×Iaokan LungNow a systems biologist at Harvard University, he used mass cytometry to study intracellular signaling during his graduate studies. However, although this method allows the use of metal-tagged antibodies to query up to 50 unique proteins with limited overlap, detection requires high signal generation, making it difficult to study low-abundance proteins. becomes complicated. As a postdoctoral researcher in Peng Ying’s lab, Lun and his colleagues developed a new approach that uses DNA molecules to enhance signals from these targets. technique, Amplification by circulatory expansion (ACE) is suitable for in vitro mass cytometry analysis and imaging mass cytometry (IMC) studies.1
What motivated you to develop the ACE technique?
One of the limitations of mass cytometry is its low sensitivity. Multiple copies of the antibody tag must be bound to the target to reach the detection limit, making it difficult to study low-abundance targets such as whole proteins or post-translational modification (PTM) sites. Our group previously another way Although it is a method to amplify signals from cells and tissues in situ, it was not effective for in vitro mass cytometry samples.2
What happens with this approach?
Unlike traditional mass cytometry, where a metal tag is attached directly to the antibody that binds to the target, short DNA polymers were added to the antibody over a series of amplification steps. We conjugated metal-binding detection polymers to oligonucleotide strands that recognize these extender sequences to tag these amplified DNA polymers. This process amplified the specific target signal by more than 500 times.
DNA extenders conjugated to target-specific antibodies provide more binding sites for the final detector, thus overcoming the signal-to-background problem of low-abundance targets. This not only helps identify low-abundance targets such as PTMs at the single-cell level, but also extends mass cytometry to the study of small cells that cannot be detected using traditional methods. We also showed that ACE can be applied to IMCs to overcome the limited antibody signal encountered in conventional IMCs while offering the advantage of mass cytometry that avoids autofluorescence in standard fluorescence microscopy.
To enhance signal detection of low-abundance proteins by mass cytometry, researchers developed a method called circular extension amplification (ACE). They conjugated a unique DNA oligonucleotide sequence to a target-specific antibody, incubated the sample with the DNA-bound antibody, and removed unbound proteins by washing (1). The researchers performed multiple rounds of amplification to extend the DNA strand bound to the antibody using an oligonucleotide and a polymerase containing two consecutive sequences complementary to the sequences on the DNA-bound antibody (2 ). After amplification, the researchers added a detection oligonucleotide bound to the metal ion. The sequences of these detectors were complementary to the DNA-binding antibodies. The detector also contained a nucleic acid that cross-linked with opposite adjacent bases when exposed to ultraviolet light (3). Metal ion-binding amplified DNA antibody tags provided improved signals in both suspension and imaging mass cytometry samples (4).
Adapted from Imran Chowdhury, Lun X et al.
What were the challenges in developing ACE?
One of the challenges was to prevent the DNA stretches from denaturing during the 200 degree Celsius heating step to evaporate the sample. The inclusion of a cross-linking step increased the stability of the DNA during this step. Additionally, a problem occurred during the sample introduction step. It turns out this is because the device’s tube is made of silica, which binds to DNA. Replacing this tube with non-DNA-binding plastic solved this problem.
What are the future directions for this technology in your lab?
One of our future projects in the lab is to simplify the antibody conjugation protocols we currently use and make them more accessible. We are also interested in applying this signal amplification to other methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot.
Conflict of Interest Disclosure: Xiaokang Lun, Peng ying, and another study co-author have filed a patent related to the ACE method.
