Introduction:: Magnetic Resonance Imaging (MRI) is a powerful imaging modality that can non-invasively visualize anatomical detail of the human body with high spatial and temporal resolution. Gadolinium (Gd)-based contrast agents (CA) are widely used to enhance anatomical details in magnetic resonance imaging (MRI). Significant research has expanded the field of CAs into bioresponsive CAs by modulating the signal to image and monitor biochemical processes, such as pH. One approach to enhance the qualitative and quantitative bioresponsive MR signal and address the lack of signal sensitivity is the use of nanotechnology. DNA nanomaterials have drawn great attention due to their biocompatibility, reproducibility, and flexible functionalization. DNA possesses high binding specificity, thermodynamic stability, and can be assembled into a wide range of tailored structures. As a result, the programmability of DNA nanomaterials serves as a key characteristic that facilitates construction of an effective biosensor nanoplatform for MRI analysis. In this work, we introduce the modular, dynamic actuation mechanism of DNA-based nanostructures as a new way to modulate the MRI signal based on rotational correlation time, τR. We combined a pH-responsive oligonucleotide (i-motif) and a clinical standard CA (Gd-DOTA), to develop a pH-responsive MRI CA. The i-motif folds into a quadruplex in acidic conditions and incorporated onto larger nanoplatforms to achieve increased relaxivity, r1, compared to unbound i-motif. This approach paves a path for novel programmable, dynamic DNA-based complexes for τR-modulated bioresponsive MRI CAs.
Materials and Methods:: To tailor the sensor for actuation in a dynamic range, we chose a reported i-motif sequence which undergoes a change in folding from 5 to 95 % between pH 6.26 – 5.90.
Gd-DOTA & Oligonucleotide Coupling Preparation. Custom amine/thiol-functionalized single-strand DNA oligonucleotides were purchased from MilliporeSigma. Gd-DOTA was conjugated onto the DNA oligonucleotide via thioamide coupling on the amine terminal end of the DNA oligonucleotide. DNA-Gd-DOTA conjugates were purified with ethanol precipitation and then with Illustra NAP-10 column to remove excess Gd-p-SCN-Bn-DOTA. The conjugates were eluted in nanopure water and then lyophilized overnight.
pH Titration (1.4 T NMR). pH-responsive CAs and control CAs were dispersed in buffer at pH = 9. The pH titration was carried out by adding 1 – 3 µL increments of 0.1 M HCl. With each addition, the solution was vigorously vortexed, the pH was measured using a calibrated pH meter and T1 measurements were obtained using 1.4 T Bruker minispec mq60 NMR analyzer at 37°C. The relaxivity (r1) at each point was calculated.
T1 values were measured in the pH range of 4.5 – 7.5. Relaxivity values were obtained by using Eqn 1 and plotted against pH, fitted to a dose-response curve.
Eqn1: ri= (1/Ti-1/(Ti,o))/[CA]
Results, Conclusions, and Discussions:: From the 1.4 T NMR 37 °C, relaxivity profiles of i-motif-based CAs as a function of pH were obtained between pH 4.50 – 7.50 with a calculated. The profiles of the i-motif-based CAs were shown to be sensitive with a narrow response range. The per metal center relaxivities resulted in significantly higher values than the standard Gd-DOTA agent (∼3.11 mM–1 s–1 at 1.4 T). For the control CA, there was no significant change in r1 in vitro at 1.4 T over range of pH 4.50 – 7.50. When comparing the percent change in r1 between i-motif-based CA vs the control-CA, there is a clear increase in signal change for i-motif-based CA. In summary, the desirable characteristics of the pH-responsive i-motif-based CA for MR analysis enable another step towards translation of more bioresponsive CAs into future in vivo studies. By utilizing the rotational motion of a stimuli-responsive DNA-based i-motif, combined with an FDA approved CA, this platform presents an unparalleled opportunity to develop bioresponsive CAs for in vivo translation with ease of synthesis and tunability.
