Department of Biochemistry, University of Colorado, Boulder, CO 80309-0596, USA.
Nucleic Acids Res. 2020 Sep 25;48(17):e101. doi: 10.1093/nar/gkaa673.
Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.
最近在生物工程方面的努力使得使用基于 CRISPR 的方法可以更快速、廉价和灵敏地检测样本中的核酸。我们扩展了这些基于 Cas13a 的方法之一，以在单次反应中检测小分子。使用基于 SHERLOCK 的体外转录分析 (SPRINT)，体外转录的 RNA 序列特异性地触发 Cas13a 的核糖核酸酶活性。该事件激活了其非特异性核糖核酸酶活性，从而使带有猝灭剂/荧光团对标记的 RNA 寡核苷酸发生切割，从而使荧光团去猝灭。可以测量这种荧光输出以评估转录输出。利用核糖开关或蛋白质通过特定效应分子来调节转录，作为一种偶联测定，将效应物浓度转化为荧光强度。通过这种方式，我们定量了包括辅因子、核苷酸、氨基酸代谢物、四环素和单价离子在内的八种不同化合物在样品中的浓度。通过这种方式，可以在几个小时内轻松定量数百个反应。这种提高的通量还可以实现转录调节剂、抑制转录的合成化合物或其他偶联酶反应的详细表征。这些 SPRINT 反应很容易适应便携式格式，因此可用于现场或护理点情况下分析物的检测。
