Communication pubs.acs.org/JACS
DNA “Nano-Claw”: Logic-Based Autonomous Cancer Targeting and Therapy Mingxu You,†,‡ Lu Peng,‡ Na Shao,‡ Liqin Zhang,‡ Liping Qiu,† Cheng Cui,‡ and Weihong Tan*,†,‡ †
Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China ‡ Department of Chemistry and Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Shands Cancer Center, UF Genetics Institute, McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, United States S Supporting Information *
Some AND-gate-based bireceptor-targeting methods have been recently demonstrated, including the utilization of a proximity-based ligation probe,6 bispecific antibodies,7 chimeric costimulatory antigen receptors,8 and a logic-gated DNA origami robot.9 However, considering the large population of similar cells in biological systems, a reliable approach capable of examining more complex cellular configurations is still needed. DNA has been widely used to construct devices performing intelligent tasks, such as sensing10 and computation.9,11−17 Among these, aptamers are DNA/RNA strands that are able to selectively recognize a wide range of targets, from small organic/ inorganic molecules to proteins.18−21 Recently, a panel of aptamers has been selected for cell membrane proteins using a process called cell-SELEX.22 These aptamers demonstrate the ability to identify different expression patterns of the membrane receptors in a variety of cell types.18 Our goal is to use aptamers as building blocks for a molecularly assembled logic robot, called the “Nano-Claw”, which can recognize the expression levels of multiple cell membrane markers and autonomously induce therapeutic operations. Structurally, this logic robot consists of an oligonucleotide backbone as the scaffold,23,24 several structure-switchable aptamers as “capture toes”, and a logic-gated DNA duplex as the “effector toe” (Figure 1). The “capture toes” have two functions: first targeting each cell-surface marker and then generating the respective barcode oligonucleotide for activation of the “effector toe”. Finally, the “effector toe” analyzes these barcode oligonucleotides and autonomously makes decisions in generating a diagnostic signal (such as fluorescence) and therapeutic effect. The functions of the “capture toes” were achieved on the basis of structure-switchable aptamers.25,26 In the absence of target, the aptamer binds to a piece of complementary DNA (cDNA) to form a duplex structure. However, when a target is introduced, the structures are induced to switch from aptamer/cDNA duplex to aptamer/target complex and release the cDNA as an output.10,25 Three aptamers, Sgc8c, Sgc4f, and TC01, were chosen to respectively target three overexpressed markers (PTK7 for Sgc8c; Sgc4f and TC01 targets not yet identified) on the surface of cancer cells, such as human acute lymphoblastic leukemia cells (CCRF-CEM). Several 11−19 nt-long candidate
ABSTRACT: Cell types, both healthy and diseased, can be classified by inventories of their cell-surface markers. Programmable analysis of multiple markers would enable clinicians to develop a comprehensive disease profile, leading to more accurate diagnosis and intervention. As a first step to accomplish this, we have designed a DNAbased device, called “Nano-Claw”. Combining the special structure-switching properties of DNA aptamers with toehold-mediated strand displacement reactions, this claw is capable of performing autonomous logic-based analysis of multiple cancer cell-surface markers and, in response, producing a diagnostic signal and/or targeted photodynamic therapy. We anticipate that this design can be widely applied in facilitating basic biomedical research, accurate disease diagnosis, and effective therapy.
A
t the boundaries of eukaryotic cells, different cell-surface receptors, together with other proteins, lipids, and carbohydrates, function in cascade and participate in the communication between the cell and the outside world. However, in cancer cells, alterations in the expression level and/or function of the cell membrane receptors can lead to systemic dysfunction, such as aberrant cellular metabolism, signaling and proliferation.1,2 In recent decades, advances in biomedicine have expanded our knowledge of the molecular signatures of diseases. By profiling the high or low expression levels of multiple membrane markers, medical practitioners will more precisely target diseased cells and provide more accurate disease therapy. Most identified cell-surface markers are not exclusively expressed on the target population of diseased cells. A marker overexpressed in cancer cells is often also expressed at a lower level in some normal cells. In diseases such as leukemia, both healthy and diseased subpopulations of white blood cells display surface markers that are indistinguishable by the current singlereceptor antibody therapy, potentially leading to serious complications and even death by the indiscriminate invasion of the host defense system.3 In comparison, a more practical and less risk-prone approach would simultaneously assess multiple surface receptors to pinpoint specific disease cells and enhance diagnostic accuracy in differentiating similar cells.4,5 © 2013 American Chemical Society
Received: November 16, 2013 Published: December 24, 2013 1256
dx.doi.org/10.1021/ja4114903 | J. Am. Chem. Soc. 2014, 136, 1256−1259
Journal of the American Chemical Society
Communication
Figure 1. Symbols and construction schemes are shown for (a) twoinput trivalent “Y”-shaped Nano-Claw and (b) three-input tetravalent “X”-shaped Nano-Claw. (c) Flow cytometry experiment to determine the best cDNA sequences with a high Cy5.5 fluorescence signal (from biotin-labeled TC01, Sgc4f or Sgc8c aptamer) and low FITC fluorescence signal (labeled on the candidate strands).
Figure 2. (a) Experimental scheme of aptamer-switch AND gate. (b) Adjusting the gating properties by changing the DNA sequence design, CEM and HeLa cells can be either dually targeted or distinguished. (c) Cell viability test for the AND and INH gates after visible irradiation for 3 h and subsequent growth for 48 h (*: p-value
DNA “Nano-Claw”: Logic-Based Autonomous Cancer Targeting and Therapy Mingxu You,†,‡ Lu Peng,‡ Na Shao,‡ Liqin Zhang,‡ Liping Qiu,† Cheng Cui,‡ and Weihong Tan*,†,‡ †
Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China ‡ Department of Chemistry and Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Shands Cancer Center, UF Genetics Institute, McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, United States S Supporting Information *
Some AND-gate-based bireceptor-targeting methods have been recently demonstrated, including the utilization of a proximity-based ligation probe,6 bispecific antibodies,7 chimeric costimulatory antigen receptors,8 and a logic-gated DNA origami robot.9 However, considering the large population of similar cells in biological systems, a reliable approach capable of examining more complex cellular configurations is still needed. DNA has been widely used to construct devices performing intelligent tasks, such as sensing10 and computation.9,11−17 Among these, aptamers are DNA/RNA strands that are able to selectively recognize a wide range of targets, from small organic/ inorganic molecules to proteins.18−21 Recently, a panel of aptamers has been selected for cell membrane proteins using a process called cell-SELEX.22 These aptamers demonstrate the ability to identify different expression patterns of the membrane receptors in a variety of cell types.18 Our goal is to use aptamers as building blocks for a molecularly assembled logic robot, called the “Nano-Claw”, which can recognize the expression levels of multiple cell membrane markers and autonomously induce therapeutic operations. Structurally, this logic robot consists of an oligonucleotide backbone as the scaffold,23,24 several structure-switchable aptamers as “capture toes”, and a logic-gated DNA duplex as the “effector toe” (Figure 1). The “capture toes” have two functions: first targeting each cell-surface marker and then generating the respective barcode oligonucleotide for activation of the “effector toe”. Finally, the “effector toe” analyzes these barcode oligonucleotides and autonomously makes decisions in generating a diagnostic signal (such as fluorescence) and therapeutic effect. The functions of the “capture toes” were achieved on the basis of structure-switchable aptamers.25,26 In the absence of target, the aptamer binds to a piece of complementary DNA (cDNA) to form a duplex structure. However, when a target is introduced, the structures are induced to switch from aptamer/cDNA duplex to aptamer/target complex and release the cDNA as an output.10,25 Three aptamers, Sgc8c, Sgc4f, and TC01, were chosen to respectively target three overexpressed markers (PTK7 for Sgc8c; Sgc4f and TC01 targets not yet identified) on the surface of cancer cells, such as human acute lymphoblastic leukemia cells (CCRF-CEM). Several 11−19 nt-long candidate
ABSTRACT: Cell types, both healthy and diseased, can be classified by inventories of their cell-surface markers. Programmable analysis of multiple markers would enable clinicians to develop a comprehensive disease profile, leading to more accurate diagnosis and intervention. As a first step to accomplish this, we have designed a DNAbased device, called “Nano-Claw”. Combining the special structure-switching properties of DNA aptamers with toehold-mediated strand displacement reactions, this claw is capable of performing autonomous logic-based analysis of multiple cancer cell-surface markers and, in response, producing a diagnostic signal and/or targeted photodynamic therapy. We anticipate that this design can be widely applied in facilitating basic biomedical research, accurate disease diagnosis, and effective therapy.
A
t the boundaries of eukaryotic cells, different cell-surface receptors, together with other proteins, lipids, and carbohydrates, function in cascade and participate in the communication between the cell and the outside world. However, in cancer cells, alterations in the expression level and/or function of the cell membrane receptors can lead to systemic dysfunction, such as aberrant cellular metabolism, signaling and proliferation.1,2 In recent decades, advances in biomedicine have expanded our knowledge of the molecular signatures of diseases. By profiling the high or low expression levels of multiple membrane markers, medical practitioners will more precisely target diseased cells and provide more accurate disease therapy. Most identified cell-surface markers are not exclusively expressed on the target population of diseased cells. A marker overexpressed in cancer cells is often also expressed at a lower level in some normal cells. In diseases such as leukemia, both healthy and diseased subpopulations of white blood cells display surface markers that are indistinguishable by the current singlereceptor antibody therapy, potentially leading to serious complications and even death by the indiscriminate invasion of the host defense system.3 In comparison, a more practical and less risk-prone approach would simultaneously assess multiple surface receptors to pinpoint specific disease cells and enhance diagnostic accuracy in differentiating similar cells.4,5 © 2013 American Chemical Society
Received: November 16, 2013 Published: December 24, 2013 1256
dx.doi.org/10.1021/ja4114903 | J. Am. Chem. Soc. 2014, 136, 1256−1259
Journal of the American Chemical Society
Communication
Figure 1. Symbols and construction schemes are shown for (a) twoinput trivalent “Y”-shaped Nano-Claw and (b) three-input tetravalent “X”-shaped Nano-Claw. (c) Flow cytometry experiment to determine the best cDNA sequences with a high Cy5.5 fluorescence signal (from biotin-labeled TC01, Sgc4f or Sgc8c aptamer) and low FITC fluorescence signal (labeled on the candidate strands).
Figure 2. (a) Experimental scheme of aptamer-switch AND gate. (b) Adjusting the gating properties by changing the DNA sequence design, CEM and HeLa cells can be either dually targeted or distinguished. (c) Cell viability test for the AND and INH gates after visible irradiation for 3 h and subsequent growth for 48 h (*: p-value
