Ulf Landegren's projects in molecular techniques
Super rolling circle amplification and applications for ctDNA mutation detection
Using a technique I have developed it is now possible to locally amplify individual detected nucleic acid or protein molecules with extreme specificity to easily detected levels. The techniques offer radically new opportunities to enhance visualization in situ, obtain digital readout of multiplex biomarker assays, or clone DNA molecules at 100 % efficiency and with no need for bacterial transformation. In a longer perspective, the technique is promising for demanding detection reactions at the point of care.
I am currently using the technique to investigate the presence of tumour-specific mutant DNA in plasma from patients treated for cancer. Using flow cytometric read-out we are able to find single mutant DNA sequences in the presence of 100,000 wild-type fragments, and we can detect multiple mutations from single patient sample using a multiplex approach.
Molecular tools for analysis of drug binding characteristics
Structural similarities in active sites of drug targets lead to risks of poor selectivity and unwanted side effects in rational drug design. There is a great need for more accurate techniques to monitor selective binding and correct localization of a candidate drug and its target interaction in healthy or pathological clinical specimen in the process of drug discovery in preclinical studies.
In a first phase, we have developed very sensitive and specific in situ drug-target interaction detection methods TE-MA (Target Engagement–Mediated Amplification), where target binding by DNA-linked kinase inhibitors were visualized and quantified in cells and tissues by rolling-circle amplification (RCA) and Pharma-PLA, using the proximity ligation assays mechanism.
The methods serve to investigate selective target binding and correct localization of candidate drug in relevant clinical specimen during lead optimization in preclinical drug discovery. Another on-going effort is aimed to combine the cellular thermal shift assay (CETSA) with multiplex proximity extension assays (PEA) for quantitative drug proteins interaction analysis. Preliminarily we have developed CETSA-PEA assay in cell extracts and next aim apply this novel approach in consecutive fresh frozen samples of nucleated blood cells from leukemia patients before and after initiation of targeted therapy.
A platform for sensitive protein detection
There is a great need for protein detection at improved sensitivity, in particular since ultrasensitive protein detection greatly expands the potential ranges for biomarkers, and it may translate to earlier diagnosis of disease processes, which in turn can improve chances for successful treatment outcomes. I am developing protein assay formats that enhance specificity of detection, reduce nonspecific background, and permit strongly amplified detection signals even using standard assay formats and instrumentation readily available in hospitals and research labs.
In one approach, PlaRca, proteins are captured from biological samples via antibodies immobilized in microtiter wells. The proteins are then detected via two further antibodies that have been modified with oligonucleotides such that they can template the formation of a circular reporter DNA strand for amplified detection via rolling circle amplification.
A variant of this assay takes advantage of reagents already developed for proximity extension assays, but combines these with capture probes that permit analysis of larger sample volumes, removal of extraneous components through washes, and that increase the specificity of recognition, just as in PlaRca, via the need for triple recognition of target molecules. I demonstrate the added value of these assay formats by exploring analyses of clinically relevant, but weakly expressed biomarkers.
Precise mapping of cell signaling pathways in cells and tissues
In this project I am optimizing both PLA and PEA to measure large numbers of proteins, post-translational modifications, and protein-protein interactions in parallel in fixed cells or tissues. The goal of the project is to develop methods to screen biomarkers or to examine complex signatures of protein and modifications to better define cellular states and responses.
In addition, I also combine these molecular tools with classical biochemical assays to precisely map dynamics of cell signalling pathways and to bring new insights about how post-translational modifications and interactions are regulated. The above approaches can greatly improve opportunities to investigate cellular functions in health and disease, and in responses to experimental or established molecularly targeted therapies.
Dried blood spots for easy sample handling and RCA Reporters for simplified and improved RCA based detection assays
Capillary dried spots of blood or plasma, sampled from a finger prick offers many important advantages over venous sampling. These advantages include no need for trained personnel during sampling, no transportation regulation enabling sampling at home, and inexpensive storage of even very large biobanks and routine testing for wellness.
A major limitation with dried blood spots is the limited sample amount. Methods developed in our lab (proximity assays for protein detection) consume minute amount of sample and we have demonstrated excellent correlation between wet and dried samples also in highly multiplex protein measurement.
RCA Reporters are new tools, currently under development in our lab, for highly specific and sensitive rolling circle amplification (RCA)-based detections with a single step protocol. Preformed circular RCA templates are added to the sample together with all other necessary components. Only in the presence of specific target molecules does amplification occur that generates an easily detectable signal.
The simplicity of RCA Reporters potentially makes them suitable for point of care applications for detection of either nucleic acids or proteins. Another possible implementation of RCA Reporters is to increase the power of any RCA method simply by adding RCA Reporters to the RCA mix. This will drastically reduce incubation times, or increase the size of the original rolling circle products making them large enough to be detected by e.g. regular flow cytometers or perhaps the naked eye.
Molecular tools for sensitive point of care infectious diagnostics
The turn-around time of an infectious diagnostic test is an important parameter in controlling disease spread and choosing appropriate treatment regiments. Current molecular methods with quick turn-around times require costly equipment and skilled technicians to operate, making them unsuitable for use in low resource environments where rapid infectious diagnostics are needed. Thus, using molecular tools previously developed in our lab such as ExCirc probes and the Proximity assays, we aim to develop rapid point of care diagnostics systems for infectious diagnosis in low resource settings.
ExCirc probes are nucleic acid amplification probes that require multiple hybridization and enzymatic events to yield circular DNA molecules that can be amplified through rolling circle amplification. These probes offer increased specificity by requiring multiple recognition events while isothermal rolling circle amplification avoids the need for PCR equipment for amplification. Along with developing these probes we aim to use simplex and multiplex readout methods that do not require complex equipment and skilled technicians to analyse.