismatch.
Discussion
This finding suggests that while dsNB-DNA can be reduced by DNA CT alone, dsMBꞌ-DNA can be reduced either by a DNA-mediated pathway or by direct surface reduction. The trends observed in this work are consistent with the work presented here and demonstrate that DNA-mediated reduction of the MBꞌ-DNA duplex may contribute significantly to the observed signal.
Intraduplex DNA-mediated electrochemistry of covalently tethered redox-active reporters
DNA-mediated reduction of freely diffusible and covalently bound redox-active reporters has long been established on these DNA-modified electrodes (23-25). Here we demonstrate the generality of DNA-mediated reduction of covalently bound reporters by altering redox-active species and covalent linkage (Figure 3.1).
Materials and Methods
Purified amino-modified DNA was covalently modified with NHS-ester activated MB', NB' and AQ'. The same thiol-modified complementary scaffold was used for all duplex DNA to cancel out variations caused by thiol-driven DNA self-assembly.
Results and Discussion
This differential behavior of reducing and oxidizing peaks can be understood based on the low binding affinity of the reporter at. The shift in the midpoint potential of the 5' hydroxyl bond is attributed to binding via an end-capping mode as opposed to cross-linking (13). It has previously been shown that DNA-mediated reduction of MB-dT-C12-DNA is extremely sensitive to electrode assembly conditions, yielding a larger DNA-mediated signal with decreasing surface accessibility of reporters (12).
Furthermore, this demonstrates the utility of truncated dT-C8 binding as a means of decreasing surface accessibility of the reporter as it retains more DNA-mediated character under low-density conditions. The importance of neighboring duplex integrity was investigated by introducing increasing fractions of unlabeled duplex DNA with a single AC mismatch.
Conclusion
In all cases, the experimental results of incorporating mismatched DNA showed significantly less signal attenuation than predicted, consistent with a predominantly intraduplex pathway (Figure 3.11). Notably, in the case of the low-density DNA films, the effect is less than 5% of the predicted effect, suggesting that 95% of the DNA-mediated signal is generated via an intraduplex pathway. Therefore, within these films there is roughly twenty times more intraduplex reduction than interduplex reduction.
Even in the tightly packed DNA monolayers where the reduced distance between duplexes would facilitate interduplex intercalation of the reporter, the observed attenuation was still only 25% of what was predicted, indicating that intraduplex reduction was still 3 times more favored than an interduplex pathway.
DNA Sensing by Electrocatalysis with Hemoglobin
The protein shield of the heme redox center minimizes direct reduction at the electrode surface and enables assays on low-density DNA films. In addition, robust reporting by electrocatalysis depends on strict shielding of the electron sink from the electrode surface, which prevents direct reduction by the electrode and false positive results (27). Reduction of the reporter results in a reduction of the electron sink, reverting the reporter back to its oxidized form and allowing replication.
However, low-density DNA films require covalent binding of the reporter to the duplex to ensure positioning of the reporter near the top of the surface. This platform has not been widely used due to the limited solution availability of the reporter and the difficulty of imposing sufficient kinetic barriers to the reduction of ferricyanide to drive electrocatalysis.
RESULTS AND DISCUSSION
An accumulated charge of μC in the presence of freely diffusible hemoglobin binds approximately ten turns per MB-DNA. Absorption spectrum of hemoglobin (5 µM) in phosphate buffer (5 mM phosphate, 5 mM NaCl, 40 mM MgCl2, 5 mM spermidine, pH 7) before (solid) and after electrocatalysis (dotted) with MB-DNA. The sequence for both the well-matched MB-DNA (blue) and the mismatched MB-DNA (red) is shown.
The consistency in the signal suppression due to mismatch incorporation indicates that the catalytic reduction of MB-DNA occurs via DNA CT. After addition of hemoglobin, the accumulated charge after 10 seconds, measured by chronocoulometry, was examined as a function of the amount of MB DNA.
Implications
This represents a dynamic range of only three orders of magnitude; Assembly with less than 500 fmol MB-DNA results in no detectable peak from reduced MB-DNA in the CV. Amplification of the electrocatalytic signal with hemoglobin resulted in a significantly improved dynamic range, where the decrease in accumulated charge due to decreasing MB-DNA moles during monolayer formation was detectable from 500 pmol to 5 fmol, which corresponds to an extended dynamic range of five orders. of size (Figure 4.9). Electrodes were incubated with 20 µL of MB-DNA at different concentrations ranging from 25 µM to 250 pM, resulting in electrodes being exposed to 500 pmol to 5 fmol of MB-DNA during monolayer formation.
MB-DNA reduction was quantified from the reduction peak area in CV (black), without hemoglobin, and the accumulated charge after 10 seconds in chronocoulometry (red), with hemoglobin. Since electrocatalytic signal amplification by hemoglobin is a general means of improving the sensitivity of current DNA-modified electrode technologies, it should be useful for many DNA electrochemical strategies based on MB redox reporting.
Multiplexed Electrochemistry of DNA-bound Metalloproteins
Circular dichroism (CD) thermal denaturation data was fit using a non-linear least-squares regression to a simple two-state unfolding model (32), where Y is the
The electrochemistry of EndoIII on DNA-modified electrodes was determined as a function of the underlying DNA film morphology. The peak splitting for the 3+/2+ redox couple in the [4Fe-4S] cluster of EndoIII was found to be dependent on monolayer morphology, while the midpoint potential remained unchanged. This attenuation in the reduction signal due to the incorporation of perturbations to the π-stack supports the assignment of the electrochemical signal from EndoIII observed on densely packed DNA films as mediated by DNA CT.
Kinetic analysis of the signal generated by EndoIII in different DNA film morphologies is shown. The non-linearity of the signal from closely packed DNA films indicates that the signal is not diffusion rate limited.
DISCUSSION
In the case of a DNA-bound redox-active reporter, such as DNA-bound methylene blue, it has been shown that the accessibility of the redox-active moiety to the electrode surface determines whether the predominant reduction mechanism is DNA CT or direct reduction through the surface of the electrode (35). The degree of DNA CT deficiency quantified for this mutant was found to be twice as pronounced compared to previous measurements, given the overall reduced variability of the DNA-modified electrodes and, importantly, the reduced contributions of surface reduction of the [4Fe-4S] cluster due to the optimized DNA morphology. This family was found to show significant differences in DNA CT efficiency, while exhibiting very similar DNA-bound reduction potentials for the 3+/2+ redox couple of the.
For the same purposes, EndoIII mutations were prepared that reverse the electrostatics of three residues within 4 Å of the [4Fe-4S] cluster that are oriented on the opposite side of the cluster relative to the DNA. Overall, it appears that differences in DNA CT proficiency can be caused both by altering the electron transfer pathway by removing aromatics, and by more indirectly modulating the electronic coupling of the cluster by altering protein stability and probably the accessibility of solvents.
CONCLUSION
NHS-ester modified Methylene Blue Synthesis
Heat to 120 °C, collect the first 10% in the first round bottom and the rest in the second round bottom, this will be your pure product (clear/yellowish). Carboxylic acid is soluble in aqueous solvent; therefore, the reaction can be monitored by increased solubility. Carboxylic acid product is deprotonated under these basic conditions and is in the aqueous layer.
Ether is the top layer, so between each wash both the aqueous and organic layers are collected. Important: The carboxylic acid (356 g/mol), the methyl ester (370 g/mol), and the NHS ester (453 g/mol) will all be present in the MS if the activation was successful.
Probe-oligonucleotide synthesis and purification
Run a Nap5 size exclusion column on NB-modified DNA to remove small molecules prior to HPLC. Add ~420 µL phosphate buffer to move the DNA to the base of the column, but not elute it. Add 600 µl of 5.0 mM phosphate buffer to dried samples, unless it is 5′-thiol modified DNA which can be used directly from the Nap5 column.
Don't bother running assays for DMTOFF of MB and NB modified DNA on PLRPS columns as they usually don't work. Add 800 µL of phosphate buffer to elute the DNA in an HPLC vial making sure to collect the first blue band.
Multiplexed Chip Fabrication
Overall, the overall quality of the SU8 layer is the most critical component to ensure successful electrochemistry. This can be adjusted by increasing the HMDS incubation time and/or the thickness of the Ti adhesive layer. Finally, plasma cleaning chips directly prior to DNA assembly for 5 min with O2 (100 W at 1 Torr) can save the chips if Au surface quality is an issue.
Pipette the S1813 photoresist from the middle of the bottle to the center of the wafer, making sure there is enough photoresist to fully coat the features on the wafer. Pipette SU-8 2002 from the middle of the bottle onto the center of the wafer, making sure there is enough photoresist to completely coat the features on the wafer.
