Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

Homogeneous,Low-volume,Efficient, and Sensitive Quantitation of Circulating Exosomal PD-L1 for Cancer Diagnosis and Immunotherapy Response Prediction

Immunotherapy has revolutionized cancer treatment, but its efficacy is severely hindered by the lack of effective predictors. Herein, we developed a homogeneous, low-volume, efficient, and sensitive exosomal programmed death-ligand 1 (PD-L1, a type of transmembrane protein) quantitation method for cancer diagnosis and immunotherapy response prediction (HOLMES-Exo_PD-L1). The method combines a newly evolved aptamer that efficiently binds to PD-L1 with less hindrance by antigen glycosylation than antibody, and homogeneous thermophoresis with a rapid binding kinetic. As a result, HOLMES-Exo_PD-L1 is higher in sensitivity, more rapid in reaction time, and easier to operate than existing enzyme-linked immunosorbent assay (ELISA)-based methods. As a consequence of an outstanding improvement of sensitivity, the level of circulating exosomal PD-L1 detected by HOLMES-Exo_PD-L1 can effectively distinguish cancer patients from healthy volunteers, and for the first time was found to correlate positively with the metastasis of adenocarcinoma. Overall, HOLMES-Exo_PD-L1 brings a fresh approach to exosomal PD-L1 quantitation, offering unprecedented potential for early cancer diagnosis and immunotherapy response prediction.     

Photo‐Tethers for the (Multi‐)Cyclic,Conformational Caging of Long Oligonucleotides

Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne‐modified photolabile nucleosides into DNA sequences, followed by a Cu^I‐catalyzed alkyne–azide cycloaddition with bis‐azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full‐length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo‐tether strategy presented here provides a robust and versatile tool for the light‐activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.