More Like this

1. Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets

   https://doi.org/10.1021/acssynbio.3c00426  

   Tang, Anli A; Afasizheva, Anna; Cano, Clara T; Plath, Kathrin; Black, Douglas; Franco, Elisa (February 2024, ACS Synthetic Biology)

   NA (Ed.)

   The development of fluorescent light-up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and limited signal-to-background ratio both in vivo and in vitro. To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor’s performance for any given RNA target. To demonstrate the versatility of our design approach, we generated Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications. 

   more » « less
2. Fluorescent Platforms for RNA Chemical Biology Research

   https://doi.org/10.3390/genes13081348  

   Du, Jinxi; Dartawan, Ricky; Rice, William; Gao, Forrest; Zhou, Joseph H.; Sheng, Jia (August 2022, Genes)

   Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating. 

   more » « less
3. In Vitro Selection of an ATP-Binding TNA Aptamer

   https://doi.org/10.3390/molecules25184194  

   Zhang, Li; Chaput, John C. (September 2020, Molecules)

   Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability. 

   more » « less
4. Customizable Aptamer Transducer Network Designed for Feed-Forward Coupling

   https://doi.org/10.1021/acsomega.1c03146  

   Hachigian, Tim; Lysne, Drew; Graugnard, Elton; Lee, Jeunghoon (October 2021, ACS Omega)

   null (Ed.)

   Solution-based biosensors that utilize aptamers have been engineered in a variety of formats to detect a range of analytes for both medical and environmental applications. However, since aptamers have fixed base sequences, incorporation of aptamers into DNA strand displacement networks for feed-forward signal amplification and processing requires significant redesign of downstream DNA reaction networks. We designed a novel aptamer transduction network that releases customizable output domains, which can then be used to initiate downstream strand displacement reaction networks without any sequence redesign of the downstream reaction networks. In our aptamer transducer (AT), aptamer input domains are independent of output domains within the same DNA complex and are reacted with a fuel strand after aptamer–ligand binding. ATs were designed to react with two fluorescent dye-labeled reporter complexes to show the customizability of the output domains, as well as being used as feed-forward inputs to two previously studied catalytic reaction networks, which can be used as amplifiers. Through our study, we show both successful customizability and feed-forward capability of our ATs. 

   more » « less
5. Characterization of DNA aptamer–protein binding using fluorescence anisotropy assays in low-volume, high-efficiency plates

   https://doi.org/10.1039/D0AY02256J  

   Weaver, Simon D.; Whelan, Rebecca J. (March 2021, Analytical Methods)

   Aptamers have many useful attributes including specific binding to molecular targets. After aptamers are identified, their target binding must be characterized. Fluorescence anisotropy (FA) is one technique that can be used to characterize affinity and to optimize aptamer–target interactions. Efforts to make FA assays more efficient by reducing assay volume and time from mixing to measurement may save time and resources by minimizing consumption of costly reagents. Here, we use thrombin and two thrombin-binding aptamers as a model system to show that plate-based FA experiments can be performed in volumes as low as 2 μL per well with 20 minute incubations with minimal loss in assay precision. We demonstrate that the aptamer–thrombin interaction is best modelled with the Hill equation, indicating cooperative binding. The miniaturization of this assay has implications in drug development, as well as in the efficiency of aptamer selection workflows by allowing for higher throughput aptamer analysis. 

   more » « less