DNA Nanotechnology for Cell Research E-Book
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- Herausgeber: Wiley-VCH GmbH
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
- Veröffentlichungsjahr: 2024
A comprehensive and unique reference that focuses on the fundamental cell science and biomedical applications of DNA nanotechnology.
Das E-Book DNA Nanotechnology for Cell Research wird angeboten von Wiley-VCH GmbH und wurde mit folgenden Begriffen kategorisiert:
Biomolecules (DNA, RNA, Peptides, etc.), Biomoleküle (DNA, RNA, Peptide), Biotechnologie, Biotechnologie i. d. Biowissenschaften, Biotechnology, Biowissenschaften, Chemie, Chemistry, DNA, DNS, Life Sciences, Nanobiotechnologie, Nanobiotechnology, Nanotechnologie, Nanotechnology
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Table of Contents
Cover
Table of Contents
Title Page
Copyright
Preface
Part I: DNA Nanotechnology for Cellular Recognition (Cell SELEX, Cell Surface Engineering)
1 Developing DNA Aptamer Toolbox for Cell Research
1.1 Cells and Their Complexity
1.2 Features and Advantages of DNA Aptamers
1.3 On‐demand Synthesis and Screening of DNA Aptamers
1.4 Toward a Toolbox of DNA Aptamers for Cellular Applications
1.5 Summary and Outlook
Acknowledgments
References
2 Bacterial Detection with Functional Nucleic Acids:
Escherichia coli
as a Case Study
2.1 General Introduction to Bacteria
2.2
E. coli
2.3 Conventional Methods for General
E. coli
Detection
2.4 Biosensors for
E. coli
Detection
2.5 Conclusion
References
3 From Ligand‐Binding Aptamers to Molecular Switches
3.1 Aptamers Can Be Generated by SELEX
3.2 Various Subtypes of SELEX Have Been Invented
3.3 Riboswitches Are Natural RNA Aptamers Carrying Expression Platforms
3.4 Riboswitches Use Various Mechanisms to Regulate Gene Expression
3.5 Riboswitches Are Potential Drug Targets
3.6 Fusing Aptamer with Expression Platform to Construct Artificial RNA Switches
3.7 Conclusions
Acknowledgments
References
4 DNA Nanotechnology‐Based Microfluidics for Liquid Biopsy
4.1 Introduction
4.2 DNA Nanotechnology‐Based Microfluidics for Isolation of Circulating Targets
4.3 DNA Nanotechnology‐Based Microfluidics for Release and Detection of Circulating Targets
4.4 DNA‐Assisted Microfluidics for Single‐Cell/Vesicle Analysis
4.5 Summary and Outlook
References
5 Spatiotemporal‐Controlled Cell Membrane Engineering Using DNA Nanotechnology
5.1 Background
5.2 DNA Modifications on the External Cell Membrane Surface
5.3 DNA Modifications on the Internal Cell Membrane Surface
5.4 Perspectives
Acknowledgments
References
Note
Part II: DNA Nanotechnology for Cell Imaging and Intracellular Sensing
6 Metal‐Dependent DNAzymes for Cell Surface Engineering and Intracellular Bioimaging
6.1 Cellular Surface Engineering and Intracellular Bioimaging Show Great Potential in Biological and Medical Research
6.2 Metal‐Specific DNAzymes: A Suitable Choice for Artificial Manipulation of Living Cells
6.3 Cell Surface Engineering by Programmable DNAzymes
6.4 Intracellular Imaging of Metal Ions with DNAzyme‐Based Biosensors
6.5 Conclusion
Acknowledgments
References
7 DNA Nanomotors for Bioimaging in Living Cells
References
8 Illuminating RNA in Live Cells with Inorganic Nanoparticles‐Based DNA Sensor Technology
8.1 RNA Detection and Imaging
8.2 RNA Imaging Based on Direct Hybridization
8.3 RNA Imaging Based on Strand Displacement Reactions
8.4 Signal‐amplified RNA Imaging
8.5 Spatiotemporally Controlled RNA Imaging in Live Cells
8.6 Conclusion
Acknowledgment
References
9 Building DNA Computing System for Smart Biosensing and Clinical Diagnosis
9.1 DNA Computing
9.2 DNA‐Based Computing Devices for Biosensing
9.3 DNA Computing for Clinical Diagnosis
9.4 Conclusion
References
10 Intelligent Sense‐on‐Demand DNA Circuits for Amplified Bioimaging in Living Cells
10.1 DNA Circuit: The Promising Technique for Bioimaging
10.2 Nonenzymatic DNA Circuits
10.3 Intelligent Integrated DNA Circuits for Amplified Bioimaging
10.4 Stimuli‐Responsive DNA Circuits for Reliable Bioimaging
10.5 Conclusion and Perspectives
Acknowledgments
References
11 DNA Nanoscaffolds for Biomacromolecules Organization and Bioimaging Applications
11.1 Introduction
11.2 Assembly of DNA‐Scaffolded Biomacromolecules
11.3 Application of DNA Nanoscaffold for Regulation of Enzyme Cascade Reaction
11.4 DNA Nanostructures Empowered Bioimaging Technologies
11.5 Summary and Outlook
References
Note
Part III: DNA Nanotechnology for Regulation of Cellular Functions
12 Adopting Nucleic Acid Nanotechnology for Genetic Regulation
In Vivo
12.1 Introduction
12.2 Toehold‐Mediated Strand Displacement: Switching Nucleic Acids with Nucleic Acids
12.3 Toehold Riboregulators and Related Systems
12.4 Applying Nucleic Acid Nanotechnology to CRISPR and RNA Interference
12.5 Delivery of Nucleic Acid Devices,
In Vivo
Production, and Challenges for
In Vivo
Operation
12.6 Conclusion and Outlook
Acknowledgments
References
13 Cell Membrane Functionalization via Nucleic Acid Tools for Visualization and Regulation of Cellular Receptors
13.1 Nucleic Acid‐Based Functionalization Strategies: From Receptor Information to DNA Probes
13.2 Uncovering Molecular Information of Cellular Receptors
13.3 Governing Cellular Receptors‐Mediated Signal Transduction
13.4 Conclusion
Acknowledgments
References
14 Harnessing DNA Nanotechnology for Nongenetic Manipulation and Functionalization of Cell Surface Receptor
14.1 Introduction
14.2 Principle of DNA‐enabled Molecular Engineering for Receptor Regulation
14.3 DNA Nanodevices for Programming Receptor Function
14.4 Elaborate and Intelligent DNA Nanodevices Reprogramming Receptor Function
14.5 Conclusions and Perspectives
Acknowledgments
References
15 DNA‐Based Cell Surface Engineering for Programming Multiple Cell–Cell Interactions
15.1 DNA Nanotechnology: The Tool of Choice for Programming Cell–Cell Interactions
15.2 Modifying Cell Surface with DNA
15.3 Programming Cell–Cell Interactions by DNA Nanotechnology
15.4 Conclusion
Acknowledgments
References
16 Designer DNA Nanostructures and Their Cellular Uptake Behaviors
16.1 Introduction
16.2 DNA Nanotechnology
16.3 Pathways of Cell Endocytosis
16.4 Analysis of DNA Nanostructures’ Cellular Uptake Behaviors
References
Part IV: DNA Nanotechnology for Cell-Targeted Medical Applications
17 Toward Production of Nucleic Acid Nanostructures in Life Cells and Their Biomedical Applications
17.1 DNA Nanostructures
17.2 RNA Nanostructures
17.3 Applications
17.4 Conclusion
References
18 Engineering Nucleic Acid Structures for Programmable Intracellular Biocomputation
References
19 DNA Supramolecular Hydrogels for Biomedical Applications
19.1 Introduction
19.2 Classification and Preparation of DNA Supramolecular Hydrogels
19.3 Biomedical Application of DNA Supramolecular Hydrogels
19.4 Conclusions and Perspectives
References
20 Rolling Circle Amplification‐Based DNA Nanotechnology for Cell Research
20.1 Introduction
20.2 Principle and Synthetic Methods of RCA
20.3 RCA‐Based DNA Nanotechnology for Cell Separation
20.4 RCA‐Based DNA Nanotechnology for Nucleic Acid Drug Delivery
20.5 Conclusion
Acknowledgment
References
21 Precise Integration of Therapeutics in DNA‐Based Nanomaterials for Cancer Treatments
21.1 DNA‐Based Nanomaterials in Biomedicine
21.2 Strategies on Constructing DNA‐Based DDSs
21.3 Precise Integration of Therapeutics into DNA‐Based DDSs to Achieve Synergistic Cancer Treatment
References
Index
End User License Agreement
List of Tables
Chapter 7
Table 7.1 Analytical performance of DNA nanomotors.
Chapter 16
Table 16.1 A summary of examples of different DNA nanostructures with differ...
Table 16.2 A summary of examples of different DNA nanostructures with differ...
Table 16.3 A summary of examples of different DNA nanostructures with other ...
Chapter 21
Table 21.1 Stability of DNA nanostructure.
List of Illustrations
Chapter 1
Figure 1.1 Schematic illustration of the DNA aptamer tools for various appli...
Figure 1.2 Schematic representation of solid‐supported oligonucleotide synth...
Figure 1.3 Schematic representation of DNA aptamer screening by the cell‐SEL...
Figure 1.4 Schematic representation of common chemical modifications used in...
Figure 1.5 Schematic representation of (a) the logic‐gated aptamer‐signal ba...
Figure 1.6 Schematic representation of (a) the construction of aptamer–drug ...
Figure 1.7 (a) Schematic illustration of chemical modification of Sgc‐8 apta...
Figure 1.8 Schematic representation of the co‐assemblies of a multivalent ap...
Figure 1.9 Schematic illustration of the design of (a) aptamer‐functionalize...
Figure 1.10 Schematic diagram of (a) the design of a logic‐gated DNA nanodev...
Chapter 2
Figure 2.1 Cleavage and reporting mechanism of RNA‐cleaving fluorogenic DNAz...
Figure 2.2 Paper‐based biosensor that can combine the cleavage reaction and ...
Figure 2.3 Surface‐to‐surface signal enrichment‐based paper biosensor for
E.
...
Figure 2.4 DNAzyme‐based electrochemical biosensor for the detection of
E. c
...
Figure 2.5 An example of aptasensor for
E. coli
detection. (a) Cell‐binding ...
Chapter 3
Figure 3.1 The SELEX cycle for the selection of aptamers that can bind to a ...
Figure 3.2 Riboswitch classes verified so far. (a) The classes that sense nu...
Figure 3.3 Consensus sequence and secondary structures of a few riboswitch c...
Figure 3.4 Riboswitch regulation of gene expression. (a) Transcriptional reg...
Figure 3.5 SAM and AdoCbl tandem riboswitches regulating the gene‐producing ...
Figure 3.6 An example of combining aptamer with ribozyme to control gene exp...
Chapter 4
Figure 4.1 Aptamer‐functionalized nanostructured “hard substrates.” (a) Apta...
Figure 4.2 Aptamer‐functionalized nanostructured “soft substrates.” (a) The ...
Figure 4.3 Multivalent aptamer‐functionalized micro/nano‐substrates. (a) Bio...
Figure 4.4 Aptamer‐functionalized biomembrane interfaces. (a) Leukocyte memb...
Figure 4.5 DNA framework‐supported aptamer substrates. (a) Engineered aptame...
Figure 4.6 DNA framework‐supported antibody substrate. (a) Antibodies‐anchor...
Figure 4.7 Aptamer denaturation for CTC release. (a) Nuclease degradation of...
Figure 4.8 CTC release by detaching aptamers from capture interface. (a) Com...
Figure 4.9 DNA nanotechnology‐based microfluidics for CTC detection and in‐d...
Figure 4.10 DNA nanotechnology‐based microfluidics for CTC destruction and C...
Figure 4.11 DNA nanotechnology‐based signal transduction for EV detection. (...
Figure 4.12 DNA nanotechnology‐based signal amplification for EV detection. ...
Figure 4.13 DNA‐assisted microfluidics for single‐cell analysis by sequencin...
Figure 4.14 Antibody‐barcode‐based single‐cell sequence for protein analysis...
Figure 4.15 DNA‐assisted microfluidics for single‐cell analysis by spectrosc...
Figure 4.16 DNA‐assisted microfluidics for single‐vesicle analysis. (a) Apta...
Chapter 5
Figure 5.1 The left figure illustrates the involvement of the natural cell m...
Figure 5.2 Nonspecific modification strategies to incorporate DNA onto the e...
Figure 5.3 Strategies for specific DNA modification. (a) Metabolic labeling ...
Figure 5.4 Sensing and imaging applications on a cell membrane using immobil...
Figure 5.5 DNA‐mediated cell assembly and cell communication. (a) Stable mul...
Figure 5.6 DNA modifications on the internal cell membrane surface. (a) A DN...
Figure 5.7 Small‐molecule ligand targeting approach. (a) Structure of rapamy...
Figure 5.8 Cell‐penetrating peptide approach. (a) Mechanism of pHLIP inserti...
Figure 5.9 Liposome fusion‐based transport strategy. (a) Spatially controlle...
Chapter 6
Figure 6.1 Metal‐specific DNAzymes. (a) Illustration of a typical DNAzyme‐ba...
Figure 6.2 DNAzyme‐controlled cell–cell interaction. (a) Design of DNAzyme‐b...
Figure 6.3 Combination cancer treatment by using engineered DNAzyme molecula...
Figure 6.4 SPDz sensor monitoring extracellular ATP. (a) Schematic drawing s...
Figure 6.5 Catalytic beacon designs that convert DNAzymes into selective flu...
Figure 6.6 Nanoparticle‐mediated DNAzyme sensing. (a) Design of DNAzyme‐func...
Figure 6.7 Photolabile groups modified DNAzymes for cellular and
in vivo
sen...
Figure 6.8 DNA–DNA hybridization controls DNAzyme activity for intracellular...
Figure 6.9 Enzyme‐controlled metal sensing. (a) Scheme shows the I‐SceI‐cont...
Figure 6.10 Different amplification methods for sensing metal ions in cells....
Chapter 7
Figure 7.1 (A) A DNA nanomotor travels over a 1D linear track that facilitat...
Figure 7.2 DNA nanomotors propelled by various forms of energy. (a) A system...
Figure 7.3 (a) A DNA nanomotor propelled by exonuclease III for traversing 3...
Figure 7.4 (a) A bipedal nanomotor can be activated by H
+
/OH
−
and ...
Figure 7.5 The different legs of DNA nanomotors. (a) A DNase‐driven monopod ...
Figure 7.6 (a) A DNA nanomotor that travels on a 2D track.(b) Detection ...
Figure 7.7 Application of DNA nanomotor for imaging nucleic acid in a live c...
Figure 7.8 (a) Schematic illustrations of the preparation and activation of ...
Figure 7.9 Schematic diagrams of DNA nanomotors for protein imaging. (a) All...
Figure 7.10 (a) A nanomotor system for the detection of dual targets: miRNA ...
Chapter 8
Figure 8.1 (a) Schematic of the formation of sandwich‐shape hybridization st...
Figure 8.2 (a) Schematic and verification of the SDR‐based nano‐flare for se...
Figure 8.3 (A) Schematic showing the secondary structure of substrates and t...
Figure 8.4 (A) Schematic showing the secondary structure of the substrates a...
Figure 8.5 (a) Working principle and (b) application of AuNP‐QDs nanoassembl...
Figure 8.6 (A) Working principle and (B) application of mRNA‐powered DNA nan...
Figure 8.7 (a) Working principle and (b) application of UCNP‐based DNA nanos...
Chapter 9
Figure 9.1 Illustrations of basic logic gates and their truth table.
Figure 9.2 DNA logic gates based on functional DNA motifs. (a) Deoxyribozyme...
Figure 9.3 DNA computation based on TMSD reactions. (A) Scheme of TMSD react...
Figure 9.4 DNA computing devices for biosensing implemented
in vitro
. (A) El...
Figure 9.5 DNA computing devices for biosensing implemented on the cell memb...
Figure 9.6 DNA computing devices for biosensing implemented within living ce...
Figure 9.7 DNA computing for intelligent clinical diagnosis. (a) The workflo...
Chapter 10
Figure 10.1 (A) Pathway of hybridization chain reaction (HCR).(B) Fundam...
Figure 10.2 (A) Schematic illustration of the concatenated HCR system for mi...
Figure 10.3 (a) Illustration of the integrated CHA‐DNAzyme for intracellular...
Figure 10.4 (a) Schematic illustration of the microRNA‐targeting R‐HCR machi...
Figure 10.5 (a) Design of the UV light‐activated DNAzyme‐CHA system for endo...
Figure 10.6 (a) Illustration of the on‐site activation of the APE1‐responsiv...
Figure 10.7 (a) Schematic diagram of the microRNA‐unlocked M‐EDz platform fo...
Figure 10.8 (a) Schematic interpretation of the endogenous ATP‐controlled HC...
Chapter 11
Figure 11.1 Workflow of DNA‐protein complexes synthesizing. (a) Conjugation ...
Figure 11.2 Enzyme cascade. (a) Schematic representation of the GOx/HRP casc...
Figure 11.3 Artificial swinging arms facilitate the substrate channeling bet...
Figure 11.4 Scaffolded enzyme cascade within living cells. (a) Illustration ...
Figure 11.5 DNA nanotechnology expended fluorescence imaging. (a) Brain tumo...
Figure 11.6 DNA origami “signposts” for cellular membrane tagging.(a) Sc...
Chapter 12
Figure 12.1 Toehold‐mediated strand displacement (TMSD). (a) TMSD involves t...
Figure 12.2 Riboswitches and toehold riboregulators. (A) Naturally occurring...
Figure 12.3 Applications for toehold switches. (A) Toehold switches can be u...
Figure 12.4 Conditional guide RNAs. (a) CRISPR‐associated nucleases like Cas...
Figure 12.5 Conditional guide RNAs for dCas12a in mammalian (eukaryotic) cel...
Chapter 13
Figure 13.1 Installation of DNA probes on receptors via nucleic acid‐based c...
Figure 13.2 Profiling receptor expression levels via nucleic acid tools. (a)...
Figure 13.3 Profiling receptor‐specific glycosylation patterns via nucleic a...
Figure 13.4 Profiling receptor nanoscale organizations via nucleic acid tool...
Figure 13.5 Manipulation of receptor‐mediated signal transduction via antago...
Figure 13.6 Manipulation of receptor‐mediated signal transduction via nuclei...
Figure 13.7 (a) Working principle of impeding PTK‐7‐VEGF‐R1 interaction via ...
Chapter 14
Figure 14.1 Scheme illustrating DNA nanotechnology‐driven nongenetic enginee...
Figure 14.2 Schematic representation of DNA‐based bivalent aptamers mimickin...
Figure 14.3 Responsive activation of receptor dimerization on the cell membr...
Figure 14.4 Spatiotemporal regulation of receptor using light‐controlled ass...
Figure 14.5 DNA nanodevices for visualization of cell surface receptor dimer...
Figure 14.6 Automated DNA nanomachines for mechanical control over receptor‐...
Figure 14.7 Selective activation of receptors based on “scanning” and “unloc...
Figure 14.8 DNA‐enabled logical recognition to improve T cell killing specif...
Figure 14.9 DNA origami as a programmable scaffold for the spatial organizat...
Chapter 15
Figure 15.1 Approaches to modifying cell surface with synthetic DNA, includi...
Figure 15.2 DNA nanotechnology‐enabled cell–cell interactions, including (a)...
Figure 15.3 Ligand–receptor binding‐based cell–cell interactions. (a) Target...
Figure 15.4 DNA hybridization‐based cell–cell interactions. (a)
In situ
‐form...
Figure 15.5 DNA circuit‐regulated cell–cell interactions. (a) Programming re...
Chapter 16
Figure 16.1 Designs of DNA nanostructures are constructed by using a fixed n...
Figure 16.2 DNA origami structures. (a) DNA origami proposed by Rothemund an...
Figure 16.3 DNA nanostructures constructed by single‐stranded DNA tiles (SST...
Figure 16.4 Dynamic DNA nanostructures. (a) DNA origami structures used as d...
Figure 16.5 Overview of cell endocytosis pathways. There are six major pathw...
Figure 16.6 Studies on DNA nanostructures with specific sizes and shapes and...
Figure 16.7 A summary on the pathways through which DNA nanostructures enter...
Chapter 17
Figure 17.1 Strategies to construct DNA nanostructures. (a) Tile‐based self‐...
Figure 17.2 Single‐stranded DNA nanostructures and the amplification approac...
Figure 17.3 The self‐assembly of RNA nanostructures. (a) Self‐assembly of RN...
Figure 17.4 The
in vivo
production of RNA nanostructures. (a) The scheme of ...
Figure 17.5 The application of
in vivo
‐expressed RNA nanostructures. (a)
In
...
Chapter 18
Figure 18.1 Elementary logic gates and several composite logic gates, includ...
Figure 18.2 Representative toehold‐mediated strand displacement reactions (S...
Figure 18.3 Strategies for controlling the kinetics of DNA circuits. (a) Ent...
Figure 18.4 Schemes of logic algorithm‐assisted DNA robots on DNA origami pl...
Figure 18.5 Nucleic acid‐based logic devices used to measure the intracellul...
Figure 18.6 Nucleic acid‐based logic devices used to monitor intracellular b...
Figure 18.7 Nucleic acid‐based logic devices used to regulate intracellular ...
Chapter 19
Figure 19.1 Formation of pure DNA supramolecular hydrogels. (A) pH‐triggered...
Figure 19.2 Formation of hybrid DNA supramolecular hydrogels: (a) pH‐stimula...
Figure 19.3 DNA supramolecular hydrogels for biosensing: (a) the enzyme‐cage...
Figure 19.4 DNA supramolecular hydrogels for drug delivery. (a) Injectable C...
Figure 19.5 DNA supramolecular hydrogels for immunotherapy. (a) Injectable, ...
Figure 19.6 DNA supramolecular hydrogels for 3D cell culture. (a) A triggere...
Figure 19.7 DNA supramolecular hydrogels for construction of artificial tiss...
Figure 19.8 DNA supramolecular hydrogels for tissue engineering
in vivo
. (a)...
Chapter 20
Figure 20.1 Schematic illustration of the process of rolling circle amplific...
Figure 20.2 Physically cross‐linked DNA network for stem cell fishing. (a) T...
Figure 20.3 T lymphocyte‐captured DNA network for localized immunotherapy (a...
Figure 20.4 RCA‐based proton‐activatable DNA nanocomplex for the co‐delivery...
Figure 20.5 RCA‐based nanocomplexes containing cascaded DNAzymes and promote...
Figure 20.6 RCA‐based DNA‐polydopamine‐MnO
2
nanocomplex for cancer therapy. ...
Figure 20.7 RCA‐based energy‐storing DNA‐based nanocomplex for laser‐free ph...
Figure 20.8 DNA signal processor based on UCNP and RCA epitaxial assembly. (...
Chapter 21
Figure 21.1 Representative DNA architectures used for drug delivery. (a) Tet...
Figure 21.2 Schematic illustration of different mechanisms of cellular endoc...
Figure 21.3 Representative anti‐cancer therapeutics, including (a) small‐mol...
Figure 21.4 Manufacture of DNA‐based drug delivery systems utilizing non‐cov...
Figure 21.5 Incorporation of nucleoside analogs into DNA‐based DDSs. (a) Dev...
Figure 21.6 Incorporation of drugs into DNA‐based DDSs via reaction with PS ...
Figure 21.7 Syntheses of drug‐DNA conjugates by terminal conjugation on DNA....
Figure 21.8 (a) Synergistic treatment based on conceptual chemogene.(b) ...
Figure 21.9 Schematic illustrations of chemogene materials. (a) Paclitaxel‐l...
Guide
Cover
Table of Contents
Title Page
Copyright
Preface
Begin Reading
Index
End User License Agreement
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DNA Nanotechnology for Cell Research
From Bioanalysis to Biomedicine
Edited by Zhou Nie
Editor
Prof. Zhou NieHunan UniversityCollege of Chemistry and Chemical EngineeringLushan Road (S)Yuelu DistrictChangsha 410082China
Cover Image: © Vink Fan/Adobe Stock Photos
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Print ISBN: 978‐3‐527‐35173‐2ePDF ISBN: 978‐3‐527‐84079‐3ePub ISBN: 978‐3‐527‐84080‐9oBook ISBN: 978‐3‐527‐84081‐6
Preface
Nucleic acids, despite their primary role in genetic information storage and transmission, also function in synergy with other biomacromolecules to assemble intricate biological structures seen in nature, such as chromosomes and Holliday junctions. Beyond their structural capacities, these natural complexes are indispensable for many essential biological processes. This intricate intertwining of structure and function received a fresh perspective in 1982, when Nadrian C. Seeman introduced a pioneering concept that DNA could be harnessed as a programmable structural material. This groundbreaking idea has paved the way for substantial advancements in the field of DNA nanotechnology over the past four decades. The Watson‐Crick base‐pairing principle, the driving force behind the development of DNA nanotechnology, provides the foundation for the construction of static or dynamic nanostructures that offer nearly infinite programmability at the nanoscale and allow temporal control. DNA nanotechnology allows for precise spatial control in 3D space and introduces stimuli‐responsive dynamic behaviors to an array of biological molecules. Recently, we have witnessed the development of DNA‐based toolkits that have facilitated a higher‐resolution exploration of cell structure, function, and behavior with improved specificity. Moreover, DNA nanotechnology also permits the precise control and intelligent programming of cellular functions, opening new possibilities for bioengineering and therapeutic applications.
This book, “DNA Nanotechnology for Cell Research: From Bioanalysis to Biomedicine,” aims to fill a gap in current literature. Unlike previous books that focused mainly on the functionality of designer DNA nanostructures as well as their in vitro applications such as bioanalysis and biocomputing, this book presents a comprehensive overview of DNA nanotechnology and its diverse applications in cell research and engineering. We have invited active researchers with extensive expertise in the field of DNA nanotechnology to contribute their knowledge and insights to this volume. The coverage of this book ranges from the fundamental concepts of DNA nanotechnology to the most cutting‐edge studies on its applications in fundamental cell research and associated biomedical fields, including (i) cellular recognition, (ii) cell imaging and intracellular sensing, (iii) regulation of cellular functions, and (iv) cell‐targeted medical applications. We hope this book serves as an up‐to‐date, tutorial‐style resource for researchers across diverse fields, from DNA nanotechnology, cell biology, chemical biology, and analytical chemistry, inspiring them to exploit the unique merits of DNA nanotechnology to unravel cellular mysteries and develop innovative therapeutic strategies.
We would like to express our sincere appreciation to all the contributing authors, reviewers, and editorial staff involved in this book. Their tremendous efforts, coupled with insightful perspectives, have significantly enriched the content of this volume. As we look to the future, we are confident that this collective endeavor will not only stimulate advancements but also inspire further exploration and innovation in this fascinating and ever‐evolving field of research.
August 2023
Zhou NieChangsha, China
Part IDNA Nanotechnology for Cellular Recognition (Cell SELEX, Cell Surface Engineering)
1Developing DNA Aptamer Toolbox for Cell Research
Liang Yue1, Shan Wang1, and Weihong Tan1,2,3
1Hunan University, College of Chemistry and Chemical Engineering, College of Biology, Aptamer Engineering Center of Hunan Province, Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, 2 Lushan Road, Changsha 410082, China
2Chinese Academy of Sciences, Hangzhou Institute of Medicine, The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), 150 Dongfang Street Xiasha, Hangzhou 310000, China
3Shanghai Jiao Tong University, Shanghai Jiao Tong University School of Medicine, Institute of Molecular Medicine, College of Chemistry and Chemical Engineering, Renji Hospital, 800 Dongchuan RD, Shanghai 200240, China
1.1 Cells and Their Complexity
Cells are the basic building blocks of life, initially discovered by Robert Hooke in 1665 [1, 2]. Their structural and functional complexity attracts significant research interest due to their intricate composition, abundance, and interactivity [3–5]. Even the simplest unicellular organism comprises various biomolecular components, ranging from nucleic acids, proteins, and lipids to carbohydrates, which are elaborately bonded together. Moreover, cells exhibit remarkable plasticity and can proliferate and differentiate to create distinct cell populations with radically different compositions and interactions, which enables them to implement specific and varied functions [6–8]. The organization of these cell populations yields a highly collaborative living system capable of carrying out all of life’s biological processes, where cells execute their functions through molecular interactions with the external environment and other cells [9, 10]. Furthermore, cells are open systems that facilitate the exchange of information, energy, and matter between cells and their external environment [11–14]. The dissipation of energy and matter is a continuous process sustaining cellular functions, which drives the cells to operate far from equilibrium [15, 16]. The nonlinear interactions and reactions of the components further enhance the complexity of cellular systems, revealing extraordinary dynamic behaviors and functions adapting to ever‐changing environmental cues [17–19].
To understand cells and their functions in their entirety, it is essential to investigate components that interact to develop and maintain high‐order cellular structures and realize complex biochemical activities [20, 21]. Reliable and accurate molecular recognition tools are required to detect specific components from the cellular pool [22, 23]. In addition, suitable technologies and methodologies are needed to observe components’ dynamics and analyze their nonlinear behaviors in biological processes [24–28]. This field has experienced substantial advancements propelled by breakthroughs in molecular recognition tools and analytical methods. Further, researchers have devoted themselves to developing new and innovative tools to observe, measure, and manipulate cellular components and systems at unprecedented levels of detail. These progresses broaden our understanding of cellular mechanisms and pave the way to develop novel therapies and treatments for diseases associated with cellular dysfunction [29–34].
Functional nucleic acids, as synthetic oligonucleotides with unique chemical‐biological properties, sequence programmability, simplicity of modification, and facile functional manipulation, have demonstrated bioactivity, versatility, and in vivo bioavailability toward biological and biomedicine applications [35–39]. The rapid progress in DNA nanotechnology has offered remarkable opportunities for tackling the intricate nature of cellular systems [40]. In particular, introducing DNA aptamers with excellent molecular recognition ability into the modular design of molecular tools has opened new avenues in cell research, providing captivating prospects for handling complex molecular systems with precision and accuracy.
1.2 Features and Advantages of DNA Aptamers
DNA aptamers [41], termed “chemical antibodies”, are short, single‐stranded nucleic acid molecules (20–100 nucleotides) that can fold into complex 3D structures and bind with various targets (e.g. metal ions [42], small organic molecules [43], proteins [44], viruses [45], bacteria [46], cells [47], and even tissues [48]) with specificity and high affinity, Figure 1.1. Compared with natural antibodies, the core competency of DNA aptamers rests with the unique properties of synthetic nucleic acids [41], involving relatively low molecular weight, low‐cost standardization synthesis, customized modification, programmable sequences and structures, sequence‐dependent multifunctionality, etc. The size of DNA aptamers (10–30 kDa and ∼3 nm in diameter) is much smaller than that of natural antibodies (∼150 kDa and 10–15 nm in diameter), which endows DNA aptamers with fewer steric hindrance and unimpaired binding affinity in a confined environment, thus rendering a broader application [49]. For instance, Yang et al. reported a straightforward strategy for labeling and manipulating cell surface protein by utilizing DNA aptamers [50]. Specifically, an aptamer‐based logic computing reaction is used to recognize selectively and covalently conjugate immune checkpoint antagonizing aptamers (e.g. D‐aPDL1, D‐Sgc8‐PA, and D‐Sgc8‐99mTc) on the surface of cancer cells, improving the precision and robustness of immune checkpoint blockade therapy.
Figure 1.1 Schematic illustration of the DNA aptamer tools for various applications.
In contrast with the complex modifying process and unmanageable conjugation numbers and sites of natural antibodies [51], DNA aptamers are facile to be synthesized and modified as a consequence of the technological progress in phosphoramidite chemistry‐based oligonucleotide synthesis over the past decades [52]. With recent advancements in automated modular synthesizers and phosphoramidite chemistry, DNA aptamers can now be conjugated with multiple functional groups at predetermined locations [53]. This approach provides many benefits for practical applications of DNA aptamers, including aptamer–drug conjugates (ApDCs) [54] and artificial nucleobase‐expanded aptamers [55].
Furthermore, nucleic acids possess base sequence programmability and modular design characteristics, whereas natural antibodies currently do not. By integrating modular design and programmability into DNA aptamer systems, modular and programmable toolboxes can be created to meet future demands in advanced biological molecular tools and materials. Specifically, DNA aptamers with target recognition abilities can be regarded as functional modules that can be integrated, on demand, into nanostructures [56], nanodevices [57], and other functional systems [58]. These features enable researchers to engineer DNA aptamer‐based systems with a high degree of flexibility and precision at the molecular level. Further, the highly predictable base‐pairing and structural reconfiguration rules of nucleic acids provide a strong basis for designing programmable DNA structures and controlling DNA dynamics. Through their conjugation with specific functional components, DNA aptamers can respond to various environmental cues [59–61], such as pH, light, and temperature, and numerous biological targets (e.g. DNA/RNA, ATP, enzymes, and metal ions) leading to innovative nanodevices. With these superior merits, DNA aptamer‐based systems have been considered promising candidates for various cellular applications.
1.3 On‐demand Synthesis and Screening of DNA Aptamers
Highly accurate and pure DNA oligonucleotides are vital for DNA aptamer screening and their practical applications. Since its introduction by Caruthers in 1981 [62], phosphoramidite chemistry has emerged as the gold standard for the synthesis of short DNA oligonucleotides (<200 nucleotides). This innovative chemical method has revolutionized the field of DNA synthesis, which allows for the production of DNA sequences with high purity and chemical modifications customized for specific research or practical applications. To grow oligonucleotide chains using phosphoramidite chemistry via a solid‐supported oligonucleotide synthesis, the following steps are typically involved, as depicted in Figure 1.2: (1) Deprotection of resin‐supported nucleoside: The first nucleoside protected by a dimethoxytrityl (DMT) group is pre‐attached to resin. Then, the 5′‐DMT protecting group is removed by acid‐catalyzed detritylation to expose the 5′‐hydroxyl group as a reactive site for the addition of the next nucleotide; (2) Coupling: Taking tetrazole or its derivative as an activator, the diisopropylamino group of the incoming nucleoside phosphoramidite is protonated to enhance its reactivity as a leaving group. The 5′‐hydroxyl group of the support‐bound nucleoside attacks the phosphorus atom of the activated nucleoside phosphoramidite, resulting in a new phosphorus‐oxygen bond; (3) Capping: To prevent the formation of any truncated sequences, a “capping” procedure is introduced after the coupling reaction, which effectively prevents unreacted 5′‐hydroxyl groups from participating in subsequent coupling reactions. With all unreacted sites “capped,” the oligonucleotide synthesis proceeds exclusively at full‐length oligonucleotides; (4) Oxidation: The phosphite‐triester (PIII) formed during the coupling step is susceptible to acid and needs to be converted into a stable form (PV) before the implementation of next acidic detritylation step. This can be achieved through iodine oxidation in the presence of water and pyridine. The resulting phosphotriester is, essentially, the DNA backbone and is protected with a 2‐cyanoethyl group from undesirable reactions during subsequent synthesis cycles; (5) Detritylation: After the oxidation step, the DMT‐protecting group located at the 5′‐end of the resin‐bound DNA chain is removed for the following nucleotide phosphoramidite. Repeat steps 2 through 5 until the desired oligonucleotide sequence has been synthesized; (6) Deprotection and purification: Finally, all the remaining protecting groups (e.g. the protecting groups from the heterocyclic bases and phosphodiester backbone) are removed, and the oligonucleotide is purified using various methods, such as HPLC or PAGE.
Figure 1.2 Schematic representation of solid‐supported oligonucleotide synthesis.
The advancement of automated oligonucleotide synthesis technologies has significantly improved the synthetic length, speed, cost, and throughput, enabling high‐throughput, cost‐effective, and large‐scale synthesis of DNA aptamers with high efficiency and precision. Because of the precise synthesis capability, the fine control over DNA aptamers is accessible, allowing for their incorporation with different chemical modifications where the position and quantity of modification can be manipulated to alter the properties and functions of DNA aptamers. The ability to engineer the structure and specificity of DNA aptamers through synthesis and chemical modification makes them a highly flexible and customizable tool for a diverse range of biological and biomedical research areas.
Relative to antibodies evolved from the natural immune response, aptamers are produced mainly by an in vitro evolution method, the systematic evolution of ligands by the exponential enrichment (SELEX) technology, developed by Szostak and Gold in 1990 [63, 64]. This technology involves iterative rounds of selection and amplification of specific nucleic acid sequences in a large oligo pool, through which various aptamers have evolved for targeting small molecules (e.g. organic dyes [65], ATP [66], cocaine [67], and mycotoxins [68]) or proteins (e.g. thrombin [69] and tumor biomarkers [70]), with specificity and high affinity. Nevertheless, the screened DNA aptamers through SELEX processes may undergo structural reconfiguration and function loss in a physiological environment, which limits their utilization in cell research.
In 2006, our group introduced an extension of the SELEX technology called cell‐SELEX [47], aiming to select aptamers against living cells. Since then, the target has been developed into diverse living species [71, 72] (e.g. bacteria, viruses, and disease tissues). For the cell‐SELEX technology, a large‐capacity (1015–1016) oligonucleotide library needs to be engineered before the screening, and each oligonucleotide comprises random sequence regions in the middle and primer sequence‐binding regions at both ends. A typical cell‐SELEX process involves a six‐step selection and amplification cycle: (i) Incubation of target cells with a library of single‐stranded DNAs including a random domain; (ii) Collection of the target cell‐bound oligonucleotides; (iii) Amplification of the collected oligonucleotides by PCR; (iv) Counter‐selection step by using negative cells to reduce the common sequences that bind with both cells; (v) Retainment of the unbound DNAs from counter‐selection process; (vi) Amplification of the unbound DNAs to yield an enriched oligonucleotide pool for the following round selection. The process is iterated until the sequences specifically targeting the cell are highly enriched. The aptamer’s affinity to the target molecules in a living cell can be significantly improved after 10–20 rounds of screening. Finally, corresponding pools are sorted and optimized for further cloning and sequencing, as shown in Figure 1.3.
Figure 1.3 Schematic representation of DNA aptamer screening by the cell‐SELEX process.
Source: Reproduced with permission from Sefah et al. [72]. © 2010 Springer Nature.
Compared with the traditional SELEX strategy, cell‐SELEX possesses several superiorities in the complex cellular system: (i) Massive biomolecules exist with different functional states and abundance in the cell. Aptamers produced by cell‐SELEX can bind to these biomolecules naturally, conducive to understanding how these molecules interact and realize complex biochemical activities. (ii) It is unnecessary to know and purify target cells’ molecular signatures in cell‐SELEX since aptamers can target unknown molecules, and it enables the discovery of potential biomarkers. (iii) For cell research, aptamers generated from cell‐SELEX are in their active and native states without structural unfolding in a cellular environment. (iv) Accompanied by the occurrence of disease in a living system, a fundamental change at the molecular level takes place, like the emergence of cancer with unique biomarkers. By analyzing the molecular difference between disease cells and normal cells, potential biomarkers and new molecular events can be targeted by cell‐SELEX, thus distinguishing disease cells from normal cells. Cell‐SELEX has successfully screened out hundreds of aptamers against more than 80 different cell lines, opening up new avenues for cellular applications [73, 74]. For example, Wu et al. reported a DNA aptamer, XQ‐2d, which targets the transferrin receptor CD71 with high affinity and specificity [75]. CD71, a membrane glycoprotein and receptor of the iron‐transferrin complex, is abundantly expressed in various cancer cells, including those found in the brain, liver, breast, lung, and colon. It is considered a significant independent prognostic marker and a promising therapeutic target for several cancers. Among 40 clinical samples of pancreatic adenocarcinoma investigated using Aptamer XQ‐2d, CD71 is overexpressed in 82.5% of the samples. Furthermore, Aptamer XQ‐2d has been used for the targeted delivery of doxorubicin (DOX) to pancreatic cancer cells. The selective delivery of loaded DOX into PDAC cell line PL45 results in a significant antiproliferation effect, implying the XQ‐2d aptamer’s great potential for targeted pancreatic cancer therapy.
With the advent of chemical synthesis and cell‐SELEX technology, DNA aptamers have emerged as a powerful toolbox for the biological and biomedicine fields due to their unique properties, including high binding affinity, specificity, stability, and simplicity of synthesis. DNA aptamers have been widely applied for cell research to identify and validate new targets on the surface of cells [76], to investigate intracellular processes, or to control cell behaviors. Also, they have been employed as biosensors [41] to detect proteins, nucleic acids, or small molecules in real time or as therapeutic agents for targeted drug delivery [57] and gene regulation. Furthermore, DNA aptamers have assisted in the isolation and detection of rare cell populations as well as in cell imaging [56] and molecular diagnostics [77]. Overall, DNA aptamers constitute a versatile tool for cell research in favor of the elucidation of the complicated workings of cells. As research in DNA aptamers progresses, new and exciting applications are anticipated to emerge, which could significantly enhance their utility and impact on cell research. These advancements have the potential to revolutionize the field of biomedicine, resulting in improved patient outcomes.
1.4 Toward a Toolbox of DNA Aptamers for Cellular Applications
As life science continues to develop, there is a growing requirement for molecular tools that enable researchers to understand cellular compositions and precisely control their dynamic behaviors. Recent advances in molecular engineering in DNA aptamers have paved the way toward a DNA aptamer‐based toolbox for various cellular applications [39, 78–80]. This toolbox consists of a standardized set of DNA aptamers that have been extensively validated for their specificity, affinity, protocols for synthesis, modification, integration, characterization, and utility in cellular applications. We believe the ongoing development of such a toolbox will unlock the full potential of DNA aptamers for cell research.
To develop and utilize a toolbox composed of a versatile set of DNA aptamers for cell research, here are the necessary protocols we can follow: (i) Identify the tasks and select appropriate aptamers. To reach research goals, we need to identify the target (e.g. biomolecules, viruses, cells, or tissues) and then select a suitable DNA aptamer with high affinity and specificity against the target. (ii) Verification of DNA aptamers: Once the DNA aptamer candidate is selected, it is crucial to verify its binding affinity and specificity by performing enzyme‐linked oligonucleotide assay (ELONA) or surface plasmon resonance (SPR) analysis. (iii) Aptamers modification: To meet the needs in the specific application scenario, it is necessary to modify them with appropriate functional groups. This process can be realized using various chemical or integrative approaches involving post‐SELEX chemical modification, DNA nanotechnology, or supramolecular self‐assembly. (iv) Application and optimization of DNA aptamer tools: After we have delivered DNA aptamer tools into cells for various applications, it is vital to evaluate their performance, optimize them based on feedback, and create new tools to fill any gaps in the toolbox when confronted with new tasks. By following these protocols and preparing for challenges, researchers can efficiently develop and utilize DNA aptamer tools in cell research.
In this context, we discuss recent advances in engineering DNA aptamers as targeting and regulating modules and integrating them with versatile modules for a multifunctional toolbox, which allows researchers to choose the desired aptamer tools and use them for specific applications without further screening and examination. We introduce the molecular engineering approaches of DNA aptamers, mainly categorized as chemical modification and DNA nanotechnology. We highlight some examples of DNA aptamers engineered for specific cellular applications, including biological regulation, targeted delivery of therapeutic agents, biosensing, cell imaging, and biomimicry.
1.4.1 Chemical Modifications via Solid‐Supported Synthesis Strategy
DNA aptamers offer promising molecular recognition tools for cells. However, their full potential in life science has been limited by several challenges, such as poor chemical and biological stability and a narrow range of chemical diversity. To address these limitations, chemical modification of aptamers through a solid‐supported synthesis strategy has been exploited to unleash their potential as molecular tools.
Solid‐phase synthesis technology is a highly controllable and automated molecular synthesis method that can efficiently produce nucleic acids from individual phosphoramidite building blocks. This technology offers a sequence‐predesigned DNA synthesis platform enabling the direct introduction of chemical groups into oligonucleotides in a controlled manner, which significantly enhances the aptamers’ biostability and versatility [81–83]. Throughout the chemical synthesis process, aptamers can be modified at nucleotide components, including bases, sugars, or backbone groups, as shown in Figure 1.4. For instance, the backbone modifications on non‐bridging oxygen of the canonical phosphodiester linkage with a methyl or ethyl group would provide uncharged backbones for minimizing the electrostatic repulsion between nucleic acid strands [84, 85]. The sugar group of the nucleotides can be modified by introducing a 2′‐fluoro or 2′‐O‐methyl group and the locked nucleic acid [86]. It has been demonstrated that 2′‐amino and 2′‐fluoro substitutions can significantly increase the half‐life of aptamers in human serum, extending from mere seconds (eight seconds) up to 86 hours. Furthermore, recent advances in nucleic acid chemistry have promoted the development of threose nucleic acid, peptide nucleic acid, and chirally inverted mirror‐image nucleic acids (L‐DNA) with unique structures different from natural nucleic acids [87, 88]. These artificial nucleic acids have also been explored as a promising category of nuclease‐resistant and biostable aptamers that cannot be degraded by human blood and serum nucleases. As mentioned, these DNA aptamers can be readily synthesized by automated oligo synthesizer with commercially available materials, facilitating practical applications in diagnostics and therapeutics.
Figure 1.4 Schematic representation of common chemical modifications used in aptamers.
Meanwhile, this innovative approach enables the efficient synthesis of ApDCs using oligonucleotide synthesizers while preserving the biological activity of the included drugs. In 2014, Tan and coworkers developed the first ApDC phosphoramidite module, which incorporated an anticancer drug moiety and a photocleavable linker [53]. Upon the application of this module, automated and modular synthesis of ApDCs has been realized, allowing for the effective introduction of multiple drugs at predetermined positions. These ApDCs display specific targeting toward cancer cells and can be photoactivated to release drugs in a precise and controlled manner. Subsequently, various ApDCs have been exploited by using an automated DNA synthesizer. For example, Lv et al. reported a Sgc8‐5FU ApDC by modifying DNA Aptamer Sgc8 with 5‐fluorouracil (5‐FU). The internalization and subsequent transportation pathways of the ApDC were investigated by the single‐particle tracking (SPT) technique [89]. The results reveal that the ApDC predominantly entered the cells via caveolin‐mediated endocytosis, similar to the pure DNA aptamer. Besides, Xuan et al. incorporated bioorthogonal chemistry with prodrug design to develop a novel aptamer prodrug conjugate (ApPdC) [90]. In the designed ApPdC, the hydrophilic DNA aptamer functions as a tumor‐targeting subunit, whereas the hydrophobic prodrug promotes the self‐assembly of ApPdC and acts as a free radical generator to enhance the efficacy of chemodynamic therapy.
Another approach involves the introduction of nucleotide analogs like Z:P, which mimic the structures and functions of natural nucleotides, into DNA aptamers to expand their chemical composition [91]. Artificial nucleotide bases promise to improve the diversity, properties, and functionalities of aptamers, making them more practical for various application scenarios. Our group proposed the concept of aptamer‐signal base conjugates (ApSC) to develop AND‐gated molecular tools for tumor‐targeted molecular imaging [92]. As depicted in Figure 1.5a, the molecular probe consists of two segments: a DNA aptamer acting as the targeting ligand and tumor microenvironment‐responsive artificial ferrocene bases for tumor cell imaging. The resulting ApSC demonstrates targeted accumulation in the tumor cells via aptamer‐mediated molecular recognition. Meanwhile, the acidic pH and high levels of H2O2 in the tumor microenvironment trigger the in situ activation of the synthetic ferrocene bases through the Fenton‐like reaction that leads to an amplified MRI signal over normal tissues.
Figure 1.5 Schematic representation of (a) the logic‐gated aptamer‐signal base conjugate for targeted molecular imaging of metastatic cancer.
Source: Reproduced with permission from Li et al. [92]. 2022 American Chemical Society.
(b) The design of the artificial‐nucleotide‐expanded aptamer against integrin alpha3 for regulating cell activities.
Source: Reproduced with permission from Tan et al. [93]. © 2018 John Wiley & Sons.
The various synthesized nucleotides can be applied to enlarge the chemical space of the nucleic acid libraries in SELEX processes, thus increasing the diversity of DNA aptamers. Hence, an artificial‐nucleotide‐expanded SELEX approach was developed to evolve artificial nucleotide‐based aptamers [93]. The nucleic acid library of cell‐SELEX was extended with three artificial nucleotide bases: Fe base with a ferrocenyl group, F base with a trifluoromethyl group, and Z:P base pair (Figure 1.5b). And an artificial nucleotide‐expanded aptamer named ZAP‐1, targeting the integrin alpha3 (ITGA3), was screened through the artificial‐nucleotide‐expanded cell‐SELEX process. The binding of aptamer ZAP‐1 with ITGA3 can reduce the interaction between ITGA3 and its natural ligand, which inhibits the triple‐negative breast cancer (TNBC) cells’ adhesion and migration. We anticipate that the artificial‐nucleotide‐expanded aptamers evolved from this artificial nucleotide‐assisted cell‐SELEX approach would enable the elucidation of the molecular basis of biological activities and regulation of biological functions.
1.4.2 Chemical Modifications Through Covalent Conjugation
Covalent conjugation is a valuable technique that creates a solid covalent bond between the aptamer and a non‐nucleotide moiety, like a fluorophore, drug, or nanomaterial (Figure 1.4). Covalent conjugation can significantly improve the performance of DNA aptamers in vitro and in vivo, offering a predicted advantage in various applications. To achieve covalent conjugation, different chemical reactions can be used, such as thiol‐maleimide, click chemistry, or amine‐reactive crosslinking, depending on the specific properties of the DNA aptamer and the intended applications.
Small‐molecule drugs possess several advantages, such as easy storage and transportation, low immunogenicity, and oral administration [94]. However, their low specificity results in low efficacy, relatively high toxicity, and unavoidable side effects. Targeted delivery of small‐molecule drugs has been proven to enhance drug efficacy significantly, thus reducing toxicity and side effects. With the efficient molecular recognition capability, DNA aptamers can serve as ideal targeting units when coupled with small‐molecule drugs for targeted drug delivery. Upon the covalent conjugation approach, our group exploited the first ApDC in 2009 and then contributed much to the ApDC research [95]. Li et al. presented an Sgc8c‐artesunate conjugate with much higher therapeutic efficacy than artesunate alone as a consequence of the DNA aptamer‐induced accumulation of artesunate in target cells [96]. And its retention time in tumor cells is much longer than that in control cells, demonstrating the specific targeting capability of the Sgc8c‐artesunate conjugate. In addition, Huang et al. investigated the impact of linkers on the properties of ApDC [97]. Combretastatin A4 is conjugated to the Sgc8c aptamer via three different linkers, namely disulfide, phosphodiester, and carbamate bonds, for the investigation of the drug release mechanism and anticancer efficacy, as shown in Figure 1.6. The results indicate that a nucleophilic attack of glutathione can cleave the phosphodiester bond, and the repeated cleavage of the linker endows the ApDC with higher anticancer efficacy. These imply that the design of the linker unit is also critical for the efficacy of ApDC.
Figure 1.6 Schematic representation of (a) the construction of aptamer–drug conjugates through automated modular synthesis strategy. (b) Three different linkers: a phosphodiester bond (Linker 1), a disulfide bond (Linker 2), and a carbamate (Linker 3) involved in modifying the DNA aptamer.
Source: Reproduced with permission from Huang et al. [97]. 2021 American Chemical Society.
TNBC is considered one of the most malignant cancers. Developing effective targeted TNBC therapy has been regarded as an essential research topic in TNBC treatment. He et al. developed an AS1411‐triptolide‐conjugate to treat TNBC, which reveals high specificity and cytotoxicity against the MDA‐MB‐231 cell line [98]. More importantly, the AS1411‐triptolide‐conjugate has excellent in vivo anti‐tumor efficacy for TNBC and negligible side effects on healthy organs. Compared with monotherapy using a single medication, polytherapy with multiple drugs can simultaneously target multiple mechanisms associated with tumor growth. This strategy can decrease individual drug dosages, enhance therapeutic efficacy against multiple targets, and overcome resistance mechanisms. However, there are challenges to creating effective combination therapies with an accurate tune of the drug ratio. To address these challenges, Zhou et al. developed cyclic bivalent ApDCs (cb‐ApDCs) with a tunable drug ratio [99]. The resulting cb‐ApDCs, remaining the specific recognition, are stable and can be quickly taken into cells. The drug ratio in cb‐ApDCs can be easily controlled to strengthen the synergistic effect without complex chemistry. These cb‐ApDCs offer a promising strategy for targeted anticancer therapy and hold the potential to stimulate the advancement of novel drug combinations for combinatorial cancer therapy.
In addition to small molecular drugs, various functional groups, such as fluorescent dyes, nanoparticles, enzymes, antibodies, and peptides, have been conjugated into DNA aptamers, which enriches their diversity and functionality. For example, by attaching fluorescent dyes to a DNA aptamer, the binding of the DNA aptamer with its target can be visualized and quantified in real time. This approach contributes to surveying the localization and dynamics of proteins in cells and provides insights into cellular signaling pathways [60], significantly promoting our understanding of cellular processes and novel discoveries in cell biology. Also, fluorescent dye‐conjugated DNA aptamers can be used to detect specific molecules in complex systems as biosensing devices.
Figure 1.7 (a) Schematic illustration of chemical modification of Sgc‐8 aptamer with gallium‐68 radiolabel. (b) Whole‐body dynamic imaging of the gallium‐68 (68Ga) radiolabeled aptamer‐injected patient at different time points post administration.
Source: Ding et al. [100]/American Association for the Advancement of Science/CC BY 4.0.
Recently, Ding et al. developed a gallium‐68 (68Ga) radiolabeled aptamer (Figure 1.7a) to address the limited knowledge of the biosafety and metabolism patterns of DNA aptamers in the human body, which has impeded their clinical application in precision medicine [100]. The first‐in‐human pharmacokinetics study of the Sgc‐8 aptamer was conducted using state‐of‐the‐art total‐body positron emission tomography (PET) technology. As shown in Figure 1.7b, the study captured dynamic distribution patterns of the aptamer in the human body, and reveals that the radiolabeled aptamer is safe for normal organs. The majority of the aptamer accumulated in the kidneys and was subsequently eliminated from the body through urine. Furthermore, a physiologically based pharmacokinetic model for the aptamer was developed, demonstrating its potential for predicting therapeutic responses and facilitating personalized treatment strategies.
1.4.3 Self‐assembly Systems Based on Chemically Modified DNA Aptamers
Besides the simple covalent modification of aptamers, self‐assembly systems based on chemically modified aptamers hold promise to enhance the cellular uptake, stability, and specificity of aptamers toward their targets. Such systems have been exploited for their potential in targeted drug delivery and imaging applications. The conjugation of highly hydrophilic DNA aptamers with hydrophobic small‐molecule drugs results in the formation of amphiphilic ApDC. These amphiphilic conjugates can self‐assemble into DNA aptamer‐based micelle structures with a hydrophobic drug core and a hydrophilic DNA aptamer shield, suggesting a multivalent effect and an enhanced aptamer‐target binding ability [101]. Such a supramolecular assembly could be a high‐efficacy transport platform for cell imaging and drug delivery. Besides the multivalent effect induced by molecular self‐assembly, Geng et al. reported a multivalent aptamer drug conjugate (ApMDC) [102] synthesized upon the coupling of a hydrophilic DNA aptamer with a hydrophobic monodendron anchored with four anticancer drugs (Figure 1.8). Amphiphilic ApMDC and its PEG‐substituted analog synergistically assemble into nanomicelles, which display precise drug loading and tunable surface density of aptamers for optimal complementation between blood circulation and tumor‐targeting ability. The released drug from the consequent degradation of nanomicelles induced by the acidic tumor microenvironment reinforces the immunogenic cell death of tumor cells. With these merits, ApMDC nanomicelles represent a robust platform for structure‐function optimization of drug conjugates and nanomedicines.
Steadily increasing attention has been paid to the orchestration of supramolecular assemblies by using amphiphilic molecules due to their broad biological applications [103]. Upon the covalent coupling of hydrophilic DNA aptamer with hydrophobic lipids, the generated amphiphilic aptamer‐lipid conjugates can self‐assemble into DNA aptamer‐based micelles, vesicles, and other complex assemblies, exhibiting enhanced target binding ability as a result of their multivalent effect, trim sizes, and increased cell permeability and carrier capacity. Therefore, DNA aptamer‐based assemblies could be broadly applicable in nanobiotechnology, cell biology, and drug delivery systems. Our group pioneered the construction of DNA aptamer‐based micelles. In 2009, they reported the development of TDO5 aptamer diacyllipid amphiphilic micelles [104] composed of a hydrophilic DNA aptamer head and a hydrophobic lipid tail. This study implies their potential as a targeted drug delivery system and a selective detection tool. One of the advantages of DNA aptamer‐conjugated assemblies is their modularity and tunability. By altering assemblies’ composition, size, and shape as well as the sequence and orientation of DNA aptamers, assemblies’ properties can be fine‐tuned to meet specific requirements. Recently, Li et al. presented a new strategy for constructing stable and specific aptamer‐lipid micelles [105]. The DNA aptamer and lipid fragments were linked to a methacrylamide branch, which covalently crosslinks the aptamer‐lipid micelle under photoirradiation, to increase the DNA aptamer’s biostability.
Another advantage of DNA aptamer‐conjugated assemblies is their biocompatibility and biodegradability. Unlike other nanoparticle‐based systems, DNA aptamer‐conjugate assemblies are usually non‐toxic and metabolizable via normal metabolic pathways in the body, providing an ideal platform for various biomedical applications, including in vivo imaging and targeted drug delivery. For example, an aptamer‐diacyllipid conjugate was synthesized by covalent coupling of an Sgc8 aptamer with a diacyllipid tail via a PEG linker [106]. Exosomes loaded with chemotherapeutic drugs were functionalized with this aptamer‐diacyllipid conjugate, leading to aptamer‐functionalized exosomes (Apt‐Exos) for targeting delivery to cancer cells. Due to the natural delivery advantages of exosomes and specific molecular recognition properties of DNA aptamers, Apt‐Exos can act as an efficient delivery tool for targeted cancer theranostics, Figure 1.9a.
Figure 1.8 Schematic representation of the co‐assemblies of a multivalent aptamer drug conjugate (ApMDC) and its PEG‐substituted analog into nanomicelles for reinforcing the immunogenic cell death of tumor cells.
Source: Reproduced with permission from Geng et al. [102]. © 2021 John Wiley & Sons.
Figure 1.9 Schematic illustration of the design of (a) aptamer‐functionalized exosomes (Apt‐Exos) and (b) aptamer‐cholesterol‐modified vesicle for targeting delivery to cancer cells.
Source: (a) Reproduced with permission from Zou et al. [106]. 2019 American Chemical Society. (b) Reproduced with permission from Luo et al. [107]. 2019 American Chemical Society.
Beyond the natural exosomes, biomimetic liposomes were also integrated with DNA aptamers for targeted therapeutics [108]
