Fluorescent Intercalator Displacement Research Articles

Sensing of Different Human Telomeric G-Quadruplex DNA Topologies by Natural Alkaloid Allocryptopine Using Spectroscopic Techniques.

This article describes how a natural alkaloid allocryptopine (ALL) is able to differentiate two forms of biologically relevant human telomeric (htel22) G-quadruplex DNAs (GQ-DNA) depending on the presence of K+ and Na+ ions by steady-state and time-resolved spectroscopic techniques. For both interactions, predominant involvements of static-type quenching mechanism with the negligible influence of dynamic collision are established by UV-vis absorption and fluorescence emission study, which is further supported by fluorescence lifetime measurements. ALL exhibits appreciable affinity toward both GQ-DNAs. Both the mixed-hybrid (3 + 1) quadruplex structures in K+ ions and the basket-type antiparallel quadruplex structure under Na+ condition are converted to parallel types in the presence of ALL. Fluorescence intercalator displacement assay experiment revealed modest selectivity of ALL to both quadruplexes over duplex DNA along with higher selectivity for antiparallel types among the two quadruplexes via groove and/or loop binding, which is distinct from the conventional π-stacking of the ligands on external G-quartets. ALL stabilized both GQ-DNA topologies moderately. The differences in the dynamics of ALL within both DNA environments have been demonstrated vividly by time-resolved anisotropy measurements using the wobbling-in-cone model. These results suggest groove binding with antiparallel G-quartet with high affinity and moderate loop binding with mixed-hybrid G-quartet accompanied by the partial end stacking additionally in both of the cases. Our conclusions are further supported by steady-state anisotropy measurements and molecular docking. The present investigation can be used in the development of a biocompatible antitumour/anticancer agent targeting particular GQ-DNA conformation.

A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.

More is not always better: finding the right trade-off between affinity and selectivity of a G-quadruplex ligand.

Guanine-rich nucleic acid sequences can fold into four-stranded G-quadruplex (G4) structures. Despite growing evidence for their biological significance, considerable work still needs to be done to detail their cellular occurrence and functions. Herein, we describe an optimized core-extended naphthalene diimide (cex-NDI) to be exploited as a G4 light-up sensor. The sensing mechanism relies on the shift of the aggregate-monomer equilibrium towards the bright monomeric state upon G4 binding. In contrast with the majority of other ligands, this novel cex-NDI is able to discriminate among G4s with different topologies, with a remarkable fluorescent response for the parallel ones. We investigate this sensing by means of biophysical methods, comparing the lead compound to a non-selective analogue. We demonstrate that mitigating the affinity of the binding core for G4s results in an increased selectivity and sensitivity of the fluorescent response. This is achieved by replacing positively charged substituents with diethylene glycol (DEG) side chains. Remarkably, the limit of detection values obtained for parallel G4s are more than one order of magnitude lower than those of the parallel-selective ligand N-methyl mesoporphyrin IX (NMM). Interestingly, the classical fluorescent intercalator displacement (FID) assay failed to reveal binding of cex-NDI to G4 because of the presence a ternary complex (G4-TO-cex-NDI) revealed by electrospray-MS. Our study thus provides a rational basis to design or modify existent scaffolds to redirect the binding preference of G4 ligands.

Novel Naphthalimide Derivatives as Selective G-Quadruplex DNA Binders.

A new derivate of 4-bromo-1,8-naphthalic anhydride and its quaternized analogue have been prepared and characterized. The interactions of both derivatives with human telomere quadruplex-DNA and ds-DNA have been comparatively studied by UV-visible (UV-Vis), fluorescent intercalator displacement assays, competition dialysis, circular dichroism (CD), agarose gel electrophoresis, and polyacrylamide gel electrophoresis. The results show that both derivatives can stabilize G-quadruplexes DNA, and they show different binding affinities for G-quadruplexes-DNA and ds-DNA. All spectroscopic studies have shown that the derivatives have a modest selectivity for G-quadruplex versus ds-DNA.

Studies on interactions of carbazole derivatives with DNA, cell image, and cytotoxicity

DNA-binding agents have been considered as an established opportunity for the development of anticancer drugs and DNA fluorescence probes. This work reported the synthesis of two novel carbazole derivatives (1 and 2) and investigated their DNA binding properties, living cell image, and cytotoxicity. The results demonstrated that both compounds presented the higher binding affinity to G-quadruplex than to duplex DNA by means of UV–Vis absorption titration and fluorescent intercalator displacement. Continuous variation analysis indicated that their binding stoichiometries of the compound/G-quadruplex were near 2 except the compound/bcl-2. Circular dichroism spectra showed that both compounds had no influence on the conformation of G-quadruplexes. Fluorescence titrations indicated that 2 had the potential to be a G-quadruplex selective fluorescent probe, while 1 could be used as a fluorescent probe for duplex DNA. Confocal fluorescence images indicated that both compounds could enter the living HepG2 cells, and 1 mainly located in nucleus whereas 2 mainly distributed in cytoplasm. DNase and RNase digest tests indicated that both compounds could enter into the nucleus and interact with duplex DNA, especially, 2 could interact with RNA and/or G-quadruplex DNA. They also exhibited an obvious antiproliferative activity to HepG2 by using MTT assays, with IC50 values of 2.7 and 9.5 μM for 1 and 2, respectively.

Insight into the binding of a non-toxic, self-assembling aromatic tripeptide with ct-DNA: Spectroscopic and viscositic studies

The report describes the synthesis, self-association and DNA binding studies of an aromatic tripeptide H-Phe-Phe-Phe-OH (FFF). The peptide backbone adopts β—sheet conformation both in solid and solution. In aqueous solution, FFF self-assembles to form nanostructured aggregates. Interactions of this peptide with calf-thymus DNA (ct-DNA) have been studied using various biophysical techniques including ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The value of mean binding constant calculated from UV and fluorescence spectroscopic data is (2.914 ± 0.74) x 103M−1 which is consistent with an external binding mode. Fluorescence intercalator displacement (FID) assay, iodide quenching study, viscosity measurement and thermal denaturation study of DNA further confirm the groove binding mode of peptide, FFF with ct-DNA. MTT cell survival assay reveals very low cytotoxicity of the peptide toward human lung carcinoma cell line A549.

Platinum(II) and palladium(II) complexes of tridentate hydrazone-based ligands as selective guanine quadruplex binders

Several tridentate hydrazone-based ligands, synthesized by a condensation reaction of either 2-(1-methylhydrazinyl)pyridine or 2-(1-methylhydrazinyl)quinoline with an aldehyde (picolinaldehyde, 1H-pyrrole-2-carbaldehyde, 2-mercaptobenzaldehyde, 2-aminobenzaldehyde) have been reacted with palladium(II) and platinum(II) salts and precursor complexes to yield nine new metal complexes. These planar complexes were investigated with respect to their capability to interact with guanine quadruplex DNA. Two sequences (H-telo, derived from the human telomeric sequence, and c-myc, representing an excerpt of the promoter region of the c-Myc oncogene) were investigated. Circular dichroism (CD) spectroscopy, temperature-dependent CD spectroscopy, UV–Vis spectroscopy and a fluorescent intercalator displacement (FID) assay were applied in this respect. The spectroscopic data show that the complexes indeed interact with guanine quadruplex DNA. According to the FID assay, some of the complexes belong to the highest-affinity metal-containing quadruplex binders reported so far. Their affinity towards quadruplex DNA is up to 80-fold higher than to a reference double helix. These findings make the metal complexes good candidates as anticancer drugs, as guanine quadruplexes have been proposed as potential targets in anticancer drug design.

Inducement and stabilization of G-quadruplex DNA by a thiophene-containing dinuclear ruthenium(II) complex

G-quadruplex structures are attractive targets for the development of anticancer drugs, as their formation in human telomere could impair telomerase activity, thus inducing apoptosis in cancer cells. In this work, a thiophene-containing dinuclear ruthenium(II) complex, [Ru2(bpy)4(H2bipt)]4+ {bpy = 2,2′-bipyridine, H2bipt = 2,5-bis[1,10]phenanthrolin[4,5-f]-(imidazol-2-yl)thiophene}, was prepared and the interaction between the complex and human telomeric DNA oligomers 5′-G3(T2AG3)3-3′ (HTG21) has been investigated by UV-Vis, fluorescence and circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assay, polymerase chain reaction (PCR) stop assay, fluorescent intercalator displacement (FID) titrations, Job plot and color reaction studies. The results indicate that the complex can well induce and stabilize the formation of antiparallel G-quadruplex of telomeric DNA in the presence or absence of metal cations, and the ΔTm value of the G-quadruplex DNA treated with the complex was obtained to be 12.8 °C even at levels of 50-fold molar of duplex DNA (calf-thymus DNA), suggesting that the complex exhibits higher G-quadruplex DNA selectivity over duplex DNA. The complex shows high interaction ability with G-quadruplex DNA at (1.17 ± 0.12) × 107 M−1 binding affinity using a 2:1 [complex]/[quadruplex] binding mode ratio. A novel visual method has been developed here for making a distinction between G-quadruplex DNA and duplex DNA by our ruthenium complex binding hemin to form the hemin-G-quadruplex DNAzyme.

Arylsulfanyl groups - Suitable side chains for 5-substituted 1,10-phenanthroline and nickel complexes as G4 ligands and telomerase inhibitors

Guanine-rich DNA sequences can undergo self-assembly into unique G-quadruplex structures that interfere with the binding of proteins to the same DNA region. The formation of DNA G-quadruplexes requires monovalent cations (Na+ and K+) or small molecules known as G-quadruplex (G4) ligands. Phenanthroline is a type of G4 ligand scaffold known for its coordination with metal ions to form complexes with a large aromatic surface area, which aptly stack with G-quartets. In this report, we have investigated the side chain effect on G-quadruplex recognition by evaluating a series of 5-substituted phenanthroline-based metal complexes (Phen-Ni) binding to telomeric G-quadruplex DNA. Results from biophysical methods including fluorescence and circular dichroism (CD) thermal denaturation, CD titration, and the fluorescent intercalator displacement (FID) assay suggest that several Phen-Ni complexes bind to G-quadruplex DNA with submicromolar G4DC50 values. Arylsulfanyl groups at the 5 position of 1,10-phenanthroline are the best side chains regarding binding affinity and selectivity towards G-quadruplex DNA. Most of the G-quadruplex binding Phen-Ni complexes can inhibit telomerase activity in vitro as indicated by the telomeric repeat amplification protocol (TRAP) assay and such inhibition is clearly concentration dependent. Our results here provide a guidance of utilizing 5-substituted phenanthroline derivatives as a viable and facile approach to design novel G4 ligands.

Copper(I)-Phosphine Polypyridyl Complexes: Synthesis, Characterization, DNA/HSA Binding Study, and Antiproliferative Activity.

A series of copper(I)-phosphine polypyridyl complexes have been investigated as potential antitumor agents. The complexes [Cu(PPh3)2dpq]NO3 (2), [Cu(PPh3)2dppz]NO3 (3), [Cu(PPh3)2dppa]NO3 (4), and [Cu(PPh3)2dppme]NO3 (5) were synthesized by the reaction of [Cu(PPh3)2NO3] with the respective planar ligand under mild conditions. These copper complexes were fully characterized by elemental analysis, molar conductivity, FAB-MS, and NMR, UV-vis, and IR spectroscopies. Interactions between these copper(I)-phosphine polypyridyl complexes and DNA have been investigated using various spectroscopic techniques and analytical methods, such as UV-vis titrations, thermal denaturation, circular dichroism, viscosity measurements, gel electrophoresis, and competitive fluorescent intercalator displacement assays. The results of our studies suggest that these copper(I) complexes interact with DNA in an intercalative way. Furthermore, their high protein binding affinities toward human serum albumin were determined by fluorescence studies. Additionally, cytotoxicity analyses of all complexes against several tumor cell lines (human breast, MCF-7; human lung, A549; and human prostate, DU-145) and non-tumor cell lines (Chinese hamster lung, V79-4; and human lung, MRC-5) were performed. The results revealed that copper(I)-phosphine polypyridyl complexes are more cytotoxic than the corresponding planar ligand and also showed to be more active than cisplatin. A good correlation was observed between the cytostatic activity and lipophilicity of the copper(I) complexes studied here.

Indenocinnoline derivatives as G-quadruplex binders, topoisomerase IIα inhibitors and antiproliferative agents

DNA intercalating agents are a consolidated therapeutic option in the treatment of tumor diseases. Starting from previous findings in the antiproliferative efficacy of a series of indeno[1,2-c]cinnoline-11-one derivatives, we performed a suitable decoration of this scaffold by means of a simple and straightforward chemistry, aiming to a) enlarge the planar core to a pentacyclic benzo[h]indeno[1,2-c]cinnoline-13-one and b) introduce a basic head tethered through a simple polymethylene chain. In fluorescence melting and fluorescence intercalator displacement assays, these new compounds displayed fair to very good intercalating properties on different nucleic acid strands, with preference for G-quadruplex sequences. Inhibition of human topoisomerase IIα and antiproliferative assays on HeLa and MCF7 tumor cell lines outlined a multitarget antiproliferative profile for tetracyclic 6 and pentacyclic derivative 20, both bearing a N,N-dimethylamine as the protonatable moiety. Particularly, compound 6 displayed a very potent inhibition of tumor cell proliferation, while 20 returned the highest thermal stabilization in melting experiments. In summary, these results outlined a potential of such highly planar scaffolds for nucleic acid binding and antiproliferative effects.

Identification of new DNA i-motif binding ligands through a fluorescent intercalator displacement assay.

i-Motifs are quadruplex DNA structures formed from sequences rich in cytosine and held together by intercalated, hemi-protonated cytosine-cytosine base pairs. These sequences are prevalent in gene promoter regions and may play a role in gene transcription. Targeting these structures with ligands could provide a novel way to target genetic disease but there are very few ligands which have been shown to interact with i-motif DNA. Fluorescent intercalator displacement (FID) assays are a simple way to screen ligands against DNA secondary structures. Here we characterise how thiazole orange interacts with i-motif DNA and assess its ability for use in a FID assay. Additionally, we report FID-based ligand screening using thiazole orange against the i-motif forming sequence from the human telomere to reveal new i-motif binding compounds which have the potential for further development.

G-quadruplex fluorescence sensing by core-extended naphthalene diimides

BackgroundFluorescent sensing of G-quadruplex nucleic acids (G4s) is an effective strategy to elucidate their role in vitro and in vivo. Small molecule ligands have often been exploited, producing an emission light up upon binding. Naphthalene diimides (NDIs), although potent G4 binders exhibiting red-NIR fluorophores, have only been marginally exploited, as they are usually quenched upon binding. Contrary, aggregating core-extended naphthalene diimides (cex-NDIs) proved to be effective probes. MethodsWe prepared a library of eighteen cex-NDIs by organic synthesis, characterising their aggregation-dependent absorption and emission properties. Absorption and emission titrations, fluorescent intercalator displacement assay (FID) and circular dichroism (CD) analysis were performed to elucidate their behavior as G4 fluorescent sensors, selectivity and binding mode. Resultscex-NDIs aggregate under aqueous solvents and as a result, their fluorescence is mostly quenched under physiological conditions. Upon G4 binding, they disaggregate into binding monomers, producing a fluorescent light-up with anti-parallel and hybrid G4s. Contrary, with parallel G4s a light-off was recorded. For the formers a groove-like interaction was inferred by ICD signals, while for the latter an end-stacking interaction mode was hypothesized by G4-FID data. Conclusionscex-NDIs G4 sensing mechanism works via a induced disaggregation. The emission response depends on the G4 topology, which dictates the prevailing -groove or end-stacking- binding mode. General significanceThis study highlights the potential of cex-NDIs as G4 fluorescent probes. Besides being readily synthesized and conveniently emitting above 600nm, they light-up upon binding to anti-parallel and hybrid G4, complementing a number of other probes' selectivity for the parallel topology. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Hit Identification of a Novel Dual Binder for h-telo/c-myc G-Quadruplex by a Combination of Pharmacophore Structure-Based Virtual Screening and Docking Refinement.

It is well known that G-quadruplexes are targets of great interest for their roles in crucial biological processes, such as aging and cancer. Hence, a promising strategy for anticancer drug therapy is the stabilization of these structures by small molecules. We report a high-throughput in silico screening of commercial libraries from several different vendors by means of a combined structure-based pharmacophore model approach followed by docking simulations. The compounds selected by the virtual screening procedure were then tested for their ability to interact with human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and fluorescence intercalator displacement. Our approach resulted in the identification of a 13-[(dimethylamino)methyl]-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one derivative as a novel promising stabilizer of G-quadruplex structures within the human telomeric and the c-myc promoter sequences.

Characterization of Nucleic Acid Binding by the Histone-Derived Antimicrobial Peptides Buforin II and DesHDAP1

Antimicrobial peptides (AMPs), which are found in numerous living organisms, are active against a wide range of bacteria and other pathogens, making them promising candidates for alternatives to conventional antibiotics. While many AMPs inhibit bacterial growth through membrane disruption, some AMPs, including buforin II and DesHDAP1, are hypothesized to kill bacteria by binding to intracellular nucleic acids after translocating across the bacterial cell membrane without significant membrane permeabilization. To understand this lesser known mechanism, the peptide-nucleic acid binding interactions of these systems were investigated using several experimental and computational methods. The peptide-nucleic acid binding for buforin II and DesHDAP1 were measured experimentally using a fluorescence intercalator displacement (FID) assay. In these experiments, having an increased composition of basic residues that are arginine versus lysine were shown to promote binding for variants of both buforin II and DesHDAP1. When binding was tested using different combinations of DNA sequences, neither buforin II nor DesHDAP1 significantly favored particular DNA sequences. To provide structural explanations for experimental results, molecular dynamics (MD) simulations, pKa calculations, electrostatics calculations via component analysis were used. Using these methods, particular arginine residues within each peptide were found to interact more favorably with DNA. Results from MD simulations also showed that nucleic acid-peptide binding was mostly due to interactions between the peptide and phosphate backbone of nucleic acids, providing an explanation for the lack of sequence specificity observed experimentally. These insights regarding nucleic acid binding of buforin II and DesHDAP1, paired with a deeper understanding of the peptides’ structures and membrane interactions, are necessary for development of novel pharmaceutical applications using AMPs.

Selective recognition of c-MYC G-quadruplex DNA using prolinamide derivatives.

Herein we report the design, synthesis, biophysical and biological evaluation of triazole containing prolinamide derivatives as selective c-MYC G-quadruplex binding ligands. A modular synthetic route has been devised for prolinamide derivatives using a copper(i) catalyzed azide-alkyne cycloaddition (CuAAC). The Förster resonance energy transfer (FRET) melting assay indicates that prolinamide trimers can significantly stabilize G-quadruplex structures over duplex DNA compared to prolinamide dimers. The fluorescent intercalator displacement (FID) assay shows that a trimer with prolinamide side chains at the para-position of the benzene ring can discriminate between different quadruplex structures and exhibits the highest binding affinity towards the c-MYC G-quadruplex structure. Molecular modeling studies reveal that the prolinamide trimer stacks upon the terminal G-quartet of the c-MYC G-quadruplex. Atomic force microscopy (AFM) analysis reveals that the tris-prolinamide ligand can be used to regulate the assembly of novel supramolecular nanoarchitectures. Further, in vitro cellular studies with human hepatocellular carcinoma (HepG2) cells indicate that the tris-prolinamide derivatives can inhibit cell proliferation and reduce c-MYC expression in cancer cells.

Quadruplex DNA-Stabilising Dinuclear Platinum(II) Terpyridine Complexes with Flexible Linkers.

Four dinuclear terpyridineplatinum(II) (Pt-terpy) complexes were investigated for interactions with G-quadruplex DNA (QDNA) and duplex DNA (dsDNA) by synchrotron radiation circular dichroism (SRCD), fluorescent intercalator displacement (FID) assays and fluorescence resonance energy transfer (FRET) melting studies. Additionally, computational docking studies were undertaken to provide insight into potential binding modes for these complexes. The complexes demonstrated the ability to increase the melting temperature of various QDNA motifs by up to 17 °C and maintain this in up to a 600-fold excess of dsDNA. This study demonstrates that dinuclear Pt-terpy complexes stabilise QDNA and have a high degree of selectivity for QDNA over dsDNA.

Interaction of metallacrown complexes with G-quadruplex DNA

Interactions of the G-quadruplex (GQ) DNA with two pentacoordinate lanthanide (III) metallacrown (MC) complexes containing phenylalanine hydroxamic acid (pheHA) and copper(II) ions of the formula Eu 15-[MCCu,pheHA]-5 (1) and Tb 15-[MCCu,pheHA]-5 (2) were investigated. Binding of both metallacrowns to human telomeric G-quadruplex DNA was followed using CD spectroscopy, DNA melting profiles, and fluorescent intercalator displacement (FID) assay. A new G-quadruplex binding assay based of quenching of Tb(III)–GQ luminescence was proposed and evaluated. All performed tests confirmed interactions of MCs with studied GQ structure. Binding affinities of MCs were appreciable (KMC ~2–5×105M−1). Higher concentration of MCs (the ratio of GQ:MC above 2.5) caused destabilization of tetraplex structure of GQ as evidenced by CD spectroscopy, melting temperatures, and Tb(III)–GQ luminescence quenching results.

Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay.

This work was the first to report that the kanamycin-binding DNA aptamer (5′-TGG GGG TTG AGG CTA AGC CGA-3′) can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA–TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Effects of Cationic Residues and Base Sequence in Nucleic Acid Binding of Histone-Derived Antimicrobial Peptides

Antimicrobial peptides (AMPs), which are found in numerous living organisms, are active against a wide range of bacteria and other pathogens. While many AMPs inhibit bacterial growth through membrane disruption, certain AMPs, including buforin II and DesHDAP1, are hypothesized to kill bacteria by binding to intracellular nucleic acids after translocating across the bacterial cell membrane. To understand this lesser known mechanism, the nucleic acid-peptide binding interactions of these systems were investigated using both experimental and computational methods. The nucleic acid-peptide binding for buforin II and DesHDAP1 were measured experimentally using a fluorescence intercalator displacement (FID) assay. In these experiments, having an increased composition of basic residues that are arginine versus lysine were shown to promote binding for variants of both buforin II and DesHDAP1. When binding was tested using nucleic acid sequences, neither buforin II nor DesHDAP1 significantly favored particular DNA or RNA sequences. To provide structural explanations for these results, molecular dynamics (MD) simulations and electrostatics calculations were used. Through these computational analyses, particular arginine residues within each peptide were found to interact more favorably with nucleic acids. Results from MD simulations also showed that nucleic acid-peptide binding was mostly due to interactions between the peptide and phosphate backbone of nucleic acids. Since these phosphate groups are identical for any DNA or RNA sequence, the prevalence of those interactions explained the lack of sequence specificity observed experimentally. These insights regarding nucleic acid binding of buforin II and DesHDAP1, paired with a deeper understanding of the peptides’ structures and membrane interactions, are necessary to develop AMPs for novel pharmaceutical applications.