The G-quadruplex structural motif of DNA has emerged as a novel and exciting target for anticancer drug discovery. The human telomeric G-quadruplex consists of a single strand repeat of d [AGGG(TTAGGG) 3 ] that can fold into higher-order DNA structures. Small molecules that selectively target and stabilize the G-quadruplex structure(s) may serve as potential therapeutic agents and have garnered significant interest in recent years. In the work presented here, the anticancer agent, actinomycin D, is demonstrated to bind to and induce changes in both structure and stability to both the Na + and K + forms of the G-quadruplex DNA. The binding of actinomycin D to the G-quadruplex DNAs are characterized by intrinsic association constants of approximately 2 × 10 5 M −1 (strand), 2:1 molecularity, and are shown to be enthalpically driven with binding enthalpies of approximately −7 kcal/mol. The free Na + or K + forms of the quadruplex structures differ in melting temperatures by approximately 8°C (60 and 68°C, respectively), whereas both forms, when complexed with actinomycin D are stabilized with melting temperatures of approximately 79°C. The induced CD signals observed for the actinomycin D-G-quadruplex complexes may indicate that the phenoxazone ring of actinomycin D to be stacked on the G-tetrad rather than intercalated between adjacent G-tetrads. Complex formation with actinomycin D results in changes to both the Na + or K + structural isoforms to "ligand-bound" complexes having similar structural properties and stabilities.The DNA structural motif known as the G-quadruplex has recently emerged as a novel and exciting target for the discovery and design of new classes of anticancer agents (1-3). DNA sequences that can form G-quadruplex structures are found extensively throughout the genome and are located in biologically relevant regions. These sequences and their corresponding quadruplex structures were first observed to exist in telomeric regions of DNA, located at the terminal ends of chromosomes (4-6). More recently, G-quadruplex forming sequences have been mapped to the promoter regions of a number of genes and oncogenes (7)(8)(9). It is postulated that G-quadruplex structures may serve important biological functions in the regulation of gene expression (10)(11)(12). Hence, these findings have led to an increased interest in the structural and functional features of G-quadruplex structural motif and offers potentially novel targets for the development of small molecules that could selectively target and stabilize the quadruplex structure.The G-quadruplex consists of stacked G-tetrads connected by lateral, diagonal, or external loops and has been shown to possess a wide range of structural polymorphism within guanine rich sequences that exhibit the motif. Burge and coworkers and Dai and coworkers recently published extensive reviews of the topologies of quadruplex DNA structures (13,14). The observed polymorphism(s) found for G-quadruplex structures include differences in molecularity, strand orienta...

The surprising observation that a 10 residue class G* peptide from apolipoprotein J, [113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high resolution structure of [113-122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle this peptide adopts amphipathic α helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (-16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (-3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol %) in DPC micelle also results in the lowering of peptide binding enthalpy (-13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α helical structure of [113-122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1–G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na+ and K+ G-quadruplex–UP1 complexes (ΔH values of −43 and −19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na+ form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 108 M–1 (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 106 M–1 (strand)]. Circular dichroism spectroscopy reveals the Na+ form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure.

G-quadruplexes are higher order DNA structures that play significant roles in gene transcription and telomeric maintenance. The formation and stability of the G-quadruplex structures are under thermodynamic control and may be of biological significance for regulatory function of cellular processes. Here, we report the structural influence and energetic contributions of the adenine bases in the loop sequences that flank G-repeats in human telomeric DNA sequence. Spectroscopic and calorimetric techniques are used to measure the thermal stability and thermodynamic contributions to the stability of human telomeric G-quadruplexes that have been designed with systematic changes of A to T throughout the telomeric sequence. These studies demonstrate that the thermal stability of the G-quadruplex structure is directly related to the number and position of the adenines that are present in the telomeric sequence. The melting temperature (Tm) was reduced from 59 °C for the wild-type sequence to 47 °C for the sequence where all four adenines were replaced with thymines (0123TTT). Furthermore, the enthalpy required for transitioning from the folded to unfolded G-quadruplex structure was reduced by 15 kcal/mol when the adenines were replaced with thymines (37 kcal/mol for the wild-type telomeric sequence reduced to 22 kcal/mol for the sequence where all four adenines were replaced with thymines (0123TTT)). The circular dichroism melting studies for G-quadruplex sequences having a single A to T change showed significantly sloping pretransition baselines and their differential scanning calorimetry (DSC) thermograms revealed biphasic melting profiles. In contrast, the deoxyoligonucleotides having sequences with two or more A to T changes did not exhibit sloping baselines or biphasic DSC thermograms. We attribute the biphasic unfolding profile and reduction in the enthalpy of unfolding to the energetic contributions of adenine hydrogen bonding within the loops as well as the adenine stacking to the G-tetrads of the G-quadruplex structure.

Buprenorphine is a commonly prescribed medication for the treatment of opioid-use disorder. As prescriptions increase in North Carolina, buprenorphine is more frequently encountered statewide in routine postmortem casework. Between 2010 and 2018, there were 131 select cases investigated by the Office of the Chief Medical Examiner where buprenorphine was detected in peripheral blood and considered a primary cause of death, with no other opioids present and no other non-opioid substances found in the lethal range. The decedents ranged in age from 14 to 64 years, with 67% male. The mean/median peripheral blood concentrations were 4.1/2.1 ng/mL for buprenorphine and 7.8/3.4 ng/mL for the metabolite, norbuprenorphine. These postmortem blood concentrations overlap antemortem therapeutic concentrations in plasma reported in the literature for opioid-dependent subjects receiving sublingual maintenance therapy. The pathologist considered scene findings, prescription history, autopsy findings, toxicological analysis, and decedent behavior prior to death to conclude a drug-related cause of death. Many of the deaths were complicated by the presence of other central nervous system depressants along with contributory underlying cardiovascular and respiratory disease. The three most prevalent additive substances were alprazolam, ethanol, and gabapentin, found in 67, 36, and 32 cases out of 131, respectively. Interpreting buprenorphine involvement in a death is complex, and instances may be under-estimated in epidemiological data because of the lack of a defined toxic or lethal range in postmortem blood along with its good safety profile. As expansion to access of opioid-use disorder treatment becomes a priority, awareness of the challenges of postmortem interpretation is needed as increased use and diversion of buprenorphine are inevitable.