Next-Generation DNA-Functionalized Quantum Dots as Biological

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Next-Generation DNA-Functionalized Quantum Dots as Biological Sensors Ganglin Wang, Zhi Li, and Nan Ma ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00887 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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ACS Chemical Biology

Next-Generation DNA-Functionalized Quantum Dots as Biological Sensors Ganglin Wang, Zhi Li, Nan Ma* The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China

Abstract DNA-functionalized quantum dots (DNA-QDs) have found considerable applications for biosensing and bioimaging. Different from the first generation (I-G) DNA-QDs prepared via conventional bioconjugation chemistry, the second generation (II-G) DNA-QDs prepared via one-step DNA-templated QD synthesis features defined number of DNA valencies (usually monovalency), which is preferable for controlled assembly and biological targeting. In this review we summarize recent progress in designing QD probes based on II-G DNA-QDs for advanced sensing and imaging applications. It opens up new avenues for high-sensitive and intelligent sensing of a range of diseaserelevant biomolecules in vitro and in living cells. Quantum dots (QDs), an important class of inorganic semiconductor nanocrystals, have been extensively applied to biosensing and bioimaging studies during the past twenty years.1-10 Compared to traditional organic fluorophores, QDs feature broad absorption spectra, large extinction coefficient, narrow and symmetric photoluminescence (PL) spectra, high quantum yield, tunable emission wavelength, and robust photostability,11-13 which enable a range of practical applications such as multiplex detection and imaging,14-18 fluorescence resonance energy transfer (FRET)-based biosensing,19-22 long-term cell imaging,23-26 single molecule tracking,27-30 and near-infrared (NIR) in vivo imaging.31-34 Surface functionalization is a crucial step to afford QDs high specificity towards a variety of biological targets. Conventional QD functionalization strategy is a multi-step process which usually involves ligand exchange/encapsulation35-40 and bioconjugation.41-44 These protocols could generate stable biofunctionalized QDs with bright PL for bioimaging. However, aside from its complexity, little control over QDs avidity is achieved via conventional strategies,45 which is problematic for precise modulating QD assembly and QD-biotarget interactions. The next generation

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QDs should feature both defined number of QD valencies for assembly and biotargeting, as well as generalizability for interacting with different types of biological targets. Besides metal chalcogenide QDs, metal nanoclusters such as DNA-stabilized silver nanoclusters are another type of widely used inorganic fluorophores for biosensing applications.46-48 The related topic has been discussed in detail in an excellent review article.49 DNA-Functionalized QDs: From first generation to second generation DNA molecules have proven to be powerful and versatile molecules for biotargeting.50,51 DNA aptamers could specifically bind to a range of biomolecules including nucleic acids,51 proteins,52,53 enzymes,54,55 cell surface receptors,56,57 and growth factors58,59 with high affinities. DNA aptamers possess several advantages such as high stability, small size, easy modification, and low cost. It is expected that DNA aptamers toward any biological targets could be generated through in vitro selection from a DNA library.60-62 Therefore, DNA-functionalized QDs could potentially serve as universal sensing and imaging probes by rationally tailoring the bio-recognition sequence.63-65 The first generation DNA-functionalized QDs (I-G DNA-QDs) are prepared via traditional bioconjugation chemistry using DNA molecules with 5’ or 3’-end modifier of amine, thiol or carboxylate groups.66-69 It produces a DNA-QD spherical nanostructure with numerous DNA molecules attached on the centered QD. The number of DNA on each QD is not uniform but follows a Poisson distribution.70 Additionally, the QDs prepared via conventional synthetic method usually have a thick protecting layer from either the inorganic or the polymeric shell,71,72 which may lead to a relatively large separation distance between FERT donor and acceptor to impede efficient energy transfer of FRET-based QD probes.73 The second generation DNA-functionalized QDs (II-G DNA-QDs), which is pioneered by Kelley group and Sargent group at University of Toronto, is prepared through a one-step approach using DNA molecules as templates to direct QD growth.74 Specifically, a chimeric DNA molecule containing a phosphorothioate (ps) domain and a natural phosphate (po) domain is used as a template for aqueous synthesis of QDs (Figure 1a). The prepared QDs are found to be preferentially associated with the ps domain because of the high affinity between sulphur atom and cadmium ions; and the phosphate domain is left free on the QD surface for biotargeting. Meanwhile, additional small thiol-


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containing molecules are introduced as co-ligands during the synthesis to passivate the unoccupied surface metal atoms to improve QD stability and quantum yield. The as-prepared II-G DNA-QDs could be directly used to target nucleic acids molecules or further functionalized with DNA aptamers through DNA hybridization to target protein or cell surface receptors (Figure 1b). Importantly, these II-G DNAQDs are mostly homogeneous with a single DNA molecule on each QD (monovalency). A later milestone work shows that the QD valency (the number of DNA molecules on each QD) could be precisely tuned between one and five by tailoring the length of the ps domain or the size of QDs.75 These DNA-QDs could be assembled to higher order QD nanostructures with defined patterns. Another fascinating aspect of nucleic acids-templated QD growth is that the QD size and emission wavelength could be rationally tuned using different DNA sequences or three-dimensional structures of RNA molecules.75-79 These DNA-QDs exhibit high biocompatibility and low cytotoxicity, ensuring their applicability for biosensing and bioimaging applications.74,79-86 The monovalent DNA-QDs represent the basic form of a II-G DNA-QD probe for biotargeting and biosensing. In many cases a series of derivatives are needed for specific applications. For example, a polyvalent DNA-QD probe is constructed by co-polymerization of DNA-QDs and DNA aptamers via hybridization chain reaction (Figure 1c).84 These polyvalent aptamer-QD polymers have a length scale of a few hundred nanometers for multivalent binding with cancer cell surface receptors. These polymeric QDs possess much higher binding affinity with cancer cells than monovalent QDs, which makes it possible to light up cancer cells with high selectivity at very low QD concentration. Heterobivalent DNA-QDs are constructed using a po-ps-po DNA template for QD synthesis (Figure 1c).82 These heterobivalent DNA-QDs could be used to sequentially target two types of spatially isolated cancer markers of live cancer cells. Ternary QDs assembly is constructed by assembling three DNA-QDs with distinct emission wavelengths (blue, green, red) onto a DNA template (Figure 1c). These ternary QDs assembly has been applied for molecular computation–based bioanalysis.87 DNAtemplated gold nanoparticle (GNP) – QDs complex is constructed by assembling DNA-QDs and DNAGNP onto a DNA linker, which yields a satellite nanostructure with multiple QDs tethered to one GNP (Figure 1c). These GNP-QDs complex has been applied for catalytic sensing and imaging of lowabundant miRNA molecules.83,86 The DNA-QDs could also self-assemble into quantum dot DNA hydrogels (QDH) with the aid of Y-shape DNA interconnectors. The QDH could be further conjugated



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with cancer therapeutics such as doxorubicin and siRNA for simultaneous cancer imaging and cancer therapy with greatly enhanced potencies.85 FRET-Based Intracellular Biosensing In situ imaging of biological targets in living cells requires unambiguous signals that could be easily distinguished from background. While this could be easily achieved for cell surface targets by washing off unbound QD probes, it remains a challenge to image intracellular targets since the free intracellular QD probes could not be simply removed. To this end FRET-based QD probes are designed for imaging of intracellular biological targets whereby the QD-target binding event triggers fluorescence signal changes. The II-G DNA-QDs possess a minimal sized protection layer due to direct passivation of DNA molecule on QD surface, and as a result the distance between FRET donor and acceptor could be minimized (< 5 nm) for efficient FRET. So far a series of DNA-templated QD FRET probes have been developed, which could be categorized into QD-fluorophore FRET probe,82 QD-QD FRET probe,87 and QD-GNP FRET probe (Figure 2).83,86 The QD-fluorophore FRET probe has the QD as FRET donor and the fluorophore as FRET acceptor. A large overlap between QD emission spectrum and fluorophore absorption spectrum is a prerequisite for efficient FRET. Upon ultraviolet (UV) excitation the energy absorbed by the QD could partially transfer to the adjacent fluorophore through non-radiative resonance to allow the fluorophore to emit light (Figure 2b). In most cases the fluorophore is not directly excited, and thus dissociation of fluorophore from the QD leads to complete signal off of fluorophore (Figure 2b).82 A limitation of QD-fluorophore FRET pair is the small Stokes shift of the fluorophore molecule, which inevitably leads to partial emission overlap between QD and fluorophore in FRET spectrum. The QD-QD FRET probe contains two or more QDs with different emission wavelengths. The QD with shorter emission wavelength serves as FRET donor and the QD with longer emission wavelength serves as FRET acceptor. Efficient FRET could occur between QDs with large separation of emission spectra because of the broad absorption spectrum of QDs. Disassembly of the QDs leads to recovery of donor QD PL and partial decrease of acceptor QD PL due to direct excitation of acceptor (Figure 2c).87 The GNP-QD complex has QDs as FRET donors and the GNP as FRET acceptor. QD PL is efficiently quenched by the GNP within the complex due to the large extinction coefficient of GNP. Disassembly of QDs and GNP leads to significant recovery of QD PL, which provides a platform with very low signal background for high-sensitive biomolecule sensing (Figure 2d).83


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Intracellular sensing of cancer biomarkers (e.g. mRNA) requires efficient delivery of QD probes into cytosol. This challenge has been tackled using a heterobivalent QD probe capable of targeting cell surface nucleolin and intracellular mRNA.82 Targeting nucleolin with AS1411 aptamer results in efficient macropinocytosis, and the internalized QD probes in leaky macropinosome subsequently translocate to cytosol for mRNA sensing (Figure 3). This strategy could bypass lysosomal sequestration, a main route identified for endocytosis-mediated nanoparticle uptake. The mRNAtargeting motif of the QD probe contains a FRET pair of CdTe QD and Cy5. The mRNA target could displace the short Cy5-labeled DNA pre-hybridized on the DNA template through strand displacement reaction (SDR) to turn off the FRET signal. Confocal microscopy is used to monitor the intracellular QD-Cy5 FRET signal for mRNA sensing. Additionally, other elegant approaches using organic fluorophore reporters have been applied to mRNA sensing in living cells.88-92 For example, a nucleaseresistant molecular beacon was used to visualize the location and movements of mRNA in living cells.88 DNA-functionalized gold nanoparticles were used as nanoflares for multiplex mRNA imaging in living cells.90,91 Signal amplification-based strategy was developed to image mRNA in living cells with improved sensitivity.92 These strategies could be potentially adapted to II-G DNA-QDs to achieve sensitive, multiplexed, and long-term tracking of mRNA molecules in living cells. Catalytic Biosensing Intracellular sensing of low-abundance cancer biomarkers such as miRNA molecules necessities DNA-QD probes with high detection sensitivity. Conventional molecular beacon based on one-to-one probe-target binding has a limit of detection in nanomolar range,93-95 which is not capable of detecting nucleic acids with picomolar intracellular concentrations. To resolve this challenge, a GNPQD complex is constructed for catalytic detection of low-abundance miRNA molecules in live cancer cells with a detection sensitivity three orders of magnitude higher than that of the conventional molecular beacons.83 In this strategy the miRNA target serves as a catalyst to sequentially disassemble multiple QDs from the centered GNP through entropy-driven DNA strand displacement reactions (SDR) (Figure 4a). In the first SDR the miRNA binds to the end toehold and displaces the DNA-QD from the DNA linker, during which the QD PL is recovered. In the second SDR fuel DNA binds to the middle toehold and displaces both DNA-GNP and miRNA from the DNA linker. The released miRNA then participates in the next round of SDRs (Figure 4b). On the basis of this strategy, high-sensitive



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detection of miRNA molecules in vitro and in live cancer cells is accomplished. This strategy was later adopted by different research groups for sensitive imaging of miRNA in living cells.96,97 Additionally, other DNA-based signal amplification strategies such as DNAzyme and hybridization chain reaction were applied for miRNA imaging in living cells.98,99 While most of these strategies utilize organic fluorophores as reporters, integration of II-G DNA-QDs into these systems could potentially offer stronger and persistent fluorescence signals to improve detection sensitivity and enable long-term imaging. Intelligent Biosensing In many cases a disease state is determined by the abnormal expression levels (either high or low) of a few disease-relevant nucleic acids molecules (e.g. mRNA and miRNA) that act synergistically.100-103 Simultaneous detection of multiple nucleic acids targets could improve the diagnosis accuracy in comparison with single target detection.103-105 Conventional strategy relies on measuring the expression level of each nucleic acids target, and then analyzing the overall outcome manually to conclude the risk of a disease. However, it is usually time- and labor-intensive, and may cause significant bias due to personal error. Intelligent molecular diagnostics has been established to automate the diagnostic process by integrating Boolean logic computing function into a molecular probe.106 For example, an AND logic gate probe could simultaneously detect two targets with high expression levels (i.e. input: 1, 1) and generate a positive decision (i.e. output: 1). DNA-QDs turn out to be superior candidates for intelligent diagnostics. It benefits from the powerful DNA computing capability and the unique optical properties of QDs. A complete set of seven elementary logic gates (OR, AND, NOR, NAND, INH, XOR, XNOR) has been realized using a series of binary and ternary QD complexes operated by SDRs.87 The QD complex is constructed by assembling two or three DNA-QDs with distinct emission wavelengths (blue (B): ZnCdSe QD; green (G): CdTe QD; red (R): ZnHgSe QD) on a DNA template. A toehold is inserted into each DNA-QD to enable SDR. Addition of certain nucleic acids target would cause disassembly or reassembly of QD complex, which is accompanied by QD FRET signal changes (Figure 5a). This DNA-programmed dynamic assembly of QDs provides a general platform for molecular computation based on elementary or integrated logic gates using nucleic acids molecules as inputs and QD PL as outputs. The ultimate goal is to generate a QD nanocomputer with integrated logic gates to perform multiplexed intelligent


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diagnostics in situ (Figure 5b). Conclusions and Outlook Since developed ten years ago, II-G DNA-QDs have found considerable applications for sensing and imaging of clinical-relevant biological targets in vitro and in living cells. It solves the longstanding challenges of controlling QD valencies that dictate their assembly and interaction with biological targets. So far the research has been mainly focused on sensing overexpressed cell surface receptors and cancer-relevant RNA molecules. It could be potentially applied to detect other types of important biomolecules using specific DNA aptamers. One future direction is to develop near-infrared (NIR) light emitting DNA-QDs for in vivo sensing of biological targets. Another direction, which might be more challenging, is to develop DNA-QD-based autonomous nanodevices that is capable of performing intelligent diagnostics and delivering therapeutics on demand for personalized medicine. Keywords Quantum dot: semiconductor nanoparticle (typically 1~10 nm) with size-dependent optical and electronic properties different from those of bulk materials. DNA: deoxyribonucleic acid, a natural biopolymer that carries genetic information. Valency: the number of complexation sites available on the surface of a given quantum dot. Biosensing: the use of an analytic device to measure physiological activity or detect biomolecules. Photoluminescence: light emission from a matter after the absorption of photons. Quantum yield: number of emitted photons occurring per number of absorbed photons. mRNA: messenger RNA, a family of RNA molecules that convey genetic information from DNA to the ribosome. miRNA: microRNA, a family of small non-coding RNA molecules (~22 nucleotides) that act as crucial regulators of gene expression. Molecular computation: the use of molecules to operate Boolean logic gates and build computer programs.



Figure 1. Schematic illustration of II-G DNA-QD and its derivatives. (a) Chemical structure of phosphorothioate linkage and phosphate linkage of the chimeric DNA template for QD synthesis. (b) Binding of II-G DNA-QD with complementary DNA target or specific protein target. (c) Schematic illustration of II-G DNA-QD derivatives including heterobivalent DNA-QD, polyvalent DNA-QD, ternary QDs assembly, and GNP-QDs assembly.


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Figure 2. Optical spectra of QDs and QD-based FRET probes. (a) Absorption and photoluminescence spectra of DNA-CdTe QDs under UV excitation. (b-d) FRET and non-FRET emission spectra of (b) CdTe QD-Cy5 FRET probe; (c) ZnCdSe QD-CdTe QD FRET probe; (d) CdTe QD-GNP FRET probe under UV excitation.



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Figure 3. Schematic illustration of the delivery route of the heterobivalent DNA-QD probe for imaging cell surface nucleolin and intracellular mRNA.


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Figure 4. Schematic illustration of catalytic detection of miRNA molecules using DNA-templated GNP-QD complex. (a) Catalytic disassembly of QDs and GNP using miRNA as a catalyst. (b) Mechanisms of the entropy-driven two-step SDRs for catalytic disassembly. Reproduced with permission from ref. 83 and 86.



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Figure 5. Schematic illustration of DNA-templated QDs complex for molecular computation. (a) DNA-programmed dynamic assembly of a ternary QDs complex for nucleic acids detection. Reproduced with permission from ref. 87. (b) Intelligent sensing of multiple nucleic acids targets using an integrated QD nanocomputer for disease diagnostics.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Acknowledgment Work in Nan Ma group is supported by the NSFC (21175147, 91313302, 21475093, 21522506), the National High-Tech R&D Program (2014AA020518), 1000-Young Talents Plan, PAPD, and startup funds from Soochow University.

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