Aggarwal Tushar, Wang Liming, Gutierrez Bryan, Guven Hakan, Erguven Huseyin, Cho Sarah, Izgu Enver Cagri
Department of Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ-08854, USA.
Cancer Institute of New Jersey, Rutgers University, New Brunswick, NJ-08901, USA.
Angew Chem Int Ed Engl. 2025 Mar 17;64(12):e202421936. doi: 10.1002/anie.202421936. Epub 2024 Dec 20.
Fluorescent light-up aptamer (FLAP) systems are promising (bio)sensing platforms that are genetically encodable. However, FLAP-mediated detection of each distinct target necessitates either in vitro selection or engineering of nucleic acid sequences. Furthermore, an aptamer that binds an inorganic target or a chemical species with a short lifetime is challenging to realize. Here, we describe a small-molecule approach that makes it possible for a single FLAP system to detect chemically unique, non-fluorogenic, and reactive inorganics. We developed functionalized pre-ligands of RNA aptamers that bind benzylidene imidazolinones (Baby Spinach, Broccolli, Squash). Reactive inorganics, hydrogen sulfide (HS/HS) and hydrogen peroxide (HO), can specifically convert these pre-ligands into native ligands that fluoresce with FLAPs. Adaptation of this platform to live cells opened an opportunity for constructing whole-cell sensors: Escherichia coli transformed with a Baby Spinach-encoding plasmid and incubated with pre-ligands generated fluorescence in response to exogenous HS/HS or HO. Leveraging the functional group reactivity of small molecules eliminates the requirement of in vitro selection of a new aptamer sequence or oligonucleotide scaffold engineering for distinct molecular targets. Our method allows for detecting inorganic, short-lived species, thereby advancing FLAP systems beyond their current capabilities.
荧光点亮适体（FLAP）系统是很有前景的可遗传编码的（生物）传感平台。然而，FLAP介导的对每个不同靶标的检测需要进行体外筛选或核酸序列工程。此外，要实现一种能结合无机靶标或寿命较短的化学物质的适体具有挑战性。在此，我们描述了一种小分子方法，该方法使单个FLAP系统能够检测化学性质独特、无荧光且具有反应性的无机物。我们开发了与亚苄基咪唑啉酮（如小菠菜、西兰花、南瓜）结合的RNA适体的功能化前体配体。具有反应性的无机物，硫化氢（HS⁻/H₂S）和过氧化氢（H₂O₂），能够特异性地将这些前体配体转化为能与FLAP发出荧光的天然配体。将该平台应用于活细胞为构建全细胞传感器提供了契机：用编码小菠菜的质粒转化的大肠杆菌，与前体配体一起孵育后，对外源的HS⁻/H₂S或H₂O₂产生荧光响应。利用小分子的官能团反应性消除了针对不同分子靶标进行新适体序列的体外筛选或寡核苷酸支架工程的需求。我们的方法能够检测无机的、寿命短暂的物质，从而使FLAP系统超越其当前的能力。
