Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury Previously, we reported that fluorescent peptide dansylHPHGHWNH2 (dH3w), designed on the repeats of the human histidinerich glycoprotein, shows a turnon response to Zn(II) and a complex response to Hg(II) characterized by a turnoff phase at low Hg(II) concentrations and a turnon phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the Cterminus was changed to alanine, and AcH3w, in which the Nterminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).

Short Duplex Module Coupled to GQuadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a Gquadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dyebinding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with twomodule structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTRIII, and Tel23a G4 structures and propose that such architecture of short duplexG4 signaling elements will enforce the development of improved aptasensors.