A two-armed probe for in-cell DEER measurements on proteins

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed GdIII complexes, GdIII-CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites

Corrigendum: a two-armed probe for in-cell DEER measurements on proteins (vol 26, pg 17128, 2020)

CORRIGENDUM Q. Miao, E. Zurlo, D. de Bruin, J. A. J. Wondergem, S. P. Skinner, M. Timmer, A. Blok, D. Heinrich, M. Overhand, M. Huber,* M. Ubbink* 17128–17133 A Two-Armed Probe for In-Cell DEER Measurements on Proteins Chem. Eur. J., 2020, 26 DOI: 10.1002/chem.202002743 All authors have agreed that Dr. Simon P. Skinner has made a significant contribution to this work by performing experiments and analyzing data and that his name should have been included in the list of authors. The corrected list of authors therefore reads: Dr. Qing Miao, Dr. Enrico Zurlo, Donny de Bruin, Joeri A. J. Wondergem, Dr. Simon P. Skinner, Monika Timmer, Anneloes Blok, Prof. Dr. Doris Heinrich, Dr. Mark Overhand, Dr. Martina Huber, Prof. Dr. Marcellus Ubbink The relevant affiliations for Dr. Skinner are (1) Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC Leiden, The Netherlands and (2) School of Molecular and Cellular Biology and Astbury Centre, University of Leeds, Leeds LS2 9JT, UK. The Acknowledgement section should not contain the sentence “and Dr. Simon Skinner for CLaNP5 labeled T4lys NMR data.Macromolecular Biochemistr

Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides

Abstract Background Short nucleic acid oligomers have found a wide range of applications in experimental physics, biology and medicine, and show potential for the treatment of acquired and genetic diseases. These applications rely heavily on the predictability of hybridization through Watson–Crick base pairing to allow positioning on a nanometer scale, as well as binding to the target transcripts, but also off-target binding to transcripts with partial homology. These effects are of particular importance in the development of therapeutic oligonucleotides, where off-target effects caused by the binding of mismatched sequences need to be avoided. Results We employ a novel method of probing DNA hybridization using optically active DNA-stabilized silver clusters (Ag-DNA) to measure binding efficiencies through a change in fluorescence intensity. In this way we can determine their location-specific sensitivity to individual mismatches in the sequence. The results reveal a strong dependence of the hybridization on the location of the mismatch, whereby mismatches close to the edges and center show a relatively minor impact. In parallel, we propose a simple model for calculating the annealing ratios of mismatched DNA sequences, which supports our experimental results. Conclusion The primary result shown in this work is a demonstration of a novel technique to measure DNA hybridization using fluorescent Ag-DNA. With this technique, we investigated the effect of mismatches on the hybridization efficiency, and found a significant dependence on the location of individual mismatches. These effects are strongly influenced by the length of the used oligonucleotides. The novel probe method based on fluorescent Ag-DNA functions as a reliable tool in measuring this behavior. As a secondary result, we formulated a simple model that is consistent with the experimental data

Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications

DNA-stabilized silver clusters (Ag-DNA) show excellent promise as a multi-functional nanoagent for molecular investigations in living cells. The unique properties of these fluorescent nanomaterials allow for intracellular optical sensors with tunable cytotoxicity based on simple modifications of the DNA sequences. Three Ag-DNA nanoagent designs are investigated, exhibiting optical responses to the intracellular environments and sensing-capability of ions, functional inside living cells. Their sequence-dependent fluorescence responses inside living cells include (1) a strong splitting of the fluorescence peak for a DNA hairpin construct, (2) an excitation and emission shift of up to 120 nm for a single-stranded DNA construct, and (3) a sequence robust in fluorescence properties. Additionally, the cytotoxicity of these Ag-DNA constructs is tunable, ranging from highly cytotoxic to biocompatible Ag-DNA, independent of their optical sensing capability. Thus, Ag-DNA represents a versatile live-cell nanoagent addressable towards anti-cancer, patient-specific and anti-bacterial applications

MOESM1 of Measuring DNA hybridization using fluorescent DNA-stabilized silver clusters to investigate mismatch effects on therapeutic oligonucleotides

Additional file 1: Table S1. DNA sequences used in the form of synthetic oligonucleotides in the presented DNA-DNA hybridization experiments. The designation ‘MMx’ refers to a single nucleotide mismatch on the xth location from the 5’ end of the AON sequence