A cryptophane-based “turn-on” ¹²⁹Xe NMR biosensor for monitoring calmodulin

Abstract

We present the first cryptophane-based “turn-on” 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.

Cryptophane–xenon complexes for 129Xe MRI applications

“…The eld of cryptophane biosensing has greatly evolved since the conception of the rst xenon biosensor two decades ago. We have reviewed examples of the ultrasensitive detection and monitoring of proteins, 35,41,42,45,46,55,56,61,73,78,79,92,97 ions, [47][48][49][50]82,88 nucleic acids, 51 biothiols, 54,72 and even environmental conditions such as pH 52,53,71 and temperature 89 using direct and indirect detection methods. We have highlighted instances of innovative biosensor design, such as the use of a singular moiety for both uorescence and 129 Xe MRI readout, 82 modulation of solubility aer target binding, 72 and loading up to thousands of individual cryptophane host molecules onto a single carrier.…”

“…Our laboratory recently reported the rst turn-on cryptophane biosensor, which consisted of a FRRIAR peptide conjugated to cryptophane for the detection of calmodulin (CaM). 61 Signal corresponding to bound 129 Xe was only observed in the presence of Ca 2+ -bound CaM.…”

Molecular Sensing with Host Systems for Hyperpolarized 129Xe

“…To make the biosensor more water soluble, a solubilizing linker such as 3-azidopropionic acid was treated with crude monopeptide CryA via a second CuAAC to yield Xe biosensors with two propionic acids and respective peptides in yields approximately between 40–70%. This approach was used to synthesize enantiopure trifunctionalized cryptophanes for various applications [ 206 ], including pH-dependent cell labeling of cancer cells [ 179 ], targeting integrins expressions in cells [ 127 ], sensing human carbonic anhydrase (CA) I and II enzymes [ 177 , 207 , 208 , 209 ] and monitoring Ca(II)-triggered peptide binding to calmodulin [ 178 ], respectively.…”

“… Exemplary cryptophane-A-based targeted 129 Xe biosensors discussed in this review. These biosensors were developed for investigating the following cellular targets: ( a ) folate [ 176 ] ( b ) carbonic anhydrase [ 177 ] ( c ) integrin [ 127 ] ( d ) matrix metalloproteinase-7 [ 148 ] ( e ) calmodulin [ 178 ] ( f ) cellular pH change response [ 179 ], and ( g ) glycans [ 128 ], respectively. Figures reproduced with permission from respective journal publishers.…”

<sup>129</sup>Xe Ultrafast Z-spectroscopy enables micromolar detection of biosensors on a 1T benchtop spectrometer

“…The approach of 129 Xe NMR-based biosensing using functionalized cryptophanes has yet been successfully applied in vitro for detection of small analytes (Tassali et al, 2014;Dubost et al, 2014;Jeong et al, 2015;Yang et al, 2016;Guo et al, 2016;Yang et al, 2017), of large biosystems (Wei et al, 2006;Chambers et al, 2009;Boutin et al, 2011;Rose et al, 2014;Taratula et al, 2015;Khan et al, 2015;Riggle et al, 2017;Milanole et al, 2017;Schnurr et al, 2020), or of change of physiological conditions: temperature (Schröder et al, 2008;Schilling et al, 2010) or pH (Léonce et al, 2018). To date, it has however never been used in vivo; only a proof of concept has been performed on rats using a non-functionalized cucurbituril (Hane et al, 2017).…”