news & updates Yingfu Li, PhD Associate Professor and Canada Research Chair in Directed Evolution of Nucleic Acids Department of Biochemistry and Biomedical Sciences and Department of Chemistry McMaster University - Research - People - Publications - Li Lab Relevant Publications from the Li Group Shen Y, Chiuman W, Brennan JD, Li Y. Catalysis and rational engineering of trans-acting pH6DZ1, an RNA-cleaving and fluorescence-signaling deoxyribozyme with a four-way junction structure. Chembiochem. 2006 Sep;7(9):1343-8. Elowe NH, Nutiu R, Allali-Hassani A, Cechetto JD, Hughes DW, Li Y, Brown ED. Small-molecule screening made simple for a difficult target with a signaling nucleic acid aptamer that reports on deaminase activity. Angew Chem Int Ed Engl. 2006 Aug 25;45(34):5648-52. More Publications

“If cancer stem cells lie at the heart of some cancers, then being able to predict the behavior of tumors and providing effective therapies against them means understanding the abnormal growth pathways within the stem cells themselves.”

- John Dick, Project Leader (UHN)

“The only reason that I'm willing to take a risk on this project is my confidence in the quality and camaraderie of my colleagues.”

- Scott Tanner, Principal Investigator (University of Toronto)

“We're examining DNA not for its well-recognized role as the genetic material to store and transmit genetic information, but rather for its less-known potential to act as a novel cancer diagnostic tool.”

- Yingfu Li, Principal Investigator (McMaster University)

“We're gaining more understanding of cell defects and how to modify them, so that treatment is no longer a shotgun approach, but more like an arrow aimed at a specific target.”

- Mark Minden, Principal Investigator (OCI)

yingfu li - mcmaster university

The principle task of the McMaster group, under the direction of Dr. Yingfu Li, is to develop and label aptamers that will bind with high affinity and high specificity to the suite of proteins that uniquely identify leukemic stem cells.

• The term "aptamer" is derived from the Latin aptus , meaning "to fit."

• DNA aptamers are artificial single-stranded DNA molecules with the ability to bind to a target of interest

Our job is to select a matching "key" when a specific "lock" is identified. The locks in this project are proteins; the keys are the DNA aptamers. The McMaster team (master locksmiths) will design methods to search for aptamers that have high affinity for the leukemia-specific proteins identified by John Dick's lab at the University Health Network. After the selection process is complete, the aptamers will be labeled with elemental tags (organometallic polymers) that can be detected by the ICP-MS based flow cytometer being developed by the team at the University of Toronto.

Aptamers are made in research labs using a technique called "in vitro selection" or SELEX ( S ystematic E volution of L igands by EX ponential enrichment). In vitro selection begins with an extraordinarily large library of DNA molecules containing up to 10 15 sequence variants. These DNA molecules assume a variety of unique shapes, some of which (like a key) can interact with a molecular target (like a lock). The process proceeds like this: at the beginning, there is one matching key among trillions upon trillions of mismatching keys. After each SELEX step, thousands of copies are made of the matching key (through a DNA amplification process called the polymerase chain reaction or PCR), while the mismatching keys are not copied. This process is repeated dozens of times; in the end, only the matching key (aptamer), now present in trillions of identical copies, remains. For each leukemic stem cell marker, the SELEX process can be used to identify a high affinity aptamer.

Eventually, we will develop a suite of aptamers that will uniquely identify leukemic stem cells within a complex mixture of cells (like a blood sample). The ability to identify the leukemic stem cells, and the cancer pathways that operate within them, will have applications in the diagnosis, prognosis and treatment of leukemia patients.

A new SELEX method

Figure 1: This method is called the structure-switching selection method. The aptamer library is bound to avidin-coated beads through duplexes formed between the library and biotinylated antisense DNA. The library is incubated with the target protein and those aptamers that bind to the target are eluted from the beads. DNA in solution is purified then amplified by PCR, and the amplification products are subjected to the next, more stringent, round of selection. Selection cycles continue until the final high-affinity aptamer species is identified.

Aptamers that bind to ATP or GTP - recently isolated using the structure-switching selection method

Figure 2: The top line shows the sequence of the original DNA library, where N represents mixed nucleotides (A, G, T or C) and the primer binding sites are not shown. "ATP aptamers" describes the sequences of three classes of DNA aptamers that bind ATP. "GTP aptamers" shows the sequences of one class of GTP-binding DNA aptamers. The lowercase x's represent deletions. The black letters in the central constant region represent mutations relative to the original sequence. The green letters represent the random domain; the red letters represent the central antisense-binding domain and the blue letters represent initially present fixed nucleotides.

Selection of Aptamers that Bind to CD34+ Cells

The McMaster University group has been working on generating aptamers against specific types of whole cells, a novel approach in aptamer biology. The availability of this type of reagent would be invaluable in the development of protocols for personalized diagnosis of diseases like leukemia.

Selections have been performed in the Li lab to identify aptamers that can discriminate whole leukemic stem cells from other cell types. Once stem cell-specific aptamers are isolated, they will be element tagged and used as affinity probes for cells being analyzed by the cytometric ICP-MS.

Figure 3: Gel-shift experiments for two DNA populations from CD34+ cell selection. DNA species isolated after multiple rounds of selection for binding to whole CD34+ cells are radioactively labeled and tested for interaction with crude membrane extracts from CD34+ (Kasumi-3) and CD34- (Kasumi-6) cells. In both A (DNA from the 13th round of selection) and B (DNA from the 19th round of selection), a DNA-protein complex is observed that is uniquely specific to the CD34+ extract.