Design of experiments for the automated development of a multicellular cardiac model for high-throughput screening.

Cardiovascular toxicity remains a major cause of drug attrition in early drug development, clinical trials, and post-market surveillance. In vitro assessment of cardiovascular liabilities often relies on single cell type-based model systems coupled with functional assays, like calcium flux and multielectrode arrays. Although these models offer high-throughput capabilities and demonstrate good predictivity for functional cardiotoxicities, they fail to consider the vital contribution of non-myocyte cells, thus limiting the potential for integrated risk assessment.

A New 1,5-Disubstituted Triazole DNA Backbone Mimic with Enhanced Polymerase Compatibility.

Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the RuAAC-catalyzed synthesis of a novel 1,5-disubstituted triazole (TL) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL has superior replication kinetics to these analogues and is accurately replicated by polymerases.

"Split-and-Click" sgRNA.

CRISPR-Cas9 gene editing is dependent on a programmable single guide RNA (sgRNA) that directs Cas9 endonuclease activity. This RNA is often generated by enzymatic reactions, however the process becomes time-consuming as the number of sgRNAs increases and does not allow the incorporation of chemical modifications that can improve or expand the functionality of CRISPR. Solid-phase RNA synthesis can overcome these issues, but highly pure full-length sgRNA remains at the limits of current synthetic methods.

Dynamics of the 4D genome during in vivo lineage specification and differentiation.

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique.

Squaramides and Ureas: A Flexible Approach to Polymerase-Compatible Nucleic Acid Assembly.

Joining oligonucleotides together (ligation) is a powerful means of retrieving information from the nanoscale. To recover this information, the linkages created must be compatible with polymerases. However, enzymatic ligation is restrictive and current chemical ligation methods lack flexibility.

Squaramide-Based 5'-Phosphate Replacements Bind to the DNA Repair Exonuclease SNM1A.

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of -oxyamide and squaramide modifications at the 5'-end of oligonucleotides as phosphate replacements and their biological evaluation using the 5'-exonuclease SNM1A.

An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA.

As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs.

Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries.

DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.

Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation.

The molecular properties of the phosphodiester backbone that made it the evolutionary choice for the enzymatic replication of genetic information are not well understood. To address this, and to develop new chemical ligation strategies for assembly of biocompatible modified DNA, we have synthesized oligonucleotides containing several structurally and electronically varied artificial linkages. This has yielded a new highly promising ligation method based on amide backbone formation that is chemically orthogonal to CuAAC "click" ligation.

Studies of G-quadruplexes formed within self-assembled DNA mini-circles.

We have developed self-assembled DNA mini-circles that contain a G-quadruplex-forming sequence from the c-Myc oncogene promoter and demonstrate by FRET that the G-quadruplex unfolding kinetics are 10-fold slower than for the simpler 24-mer G-quadruplex that is commonly used for FRET experiments.

NMR Structure of a Triangulenium-Based Long-Lived Fluorescence Probe Bound to a G-Quadruplex.

An NMR structural study of the interaction between a small-molecule optical probe (DAOTA-M2) and a G-quadruplex from the promoter region of the c-myc oncogene revealed that they interact at 1:2 binding stoichiometry. NMR-restrained structural calculations show that binding of DAOTA-M2 occurs mainly through π-π stacking between the polyaromatic core of the ligand and guanine residues of the outer G-quartets. Interestingly, the binding affinities of DAOTA-M2 differ by a factor of two for the outer G-quartets of the unimolecular parallel G-quadruplex under study.

Synthesis of chemically modified DNA.

Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation, can significantly alter DNA function, and more drastic changes, such as replacement with unnatural base pairs, could expand its function. In order to realize the full potential of DNA in therapeutic and synthetic biology applications, our ability to 'write' long modified DNA in a controlled manner must be improved.

Trianguleniums as Optical Probes for G-Quadruplexes: A Photophysical, Electrochemical, and Computational Study.

Nucleic acids can adopt non-duplex topologies, such as G-quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA-M2 as a unique fluorescence probe for such studies.

The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy.

Guanine-rich oligonucleotides can fold into quadruple-stranded helical structures known as G-quadruplexes. Mounting experimental evidence has gathered suggesting that these non-canonical nucleic acid structures form in vivo and play essential biological roles. However, to date, there are no small-molecule optical probes to image G-quadruplexes in live cells.

Molecular recognition and scavenging of arsenate from aqueous solution using dimetallic receptors.

A series of copper(II), nickel(II) and zinc(II) dimetallic complexes were prepared and their affinities towards arsenate investigated. Indicator displacement assays (IDAs) were carried out to establish the complexes with best affinities towards arsenate. A di-zinc complex (3) was selected and its arsenate-binding abilities investigated by isothermal titration calorimetry (ITC).