Samrat Dutta

Increased heating efficiency and selective thermal ablation of malignant tissue with DNA-encased multiwalled carbon nanotubes.

Nanoparticles, including multiwalled carbon nanotubes (MWNTs), strongly absorb near-infrared (nIR) radiation and efficiently convert absorbed energy to released heat which can be used for localized hyperthermia applications. We demonstrate for the first time that DNA-encasement increases heat emission following nIR irradiation of MWNTs, and DNA-encased MWNTs can be used to safely eradicate a tumor mass in vivo. Upon irradiation of DNA-encased MWNTs, heat is generated with a linear dependence on irradiation time and laser power. DNA-encasement resulted in a 3-fold reduction in the concentration of MWNTs required to impart a 10 degrees C temperature increase in bulk solution temperature. A single treatment consisting of intratumoral injection of MWNTs (100 microL of a 500 microg/mL solution) followed by laser irradiation at 1064 nm, 2.5 W/cm(2) completely eradicated PC3 xenograft tumors in 8/8 (100%) of nude mice. Tumors that received only MWNT injection or laser irradiation showed growth rates indistinguishable from nontreated control tumors. Nonmalignant tissues displayed no long-term damage from treatment. The results demonstrate that DNA-encased MWNTs are more efficient at converting nIR irradiation into heat compared to nonencased MWNTs and that DNA-encased MWNTs can be used safely and effectively for the selective thermal ablation of malignant tissue in vivo.

PT-ACRAMTU, A Platinum-Acridine Anticancer Agent, Lengthens and Aggregates, but does not Stiffen or Soften DNA

We used atomic force microscopy (AFM) to study the dose-dependent change in conformational and mechanical properties of DNA treated with PT-ACRAMTU ([PtCl(en)(ACRAMTU-S)](NO3)2, (enxa0=xa0ethane-1,2-diamine, ACRAMTUxa0=xa01-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea. PT-ACRAMTU is the parent drug of a family of non-classical platinum-based agents that show potent activity in non-small cell lung cancer in vitro and in vivo. Its acridine moiety intercalates between DNA bases, while the platinum group forms mono-adducts with DNA bases. AFM images show that PT-ACRAMTU causes some DNA looping and aggregation at drug-to-base pair ratio (rb) of 0.1 and higher. Very significant lengthening of the DNA was observed with increasing doses of PT-ACRAMTU, and reached saturation at an rb of 0.15. At rb of 0.1, lengthening was 0.6xa0nm per drug molecule, which is more than one fully stretched base pair stack can accommodate, indicating that ACRAMTU also disturbs the stacking of neighboring base pair stacks. Analysis of the AFM images based on the worm-like chain (WLC) model showed that PT-ACRAMTU did not change the flexibility of (non-aggregated) DNA, despite the extreme lengthening. The persistence length of untreated DNA and DNA treated with PT-ACRAMTU was in the range of 49–65xa0nm. Potential consequences of the perturbations caused by this agent for the recognition and processing of the DNA adducts it forms are discussed.

Multiscale Modeling of Double-Helical DNA and RNA: A Unification through Lie Groups

Several different mechanical models of double-helical nucleic-acid structures that have been presented in the literature are reviewed here together with a new analysis method that provides a reconciliation between these disparate models. In all cases, terminology and basic results from the theory of Lie groups are used to describe rigid-body motions in a coordinate-free way, and when necessary, coordinates are introduced in a way in which simple equations result. We consider double-helical DNAs and RNAs which, in their unstressed referential state, have backbones that are either straight, slightly precurved, or bent by the action of a protein or other bound molecule. At the coarsest level, we consider worm-like chains with anisotropic bending stiffness. Then, we show how bi-rod models converge to this for sufficiently long filament lengths. At a finer level, we examine elastic networks of rigid bases and show how these relate to the coarser models. Finally, we show how results from molecular dynamics simulation at full atomic resolution (which is the finest scale considered here) and AFM experimental measurements (which is at the coarsest scale) relate to these models.

Selection of bead-displayed, PNA-encoded chemicals

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab‐on‐Bead™ library, a one‐bead, one‐sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy‐aided identification of single target‐bound beads and the extraction – wet or dry – of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target‐binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target‐binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead‐based design allows for easy screening over a variety of prepared surfaces that can extend this techniques application to the discovery of diagnostic reagents and disease markers. Copyright

PROBING αIIbβ3: LIGAND INTERACTIONS BY DYNAMIC FORCE SPECTROSCOPY AND SURFACE PLASMON RESONANCE.

The interaction between platelet integrin αIIbβ3 and fibrin(ogen) plays a key role in blood clot formation and stability. Integrin antagonists, a class of pharmaceuticals used to prevent and treat cardiovascular disease, are designed to competitively interfere with this process. However, the energetics of the integrin-drug binding are not fully understood, potentially hampering further development of this class of pharmaceuticals. We integrated dynamic force spectroscopy (DFS) and surface plasmon resonance (SPR) to probe the energetics of complex formation between αIIbβ3 and cHarGD, a cyclic peptide integrin antagonist. Analysis of αIIbβ3:cHarGD DFS rupture force data at pulling rates of 14 000 pN/s, 42 000 pN/s and 70 000 pN/s yielded koff = 0.02-0.09 s-1, a dissociation energy barrier [Formula: see text] = 22-29 kJ/mol, and a potential well width x-1 = 0.5-0.8 nm. SPR kinetic data yielded an association rate constant kon = 7 × 103 L/mol-s and a dissociation rate constant koff = 10-2 s-1, followed by a slower stabilization step (τ ~ 400 s). Both DFS and SPR detected minimal interactions between αIIbβ3 and cHarGA demonstrating a key role for electrostatic interactions between the ligand aspartate and the integrin metal ion-dependent adhesion site (MIDAS). Our work provides new insights into the energy landscape of αIIbβ3s interactions with pharmacological and physiological ligands.

Calcium increases titin N2A binding to F-actin and regulated thin filaments

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A – F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A – actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A – actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin – actin binding occurs in skeletal muscle sarcomeres in vivo.

Analysis of single, cisplatin-induced DNA bends by atomic force microscopy and simulations

Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end‐to‐end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end‐to‐end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end‐to‐end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend.

Single Molecule Study of Cis-Platin DNA Interaction

Background: Cis-platin belongs to the family of inorganic metal drugs which has a success rate of ∼ 90 % against testicular and ovarian cancer. Substantial research has been done on cisplatin-DNA interaction using various techniques including atomic force microscopy AFM, however, the exact mechanical change (e.g. bend angle and ridigidty) in DNA due to its interaction with a single molecule of cis-platin is controversial. Elucidation of these parameters will enhance our understanding of how proteins interact with cis-platin and will, thus, enhance our understanding of the successes and limitations of cis-platin.Objectives & Methods: Using statistical analysis of AFM imaging and simulations based on the worm-like-chain model we investigated the interaction of cis-platin with DNA at the single molecule level to determined the cis-platin induced DNA bend angle and change in DNA mechanical properties. We used three constructs of DNA (300 bp) with a single GG, a single AG or two, in-phase GG sites in the center. We measured the contour length (L), end to end distance(R) of DNA and DNA-cisplatin complexes, and determined the persistence length (P), bend angle β, and local flexibility around the cis-platin site.Results: R/L Distribution of the DNA-cisplatin complex showed a small but significant change when compared with the normal DNA distribution; the cisplatin-induced bend angle β was measured to be ∼38°. R/L distribution of the simulated DNA molecules matched the experimental data.Conclusions: The R/L distribution of the DNA-cisplatin complex vs normal, of AFM images and simulated DNA molecules shows that the DNA is damaged locally at the platination site while retaining its global mechanical properties. Our bend angle agrees with the X-ray and the gel-electrophoresis data further confirming the specificity of our technique in capturing the DNA-cisplatin interaction.