Nanomolar Binding of an Antibiotic Peptide to DNA Measured with Raman Spectroscopy

Abstract

Immobilization of DNA to surfaces offers a convenient means of screening the binding affinity and selectivity of potential small-molecule therapeutic candidates. Unfortunately, most surface-sensitive methods for detecting these binding interactions are not informative of the molecular structure, information that is valuable for understanding the non-covalent interactions that stabilize binding. In this work, we report a method to meet this challenge by employing confocal Raman microscopy to quantify the association of a minor-groove-binding antimicrobial peptide, netropsin, to duplex DNA hairpin sequences immobilized on the interior surfaces of porous silica particles. To assess binding selectivity, particles functionalized with different sequences of DNA were equilibrated with solutions of 100 nM netropsin, and selective association was detected based on the presence of netropsin Raman scattering in the particles. The selectivity study revealed that netropsin binds to sequences of duplex DNA having AT-rich recognition regions. To quantify binding affinities, these AT-rich DNA sequences were equilibrated with a range of netropsin solution concentrations (1 to 100 nM). Raman scattering intensities of netropsin versus solution concentration were well described by single-binding-site Langmuir isotherms with nanomolar dissociation constants, in agreement with previous isothermal calorimetry and surface plasmon resonance results. Target sequence binding was accompanied with changes in netropsin and DNA vibrational modes consistent with the hydrogen bonding between the amide groups of netropsin and adenine and thymine bases in the DNA minor groove. The binding of netropsin to a control sequence lacking the AT-rich recognition region exhibited an affinity nearly 4 orders of magnitude weaker than found for the target sequences. The Raman spectrum of netropsin interacting with this control sequence showed broad pyrrole and amide mode vibrations at frequencies similar to a free solution, revealing less constrained conformations compared with the specific binding interactions observed with AT-rich sequences.

Raman Scattering Reveals Ion-Dependent G-Quadruplex Formation in the 15-mer Thrombin-Binding Aptamer upon Association with α-Thrombin

“…This study represents an advancement in experimental technique to understand surface-immobilized aptamers and factors that influence their conformations and reactivity. While the present study focused on an aptamer with a G-quadruplex secondary structure, the methodology could also be applied to aptamers that undergo hybridization changes in stem-loop regions upon analyte binding, where Raman scattering changes upon duplex formation are readily detected and interpreted. , Future applications would include measuring binding isotherms while identifying sites of interaction between the aptamer and its target through shifts in vibrational frequencies and intensities, as recently demonstrated for small-molecule binding to dsDNA . In addition to investigations of biosensors, this method should allow studies of the association of proteins with specific sequences of double-stranded DNA or modified DNA bases.…”

“…60,83 Future applications would include measuring binding isotherms while identifying sites of interaction between the aptamer and its target through shifts in vibrational frequencies and intensities, as recently demonstrated for smallmolecule binding to dsDNA. 61 In addition to investigations of biosensors, this method should allow studies of the association of proteins with specific sequences of double-stranded DNA or modified DNA bases. Examples would include studies of selective binding of repair enzymes to sequences that include oxidized bases, 84,85 or proteins that recognize DNA methylation and thereby regulate transcription.…”

“…This attribute allows the Raman scattering intensities to report the surface density of immobilized DNA, 59 determine the hybridization efficiency of fully and partially complementary DNA, 60 and quantify molecules from nanomolar solutions that bind to immobilized DNA strands. 61 In the present work, we apply this method to investigate the folding response of an immobilized 15-mer thrombin-binding DNA aptamer, its conformations in solutions of different metal-ion compositions, and how these metal ions impact changes in conformation upon binding of its target, α-thrombin. The thrombin-binding aptamer (TBA) is a guanine-rich sequence that forms a pair of stacked antiparallel G-quadruplexes in the presence of stabilizing cations (potassium and sodium), while in lithium-ion solutions, the aptamer remains in an unfolded state.…”

“…Because silica is a purely dielectric material and the internal surfaces of the porous material are randomly oriented, Raman scattering is independent of both the orientation and proximity of the oligonucleotide relative to the silica surface. This attribute allows the Raman scattering intensities to report the surface density of immobilized DNA, determine the hybridization efficiency of fully and partially complementary DNA, and quantify molecules from nanomolar solutions that bind to immobilized DNA strands …”

Deciphering biomolecular complexities: the indispensable role of surface-enhanced Raman spectroscopy in modern bioanalytical research

“…Exemplifying this, Myres and colleagues employed SERS to elucidate the intricate interactions between certain oncological therapeutic agents and DNA, thereby furnishing insights pivotal for comprehending drug mechanisms and refining pharmacological design. 39 In summary, the integration of SERS within nucleic acid analytics not only augments our grasp over genetic intricacies but also catalyzes advancements in disease diagnostics and therapeutics.…”