Enrichment technique to allow early detection and monitor emergence of KRAS mutation in response to treatment.

Sensitivity of cell-free circulating tumour DNA (ctDNA) assays is often hampered by the limited quantity of intact mutant nucleotide fragments. To overcome the issue of substrate limitation in clinical applications, we developed an enrichment method utilizing pyrrole-imidazole (PI) polyamides and their ability to bind the minor groove of B-DNA. We present here a proof-of-concept experiment to enrich specific mutant KRAS alleles with biotinylated PI polyamides. We investigated the clinical feasibility of incorporating PI polyamides to detect KRAS mutations in ctDNA from 40 colorectal cancer (CRC) patients, of whom 17 carried mutations in KRAS. After enriching ctDNA with those polyamides, we used digital PCR to detect several common KRAS codon 12 mutations. Enrichment by biotinylated PI polyamides improved the sensitivity of ctDNA analysis (88.9% vs. 11.1%, P < 0.01) in 9 non-metastatic mutation-positive patients. We observed no differences in performance for the 8 metastatic subjects (100% vs. 75%, P = 0.47). In the remaining 23/40 patients with wild type KRAS codon 12, no mutant alleles were detected with or without polyamide-facilitated enrichment. Enriching B-form of ctDNA with PI polyamides significantly improved the assay sensitivity in detecting KRAS mutations in non-metastatic CRC patient samples.

Binding of anticancer drug daunomycin to a TGGGGT G-quadruplex DNA probed by all-atom molecular dynamics simulations: additional pure groove binding mode and implications on designing more selective G-quadruplex ligands.

DNA G-quadruplex structures are emerging cancer-specific targets for chemotherapeutics. Ligands that bind to and stabilize DNA G-quadruplexes have the potential to be anti-cancer drugs. Lack of binding selectivity to DNA G-quadruplex over DNA duplex remains a major challenge when attempting to develop G-quadruplex ligands into successful anti-cancer drugs. Thorough understanding of the binding nature of existing non-selective ligands that bind to both DNA quadruplex and DNA duplex will help to address this challenge. Daunomycin and doxorubicin, two commonly used anticancer drugs, are examples of non-selective DNA ligands. In this study, we extended our early all-atom binding simulation studies between doxorubicin and a DNA duplex (d(CGATCG)2) to probe the binding between daunomycin and a parallel DNA quadruplex (d(TGGGGT)4) and DNA duplex. In addition to the end stacking mode, which mimics the mode in the crystal structure, a pure groove binding mode was observed in our free binding simulations. The dynamic and energetic properties of these two binding modes are thoroughly examined, and a detailed comparison is made between DNA quadruplex binding modes and DNA duplex binding modes. Implications on the design of more selective DNA quadruplex ligands are also discussed. Graphical abstract Top stacking and groov binding modes from the MD simulations.

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA.

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.

Docking studies on DNA intercalators.

DNA is an important target for the treatment of multiple pathologies, most notably cancer. In particular, DNA intercalators have often been used as anticancer drugs. However, despite their relevance to drug discovery, only a few systematic computational studies were performed on DNA-intercalator complexes. In this work we have analyzed ligand binding sites preferences in 63 high resolution DNA-intercalator complexes available in the PDB and found that ligands bind preferentially between G and C and between the C and A base pairs (70% and 11%, respectively). Next, we examined the ability of AUTODOCK to accurately dock ligands into preformed intercalation sites. Following the optimization of the docking protocol, AUTODOCK was able to generate conformations with RMSD values <2.00 Šwith respect to crystal structures in ∼80% of the cases while focusing on the preformed binding site (small grid box) or on the entire DNA structure (large grid box). In addition, a top ranked conformation with an RMSD < 2.00 Šwas identified in 75% and 60% of the cases using small and large docking boxes, respectively. Moreover, under the large docking box setting AUTODOCK was able to successfully distinguish between the intercalation site and the minor groove site. However, in all cases the crystal structures and poses tightly clustered around it had a lower score than the best scoring poses suggesting a potential scoring problem with AUTODOCK. A close examination of all cases where the top ranked pose had an RMSD value >2.00 Šsuggests that AUTODOCK may overemphasize the hydrogen bonding term. A decision tree was built to identify ligands which are likely to be accurately docked based on their characteristics. This analysis revealed that AUTODOCK performs best for intercalators characterized by a large number of aromatic rings, low flexibility, high molecular weight, and a small number of hydrogen bond acceptors. Finally, for canonical B-DNA structures (where preformed sites are unavailable), we demonstrated that intercalation sites could be formed by inserting an anthracene moiety between the (anticipated) site-flanking base pairs and by relaxing the structure using either energy minimization or preferably molecular dynamics simulations. Such sites were suitable for the docking of different intercalators by AUTODOCK.