Metallo-intercalators and metallo-insertors

Since the elucidation of the structure of double helical DNA, the construction of small molecules that recognize and react at specific DNA sites has been an area of considerable interest. In particular, the study of transition metal complexes that bind DNA with specificity has been a burgeoning field. This growth has been due in large part to the useful properties of metal complexes, which possess a wide array of photophysical attributes and allow for the modular assembly of an ensemble of recognition elements. Here we review recent experiments in our laboratory aimed at the design and study of octahedral metal complexes that bind DNA non-covalently and target reactions to specific sites. Emphasis is placed both on the variety of methods employed to confer site-specificity and upon the many applications for these complexes. Particular attention is given to the family of complexes recently designed that target single base mismatches in duplex DNA through metallo-insertion

A Mismatch-Selective Bifunctional Rhodium−Oregon Green Conjugate: A Fluorescent Probe for Mismatched DNA

A fluorescent metallointercalator conjugate that selectively targets DNA base mismatches has been synthesized by coupling an organic fluorophore to a bulky Rh intercalator containing the chrysenequinone diimine ligand. Ion pairing between the cationic Rh and anionic fluorophore moieties dramatically quenches the fluorescence of the conjugate in solution and in the presence of matched DNA. However, in the presence of mismatched DNA, the fluorescence of the conjugate is increased >300%. This increase in fluorescence is attributed to the loss in intramolecular quenching associated with DNA binding; intercalation of the Rh moiety into the mismatched site can lead to electrostatic repulsion of the anionic fluorophore away from the DNA phosphate backbone and Rh. Denaturing PAGE experiments with ^(32)P-labeled oligonucleotides indicate that the conjugate selectively binds the mismatched DNA with a binding affinity of 6 × 10^5 M^(-1) and, upon irradiation, cleaves the DNA backbone neighboring the mismatched site

DNA base mismatch detection with bulky rhodium intercalators: synthesis and applications

This protocol describes the syntheses and applications of two metallointercalators, Rh(bpy)_2(chrysi)^(3+) and Rh(bpy)_2(phzi)^(3+), that target single base mismatches in DNA. The complexes bind mismatched DNA sites specifically and, upon photoactivation, promote strand scission neighboring the mismatch. Owing to their high specificity and sequence context independence, targeting mismatches with these complexes offers an attractive alternative to current mismatch- and SNP-detection methodologies. This protocol also describes the synthesis of these complexes and their use in marking mismatched sites. Irradiation of 32P-labeled duplex DNA with either intercalator followed by denaturing PAGE allows the detection of mismatches in oligonucleotides. The protocol also outlines a method for efficient detection of single nucleotide polymorphisms (SNPs) in larger genes or plasmids. Pooled genes are denatured and re-annealed to form heteroduplexes; they are then incubated with either complex, irradiated and analyzed using capillary electrophoresis to probe for mismatches (SNP sites). The synthesis of the metallointercalators requires approximately 5–7 d. The mismatch- and SNP-detection experiments each require approximately 3 d

Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 1: Cysteine Residues and Glycans

Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates—both antibody-drug conjugates as well as constructs for in vivo imaging—has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities