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.

FRETBased Aptasensor for the Selective and Sensitive Detection of Lysozyme Lysozyme is a conserved antimicrobial enzyme and has been cited for its role in immune modulation. Increase in lysozyme concentration in body fluids is also regarded as an early warning of some diseases such as Alzheimerxe2x80x99s, sarcoidosis, Crohnxe2x80x99s disease, and breast cancer. Therefore, a method for a sensitive and selective detection of lysozyme can benefit many different areas of research. In this regard, several aptamers that are specific to lysozyme have been developed, but there is still a lack of a detection method that is sensitive, specific, and quantitative. In this work, we demonstrated a singlemolecule fluorescence resonance energy transfer (smFRET)based detection of lysozyme using an aptamer sensor (also called aptasensor) in which the binding of lysozyme triggers its conformational switch from a lowFRET to highFRET state. Using this strategy, we demonstrated that the aptasensor is sensitive down to 2.3 picomoles (30 nM) of lysozyme with a dynamic range extending to 2 xc2xb5M and has little to no interference from similar biomolecules. The smFRET approach used here requires a dramatically small amount of aptasensor (3000fold less as compared to typical bulk fluorescence methods), and it is cost effective compared to enzymatic and antibodybased approaches. Additionally, the aptasensor can be readily regenerated in situ via a process called toehold mediated strand displacement (TMSD). The FRETbased aptasensing of lysozyme that we developed here could be implemented to detect other protein biomarkers by incorporating proteinspecific aptamers without the need for changing fluorophorelabeled DNA strands.