Functional Nucleic Acid Nanomaterials: Development, Properties, and Applications

Abstract

Functional nucleic acid (FNA) nanotechnology is an interdisciplinary field between nucleic acid biochemistry and nanotechnology that focuses on the study of interactions between FNAs and nanomaterials and explores the particular advantages and applications of FNA nanomaterials. With the goal of building the next-generation biomaterials that combine the advantages of FNAs and nanomaterials, the interactions between FNAs and nanomaterials as well as FNA self-assembly technologies have established themselves as hot research areas, where the target recognition, response, and self-assembly ability, combined with the plasmon properties, stability, stimuli-response, and delivery potential of various nanomaterials can give rise to a variety of novel fascinating applications. As research on the structural and functional group features of FNAs and nanomaterials rapidly develops, many laboratories have reported numerous methods to construct FNA nanomaterials. In this Review, we first introduce some widely used FNAs and nanomaterials along with their classification, structure, and application features. Then we discuss the most successful methods employing FNAs and nanomaterials as elements for creating advanced FNA nanomaterials. Finally, we review the extensive applications of FNA nanomaterials in bioimaging, biosensing, biomedicine, and other important fields, with their own advantages and drawbacks, and provide our perspective about the issues and developing trends in FNA nanotechnology.

Graphical Abstract

1. Introduction

Nobel laureate Richard P. Feynman first introduced the world to nanotechnology in his famous lecture “There’s Plenty of Room at the Bottom” in 1960. Since then, we have observed major research breakthroughs and developments in nanoscale-related physics, chemistry, and biology. Nanotechnology is commonly defined as creating materials with fundamentally new properties and functions on the order of nanometers. So far, nanotechnology mainly encompasses two approaches: i) the “top-down” approach, in which large structures are reduced in size to nanoscale and ii) the “bottom-up” approach, also called “molecular nanotechnology”,^[1] in which materials are engineered from atoms or molecular components through a process of assembly or self-assembly. The two approaches of nanotechnology hold great potential for breakthroughs in medicine, food, energy, biotechnology, information technology, and national security.

With the rapid development of nanotechnology, the most important research and development trends tend to focus on four topics: i) synthesis, characterization, and structure simulation and prediction for nanomaterials; ii) functionalization of nanomaterials; iii) construction of nanoelements, which can be utilized in various kinds of sensors based on the light, electrical, or catalyzed properties of the nanomaterials used; and iv) preparation of nanomachines which utilize nanotechnology to connect different nanomaterials to make them perform mechanical work on the nanometer scale.^[2] In order to better deal with the above topics and promote the development of nanotechnology, the introduction of some new elements for nanotechnology is imperative and even some biological elements have already contributed to the new vitality of nanotechnology. In particular, the combination of nucleic acids and nanotechnology has always attracted the attention of laboratories around the world.

Based on their important functions of storing and transmitting genetic information, nucleic acids are the most basic building blocks of life. In 1953, the discovery of the principle of complementary base-pairing and the DNA double-helix structure opened the era of molecular biology and extended the research on the genetic function of DNA to the molecular level. In the 1990s, more and more non-genetic functions of nucleic acids started to attract the interest of researchers; the first representatives were RNA aptamers of organic dyes and a DNA aptamer of thrombin (Figure 1).^[3] The family of non-genetic nucleic acids continues to grow, and thus the concept of “functional nucleic acids (FNAs)” came into being. FNA is a generic name for nucleic acids and nucleic acid analogue molecules, including DNAzymes,^[4] aptamers,^[3a,b,4b,5] DNA tiles, and DNA origami,^[6] and other types of unconventional nucleic acids (Figure 1),^[3c,7] that can replace traditional proteases and antibodies, possess independent structural functions, and perform specific biological non-genetic functions.^{[3a–c,4a,b,8]} The concept of FNA was also used to generalize the non-genetic functions of nucleic acids (Figure 1).^[9] Meanwhile, research on the combination and functions of FNAs, such as DNAzymes, aptazymes, and aptamers, and nanomaterials, especially gold nanomaterials in vitro and in cells, has progressed (Figure 1).^[10] In recent years, FNAs have been applied to various biological fields such as molecular imaging, biomolecule detection, and nucleic acid self-assembly and have shown particular advantages.^[6c] Further, the well-arranged hydrophobic/hydrophilic and easily modified functional groups, flexible structures, high efficiency catalytic capacity, and the potential of constructing complex structures and patterns of various FNAs also have encouraged more and more researchers to introduce FNAs into nanotechnology, and they have laid good foundations for the combination of FNAs with nanomaterials.^[11] The functional group distribution, charge characteristics, and structural features of the nanomaterials determine how they can be combined with various FNA. In general, while nanomaterials provide stable platforms for the binding, cleavage, linkage, folding, unfolding, and delivery of FNAs, FNAs improve the targeting and delivery ability of nanomaterials, and greatly broaden their application fields. Therefore, a comprehensive review is needed to summarize and analyze all the progress in the FNA nanomaterials field, especially the achievements in the preparation and applications of FNA nanomaterials. In this Review, we first discuss the structure and group features of representative FNAs and nanomaterials. Then we summarize mechanisms and advances of combination methods for the preparation of FNA nanomaterials. Finally, widely used applications of FNA nanomaterials are discussed and an outlook of future developments is given.

Figure 1.

Some historic developments and breakthroughs in the fields of FNAs, self-assembled FNA nanostructures, FNA-nanomaterials nanostructures, and nucleic acid amplification reactions. Aptamer (1990).^[3b] Cube (1991).^[12] Peptide nucleic acids (PNAs) (1992).^[7] Thrombin aptamer (1992).^[3d] MicroRNA (1993).^[13] i-motif (1994).^[14] Cleavage DNAzyme (1994).^[4c] Molecular beacon (1996).^[15] DNA-AuNPs conjugation (1996).^[16] DNA-templated AgNCs (1998).^[17] G-quadruplex (1999).^[4b] siRNA (1999).^[7] Catalytic beacon (2000).^[18] Triplex DNA (2003).^[19] DNA-templated CuNCs (2003).^[20] DNAzyme-AuNPs combination (2003).^[10a] Link DNAzyme (2004).^[4a] Hybridization chain reaction (HCR) (2004).^[21] Dendrimer (2004).^[22] Aptazyme-AuNPs combination (2004).^[10b] Tetrahedron (2005).^[6e] In situ aptamer for cancer study (2006).^[23] Hydrogel (2006).^[24] Aptamer-AuNPs combination (2006).^[10c] 2D origami (2006).^[6c] T-Hg²⁺-T (2007).^[25] Mesoporous silica nanoparticles deliver DNA (2007).^[26] C-Ag⁺-C (2008).^[27] Graphene-based DNA biosensor (2009).^[28] “FNA” concept (2009).^[9] Split aptamer (2009).^[29] Aptamer-AuNPs delivery in cells (2009).^[10e] 3D origami (2009).^[6b] DNA-functionalized Au nanoflowers (2010).^[30] Spinach (2011).^[31] Catalyzed hairpin assembly (CHA) (2011).^[32] E. coli DNAzyme (2011).^[5] Tetrahedron delivery in cell (2011).^[33] Digital information storage in DNA (2012). DNA nanoflower (2013).^[34] DNAzyme-AuNPs delivery in cell (2013).^[10d] DNA origami-protein (2014).^[35] Unnatural base pairs (2014). Onpattro (2018).^[36]

2. Functional Nucleic Acids

Nucleic acids are biomolecules that carry the most important genetic information in nature. Since they were discovered late relative to other biological components, great efforts have been made to understand their roles in the survival and function of living systems. In addition to their part in gene-coding substances, many other interesting functions of nucleic acids were discovered, prompting further research by scientists. DNAzymes can catalyze the breakage or linkage of a DNA substrate strand at a specific site,^[3a,4a,37] while aptamers, single-stranded oligonucleotides, can specifically bind to different kinds of target molecules. Nucleic acids possess many unique properties, including their biological functions, biocompatibility,^[38] molecular recognition capacity, and controllability on the nanoscale,^[39] which facilitate the construction of a wide variety of complex inorganic and organic nanostructures.

FNAs are practically “all-powerful” due to their totipotency. FNAs come from several kinds of nucleotides and are easily synthesized. There are more than a dozen basic nucleotides, including A, G, C, T, X, and Y.^[40] As the number of artificial nucleic acids increases, the cost of synthesizing them decreases, and the technology to do so becomes more sophisticated. FNA structures are diverse and can present as single-stranded, double-stranded, triple-stranded, and quadruple-stranded DNA.^[3c,4b] FNAs are highly compatible with various targets including inorganic molecules (such as metal ions and pesticide molecules), inorganic nanomaterials (such as graphene),^[41] biomolecules (such as proteins and lipids), living systems (such as micro-organisms and cultured cells), and bionanomaterials (such as magnetosomes, liposomes, and exosomes).^[42] FNA’s totipotency is one of its most crucial traits.

2.1. DNAzymes

A DNAzyme can be defined as DNA that interacts with other molecules (e.g. metal ions) by virtue of its own secondary structure and possesses catalytic activity that is similar to that of protein enzymes.^[43] DNA- or RNA-cleaving DNAzymes typically have three main components in their secondary structures: two binding arms, a cleavage site, and an enzymatic region (Figure 2A).^[4c] Initially, the discovery of some catalytic properties of RNA in the 1980s and 1990s prompted a widespread search for DNA enzymes.^[3a,44] First, DNAzymes were created by substituting deoxyribonucleotides for ribonucleotides in the catalytic domain of the hammerhead ribozyme, but the efficiency of RNA cleavage was low.^[45] Later, a series of metal-ion- or small-molecule-dependent DNAzymes (including Pb²⁺, Mg²⁺, Zn²⁺, Ca²⁺, and histidine dependent) were obtained by in vitro selection.^[5j] To obtain DNAzymes with high efficiency and desired properties, it was necessary to optimize parameters and criteria for their selection, which in turn defined the conditions of in vitro selection.^[46] So far, many DNAzymes have been obtained through evolutionary in vitro selection processes, in which a large randomized DNA library of up to 10¹⁵ different sequences is subjected to a series of selection steps to isolate catalytically active DNA sequences. After several rounds of selection, typically between 5 and 20 rounds with PCR amplification steps between each round, DNAzymes with high affinity and selectivity for their target molecules are gradually enriched. In addition, by shortening reaction times and/or decreasing target molecule concentrations, DNAzymes with different binding affinity and sensitivity can be obtained.^[47] Owing to their controllable specificity and sensitivity, the relatively low cost of synthesis, chemical stability, and ease of functionalization through chemical modifications of DNAzymes, researchers have attached DNAzymes to nanomaterials and other molecular signaling groups to realize rapid and precise detection and imaging of various metal ions.^[48] Meanwhile, by exploiting the flexible structural features of DNAzymes, some researchers also utilized DNAzymes in the sensing of nucleic acids or non-nucleic acid targets.^[49]

Figure 2.

Various kinds of FNAs: DNAzymes, aptamers, triplex DNA assemblies, DNA tiles, DNA origami. A) Schematic illustration of the basic structure of DNAzymes including binding arms, cleavage site, and enzymatic region.^[4c] B) Some typical aptamer structures.^[3b] C) Schematic illustration of parallel and antiparallel structures and formation mechanism of triplex DNA assembly.^[60] D) Schematic illustration of some basic building elements of DNA tiles.^[75] E) Design principles for 3D DNA origami with complex curvatures.^[76]

The foremost impact of the discovery of DNAzymes is the realization of enzyme-free catalytic reactions and overcoming of the dependence on naturally occurring enzymes. As a result, DNAzymes have been widely used as switches in biosensors as well as in the field of bioimaging.

2.2. Aptamers

In 1990, two research groups reported on the use of aptamers to target proteins and small ligands with high affinity.^[3b,50] Instead of using any known sequences, the selection of aptamers was based on libraries of random RNA or DNA sequences (Figure 2B).^[3b] Similar to DNAzymes, aptamers are also obtained by an in vitro selection method called SELEX (short for Systematic Evolution of Ligands by EXponential enrichment).^[51] The selection starts with a large population of single-stranded nucleic acid molecules, followed by an iterative in vitro selection by incubating with certain targets. After subsequent amplification through polymerase chain reactions, the aptamers are enriched and have high affinity and specificity for their targets.^[52] During the past few decades, achievements have been made in the optimization of aptamers. In 2001, the process of in vitro selection was automated.^[53] In 2004, capillary electrophoresis-SELEX (CE-SELEX) was used for efficient selection of aptamers in vitro.^[54] Differences in the specific charges between components result in different electrophoretic mobilities, which are used to achieve the separation of components. Utilizing CE-SELEX technology, a methyl morpholine aptamer with good combination ability was successfully obtained.^[55] In 2005, fluorescence magnetic bead-SELEX (FluMag-SELEX) technology was invented and utilized for quantitative DNA analysis and aptamer selection. The Strehlitz group confirmed that the stem-loop structures play an important role in the target recognition by analyzing the secondary structures of the selected four types of streptavidin aptamers by FluMag-SELEX (Figure 2B).^[56] In 2010, cell-SELEX was introduced to generate aptamers that can bind specifically to a cell type of interest.^[57]

Aptamers boast advantages that distinguish them from antibodies. For example, aptamers: i) can be easily and economically obtained; ii) can be subjected to a selection process noted for the low toxicity and low immunogenicity of the targets; iii) can have better specificity and affinity than antibodies; iv) are easy to be modified chemically; v) have better thermal stability; vi) can be linked with other drugs for combination therapy; vii) possess features which can be easily tailored, for example by splitting, deleting, substitution, fusion, and elongation,^[58] to improve performance, particularly regarding realizing replacements for protein-based antibodies and the direct detection of small molecules as a result of the development of split aptamer technology; and viii) include varieties such as Mango and Spinach, which can be used to readily couple the binding of small molecules to enhance fluorescent signals. Therefore, they can be used as specific nucleic acid probes in vivo or as fluorescence switch elements in biosensors.

Since aptamers are inherently thermally stable, they can be subjected to numerous rounds of denaturation and renaturation. This allows for easy regeneration and repeated use when they are exploited in various kinds of biosensors. In the absence of targets, there is no difference between aptamers and ordinary nucleic acids. The presence of targets induces the conformational change of the aptamer. When aptamers are combined with some nanomaterials, aptamers’ conformational change may separate some groups of nanomaterials and result in a charge change and subsequent change of recorded potential. This property has been used in the electrochemical detection of various target types.^[59]

2.3. Triplex DNA

Triplex DNA, which is formed through Hoogsteen interactions with double-helix DNA,^[60] is another important member of the FNA family. Triplex DNA can be classified as parallel triplex structures formed by C-G·C⁺, T-A·T, A-T·G, and G-C·T, and antiparallel triplex structures, such as C-G·G, T-A·A, T-A·T, and C-G·A⁺ (Figure 2C). The stability of triplex DNA assemblies is affected by many parameters, such as the number and kind of the triplex base pairs, mutations or mispairings in the triplex domains, pH value, and the presence of ions or binders. For example, acidic conditions promote the protonation of C, thereby improving the stability of the C-G·C⁺ base pair, while neutral conditions improve the stability of the T-A·T base pair.^[61] The presence of Mg²⁺ affects the formation of triplex DNA because it is necessary to form the duplex structure within the triplex DNA.^[62] Phenanthrene and pyrene can be used as linkers to connect the duplex structure and the third strand to promote the formation of triplex DNA.^[63]

In the past ten years, triplex DNA has been widely used in sensing techniques. These approaches utilize triplex DNA not merely as a recognition element, but also as a functional structure-switching unit that allows the generation of the output signal upon target recognition. Therefore, the detected targets involving triplex DNA are not limited to specific nucleic acid sequences, but cover a wide array of molecular targets, including antibodies, proteins, heavy metal ions, and small molecules.^[64] In the presence of the target DNA sequence, the formation of the loop domain-target DNA duplex induces the disassembly of the triplex DNA domain and the opening of the triplex-stabilized hairpin, thus leading to an increased fluorescence as the readout signal.^[64d] This sensor’s specificity and sensitivity can be modulated by changing the number of T-A·T base pairs in the stem region.

In 2015, triplex DNA was observed directly via atomic force microscopy (AFM).^[65] Recently, nanosensors based on triplex DNA assembly demonstrated two tendencies. To start, triplex DNA assemblies combine with different metal nanoparticles to realize signal transduction. To give one example, a triplex DNA-mediated SERS sensor has been established based on a silver nanoparticle (AgNPs)-modified gold film that serves as the intensive base.^[66] Researchers built another triplex DNA-mediated SERS nanosensor, using streptavidin-coated silicon beads as SERS-active hot spots.^[67] The advantage of this strategy is that nanosensors with different signals can be set up to satisfy diverse detection demands. Additionally, triplex DNA possesses the ability to regulate the assembly and disassembly of different nanomaterials, such as origami frames, catenanes, microcapsules, and metal–organic frameworks (MOFs),^[68] based on the pH-dependence of triplex DNA base pairs. This strategy is highly promising in cell sensing and drug delivery applications because there is a pH difference between healthy cells and cancer cells.

2.4. DNA Tiles and DNA Origami

DNA tiles and DNA origami are two basic elements in nucleic acid self-assembly technology for the construction of well-organized and stable pure nucleic acid nanostructures. At the beginning exploration stage, some original DNA hybridization-based nanostructures used the principle of multiple crossover junctions to impart sufficient rigidity to achieve directional interactions. As shown in Figure 2D (top), two strand trade portions to produce a simple crossover motif. In the 1990s, scientists started to design DNA tiles with branches in order to construct interesting 2D nanostructures. Seeman and colleagues first introduced the idea by combining the sticky-end cohesion and branched DNA junctions to make geometric objects and periodic 2D lattices.^[6] By joining two double helixes together through strand exchange, they constructed a group of branched complexes called double-crossover (DX) molecules (Figure 2D bottom).^[69] With proper sticky-ended design, the DX molecules were encouraged to self-assemble into periodic 2D lattices. Following this exciting development, myriad rigid, branched DNA tile molecules were designed and constructed to self-assemble into 2D and even 3D lattices. These included triple-crossovers (TX) (Figure 2D bottom); Holliday junctions (HJ), paranemic crossovers (PX), and JX₂, all derived from double helices (Figure 2D bottom);^[70] as well as four-helix, eight-helix, and twelve-helix planar tiles; three helix and six helix bundles; parallelogram DNA junctions; cross-shaped DNA tiles; and triangular and three-point star motifs.^[71] Although there are numerous forms of these molecules, those used for DNA tile construction typically have their crossovers formed between strands of opposite polarity. Rhombohedral lattices, tetrahedrons, icosahedrons, and buckyballs were all knitted by these basic helical domains.^[72]

To date, the application of DNA tiles focuses primarily on two important aspects. First, dynamic devices based on DNA tiles are usually employed to study the interaction between proteins and DNA or between two proteins. Second, metallic, semiconductive, and magnetic nanomaterials can be organized into well-defined categories by various kinds of DNA tiles to develop or improve electronic device building blocks and chemical biosensors.

The invention of DNA tile technology greatly improved the development of DNA nanotechnology. However, because the synthesis of tile-based nanostructures involves interactions between a large number of short oligonucleotides, the yield of complete nanostructures is highly sensitive to the relative ratios of strands. Therefore, the synthesis of relatively complex nanostructures was thought to require multiple reaction steps and purifications, with the yields limited by the complexity of DNA nanostructures.^[6c] The DNA origami technique helps address some problems associated with the DNA tile method.^[6c] DNA origami is a type of DNA-based nanostructure that utilizes a long DNA strand as a molecular scaffold, along with many small staple strands. DNA origami can act as an ideal platform for sensing based on its intrinsic addressability and shape controllability and has been widely used in many fields. For example, DNA origami can help detect many kinds of targets or base-pair mismatches, such as RNA, prostate-specific antigens (PSA), and single-nucleotide polymorphisms (SNP).^[73]

Based on the construction method of 2D DNA origami, various 3D DNA origami nanostructures were also constructed and play more and more vital roles in DNA nanotechnology. The fundamental concept of 3D DNA origami with complex curvature construction methods is imagining scaffold strands being rasterized and forming suitable shape sheets (Figure 2E).^[74] 3D DNA origami nanostructures present the most advanced design concepts and relatively difficult fabrication approaches that cannot be easily duplicated. Detailed construction principles, structural features, and applications of 3D DNA nanostructures will be discussed in Section 4.5.

3. Nanomaterials That Can Bind to FNAs

3.1. Metal-Based Nanomaterials

Metal-based nanomaterials are frequently combined with FNAs. This combination give FNAs greater stability, nanoplasmonic properties, and new catalytic activities. Furthermore, FNA also give metal-based nanomaterials better biocompatibility and change their charge characteristics. The fundamental combination approach is to use metal-based nanomaterials as templates for immobilization and orientation of FNAs. In addition, the FNA-templated generation of metal nanoclusters also is an important preparation method for FNA–metal nanomaterials.^[17] Applications of FNA–metal nanomaterials are found in biosensing, bioimaging, and biomedicine. Meanwhile, FNAs also endow metal nanomaterials with precise targeting ability.

3.1.1. Au Nanomaterials

Au nanomaterials are commonly used as signal amplification labels or delivery carriers for various kinds of sensors and assays methods (Figure 3A). Based on their charge characteristics, they can form stable structures with FNAs by different approaches. Some basic combination methods for FNAs and gold nanomaterials and corresponding charge characteristics of Au nanomaterials are as follows: i) Coordinate covalent-bond bioconjugation. The formation of stable coordinate covalent bonds between Au and thiols (Au–S bonds) is based on the strong interaction between sulfur atoms and the surfaces of Au nanomaterials (Figure 4A),^[17] and the functionalization of thiol on the 5′ or 3′ end of nucleic acid strands is easy. ii) Electrostatic interactions. Au nanomaterials show reliable stability in aqueous solution, as well as high affinity with positively charged molecules and groups such as the nucleobases of DNA (Figure 4B). iii) Au–polyA interactions. The interaction between gold nanomaterials and a length of polyA sequence is powerful enough to fix FNAs on the surface of gold nanomaterials (Figure 4C);^[83] this is a novel non-labeled combination method between gold nanomaterials and FNA. iv) Etching and self-insertion. In 2010, Lu and co-workers reported that the affinity between DNA and AuNPs played an important role in the deposition and diffusion of Au atoms and morphology control of AuNPs.^[30] Meanwhile, DNA can be in situ attached to AuNP surfaces with high stability (Figure 4D). This is a novel way to functionalize AuNPs in the AuNP preparation process.

Figure 3.

Electron microscope images of various kinds of nanomaterials: A) AuNPs, B) AgNCs,^[77] C) CuNCs,^[78] D) magnetic nanoparticles,^[79] E) magnetosomes, F) Mesoporous silica nanoparticles (MSNs),^[80] G) apoferritins,^[81] H) exosomes, I,J) 2D and 3D MOFs.^[82]

Figure 4.

Interactions of AuNPs and FNAs: A) Au–S bond,^[87] B) single-strand adsorption interaction,^[88] C) AuNPs–polyA interaction,^[83] D) nucleic acid self-insertion interaction.^[30]

There are specific properties that mediate the broad applications of FNA–Au nanomaterials: i) Surface plasmon resonance (SPR). AuNP dispersion and aggregation affects its SPR and appears as a color change and movement of adsorption peak when viewed macroscopically.^[84] ii) Surface-enhanced Raman scattering (SERS). The collective oscillation of surface electrons on metal materials, especially Au nanomaterials, is leveraged for the amplification of optical processes in Raman scattering and the phenomenon of SERS has been widely used for ultrasensitive biological and chemical analysis and environmental sensing.^[85] iii) Fluorescence resonance energy transfer (FRET). When there is obvious spectrum overlap between Au nanomaterials and fluorescence groups, nanomaterials, or proteins as well as appropriate distance, Au nanomaterials can quench their fluorescence with high efficiency.^[86]

3.1.2. Ag or Cu Nanoclusters (NCs)

The principle behind nucleic acid templated generation of metal nanoclusters (NCs) is the process of nucleic acid metallization.^[89] Due to their extremely small size and high surface energy, NCs tend to aggregate, so a stabilizing agent or a matrix must be used to isolate NCs. Before the excellent work by Dickson et al. that used dendrimers as a stabilizer, metal NCs were prepared only in cryogenic noble gases or solid zeolites.^[90] Following their initial discovery, the Dickson group first demonstrated that cytosine (C)-rich DNA can also stabilize fluorescent AgNCs, which set up the connection between metal NCs and DNA.^[91] There is a strong, specific coordination between the N3 position of cytosine and Ag⁺, while C-rich DNA strands show high affinity to Ag nanomaterials. Subsequent work demonstrated that all nucleotides can participate in the binding of metal NCs. Each nucleotide has at least one metal-binding site (Figure 5A). After further research, it was concluded that AgNCs contain roughly the same amount of neutral silver atoms and silver cations, and these Ag species align in a rod-like structure rather than planar or globular structures (Figure 5B).^[83] The presence of different emission colors was attributed to the change of the rod length due to specific base/Ag interactions.

Figure 5.

Formation mechanism of FNA-templated metal NCs. A) Chemical structures of DNA nucleosides and primary metal-coordination sites.^[83] B) Rod-like model of AgNCs with neutral Ag atoms (gray balls) and Ag⁺ cations (blue balls) in poly-C DNA.^[83]

In addition to AgNCs, the formation of DNA, especially polythymine (T) strands, templated with CuNCs also holds great potential for chemical and biological applications. Compared with AgNCs, the formation of CuNCs is faster but with lower controllability. Further, researchers also found that the CuNC fluorescence might be dramatically enhanced with mismatched DNA, even with only one base mismatch.^[99] Thus, research on the generation and fluorescence intensity diversification mechanisms of CuNCs holds great potential for the detection of SNPs.

3.1.3. Bimetallic Nanoparticles (BNPs)

Bimetallic nanoparticles (BNPs) composed of two different metals such as Pt-Pd, Au-Pd, Au-Ag, and Pd-Cu^[92] have generated much interest as flexible and high-efficiency metal-based nanomaterials. In contrast to normal monometallic nanomaterials, BNPs have unique size and structure, specific optical, electronic, and thermal properties, and higher catalytic effects.^[93] In terms of functionalization with FNAs, BNPs are almost the same as monometallic nanomaterials, and the improved catalytic properties of functionalized BNPs mainly arises from FNA’s targeting ability. In BNPs, two different metals combine to show novel properties resulting from the combination. When metals such as silver, nickel, or cobalt, are combined with gold, the catalytic properties can be enhanced. Due to their greater surface area, BNPs have greater adsorption power. As a result, they are more efficient catalysts than monometallic nanoparticles and an ideal FNA bioconjugation platform. Furthermore, some BNPs such as Pd-Au BNPs also were used to discover a “DNA code” to effect the morphology of the nanomaterials.^[94]

3.2. Carbon-Based Nanomaterials

The carbon nanostructures most commonly combined with FNAs are graphene oxide (GO) (Figure 6A), carbon nanotubes (CNTs) (Figure 6B), reduced graphene oxide (rGO), graphene, and nanodiamonds. Because of their large surface area, charge characteristics, and excellent electronic transfer capability, the bases of FNAs interact with carbon-based nanomaterials primarily through van der Waals interactions (π interactions) and solvation energy.^[95] Furthermore, FNAs can be firmly fixed on the surface of various carbon-based nanomaterials. The interaction of FNAs with carbon surfaces may result in significant changes in some biosensing signals. Additionally, carbon-based nanomaterials possess a highly efficient fluorescence quenching ability. Therefore, FNA-carbon nanomaterials are ideal signal recognition elements for the construction of fluorescent biosensors.

Figure 6.

Carbon-based nanomaterials and their functional groups. A) GO with carboxyl and hydroxyl groups.^[98] B) Single-walled and multi-walled CNTs.^[99] C) CNTs with carboxyl groups.^[100] D) Fullerenes with carboxyl groups.^[100]

In common carbon-based nanomaterials, CNTs are built from C(sp²) units with hexagonal honeycomb lattices several nanometers in diameter and many microns in length. As a transducer, CNTs can strongly promote electron transfer between electroactive species and electrodes. Further, CNTs can be easily functionalized with different chemical groups such as −COOH and −OH (Figure 6C),^[96] so a variety of biomolecules are easily conjugated on its surface. Some other carbon-based nanomaterials also have similar structural and group features. For example, fullerenes can be easily functionalized with surface carboxyl groups and have excellent biocompatibility (Figure 6D).^[97]

3.3. Silicon-Based Nanomaterials

Mesoporous silica nanoparticles (MSNs) are among the most widely used kind of nanomaterial, with a porous surface and regulatable pore size and diameters between 2–50 nm. In conventional PBS, PB, or Tris-HCl buffer, MSNs have a negative surface charge.^[101] Through modification with positively charged surface groups, MSNs can easily combine with FNAs for further applications.^[12]

MSNs have desirable surface chemical properties, which make modification with polycations quite easy.^[103] For example, MSNs modified by a carbosilane dendrimer were used to deliver oligonucleotides.^[104] In addition, the abundant hydroxyl groups on the MSN surface make it easy to be functionalized to MSN-NH₂, MSN-COOH,^[105] MSN-N₃,^[106] and MSN-Cl (Figure 7A). The introduction of these groups makes the conjugation of FNAs feasible. MSNs excellent surface activity is one of its foremost qualities as a nanomaterial. The conjugation of FNAs brings forth new features of MSNs: i) The new FNA-MSN nanomaterial is an effective for drug delivery. As shown in Figure 7B, the conjugated i-motif structure and adsorbed cDNA give MSNs stable drug delivery capability.^[107] ii) The targeting ability of FNAs gives the MSN-based probe or delivery system more precise direction in vitro and in vivo.

Figure 7.

Functional groups of silicon-based nanomaterials and interactions of mesoporous silica nanoparticles and FNAs. A) Functionalization of MSN.^[110] B) Schematic illustration of proton-fueled release of guest molecules from the G-rich FNA-MSN.^[105] C) Schematic illustration of bioresponsive release system of magnetic MSNs for electrochemical detection of telomerase activity by using wrapping DNA as a molecular gate.^[107a] D) Schematic illustration of the gene loading into MSNs.^[111]

The sol–gel method is the most widely used for MSN preparation.^{[105, 108]} Meanwhile, as elaborated in Section 3.1, silica is an ideal shell material for constructing composite nanomaterials, and mesoporous silica shell is flexible due to its porosity and loading capability.^[109] As shown in Figure 7C, magnetic MSNs were used as a carrier and ssDNA was used as a wrapper to construct telomerase activity biosensors.^[107] Owing to the controllable pore size, large-pored MSNs also can be synthesized and utilized to deliver siRNA (Figure 7D).

3.4. Bionanomaterials

Exosomes are nanosized extracellular vesicles (40–150 nm) with a membrane lipid bilayer (Figure 8A) and various kinds of biomarkers on the surface and in internal compartments (Figure 8B).^[112] Exosomes can be released by all cells, and can also easily enter other cells.^[113] Exosomes originated from the fusion of multivesicular bodies with the plasma membrane of cells and were then released into the extracellular space (Figure 8B).^[114] In addition to their ability to transfer proteins, exosomes’ ability to transfer nucleic acids between cells should not be ignored. This nucleic acid delivery mechanism is paramount for cell-to-cell communication. Furthermore, exosome transportation capability also provides a theoretical basis for combination applications, such as drug delivery between exosomes and FNAs.

Figure 8.

Structural features of exosomes and apoferritin and their biological applications with FNA. A) Schematic illustration of a targeted exosome model.^[117] B) Schematic illustration of a variety of common biomarkers on the surface (e.g. tetraspanins such as CD9 and CD63 and lipid raft-associated proteins including flotillin-1) as well as the internal compartment (such as Alix and Tsg101) of exosomes.^[112] C) Structure of apoferritin.^[118] D) Preparation of enzyme-labeled FNA-apoferritin detection probe.^[123]

Most exosomes can carry characteristic molecules recognizable by certain cells facilitating selective targeting and adsorption by recipient cells. Some exosomes also show a stronger ability to abscond recognition and clearance or degradation by immune cells.^[115] Early research revealed that the small size of exosomes allows them to penetrate deep inside tissues and they are thus suitable as nanocarriers. Additionally, they display a slightly negative zeta potential for long circulation, possess a deformable cytoskeleton, and show membrane properties almost identical to those of the cell itself.^[116] The delivery of siRNA utilizing exosomes has become a leading approach in therapeutic gene modulation (Figure 8B).^[112,117] With excellent biocompatibility, no overt side effects were found in exosome-mediated gene delivery systems for in vivo application. This approach avoids nonspecific delivery, preventing immunogenicity of the delivery vehicle and reducing nucleic acid degradation.

Apoferritin is a natural iron storage protein composed of 24 polypeptide subunits that form a nearly spherical shell. With an external diameter (12 nm) and an inner cavity diameter (8 nm) (Figure 8C), it can store up to 4500 Fe³⁺ ions.^[118] In total, 14 channels are present among the subunits, with diameters of 3–4 nm. Each channel plays a large role in transfer and exchange between internal and external mass.^[119] Decomposition and assembly are the protein’s distinct features: it can be divided into 24 parts at pH 2, but the subunits reconstitute when the pH reaches 8.5.^[120] As for its unique characteristics and cavity structure, apoferritin has been used widely as a kind of nanoreactor to synthesize size-restricted bioinorganic nanomaterials.^[121]

There are many lysines and cysteines on the surface of apoferritin that have been exploited for chemical conjugation using different heterobifunctional cross-linkers with N-hydroxysuccinimide (NHS) ester and maleimide groups. This makes linking FNAs on the apoferritin surface very convenient.^[122] By linking binding aptamers on the apoferritin surface, electrochemical assays can be constructed based on the encapsulation and release of the metal ions in apoferritin (Figure 8D).^[123] Furthermore, by combination with FNAs, the natural targeting and therapy ability of apoferritin was further improved.^[122a,124]

3.5. Magnetic Nanomaterials

The structural features and application of several kinds of magnetic nanomaterials, including magnetic beads (MBs), magnetic nanozymes, magnetosomes, and other magnetic nanomaterials, are elaborated in this section.

3.5.1. Magnetic Beads (MBs)

MBs are the most widely used magnetic nanomaterial in biosensing and biomedicine. Surface coatings are an integral component of all MBs for FNA modification as well as for surface functionalization. Polymeric surface coatings play an important role in the applications of MBs with FNAs. Although not attracted to each other magnetically, owing to their superparamagnetic properties, MB nanoparticles still have a significant tendency to aggregate as a result of their high surface energy.^[125] Further, polymeric coatings provide abundant binding sites for FNAs. For example, streptavidin can be attached to the surface of MBs, and biotin-modified FNAs can be linked to MBs by the streptavidin–biotin interaction. FNA conjugation sites including amine and carboxyl groups extend out from the polymer surface.

3.5.2. Magnetic Nanozymes

Fe₃O₄ NPs were the first nanomaterials found to have an intrinsic enzymatic activity. In 2007, Gao and colleagues reported that Fe₃O₄ NPs possess an intrinsic enzyme mimetic activity similar to horseradish peroxidases, which are widely used to oxidize organic substrates in the treatment of wastewater or as detection tools. They modified the Fe₃O₄ NPs with antibodies to implement three functions: capture, separation, and detection.^[79] This discovery shifted the traditional perception that inorganic nanomaterials are biologically inert substances. Similarly, FNAs can also be combined with magnetic nanoenzymes and their functions and structures are more flexible than those of antibodies. Other kinds of iron-based metal nanoenzymes, such as Fe₂O₃, FeS, FeSe, CoFe₂O₄, BiFeO₃, and MnFe₂O₄ NPs, also have catalytic activities similar to Fe₃O₄ enzymes.^[126]

3.5.3. Magnetosomes

Bacterial magnetosomes, magnetite (Fe₃O₄), or greigite (Fe₃S₄) nanocrystals, enveloped by a phospholipid bilayer membrane, are formed by magnetosome-producing micro-organisms, including magnetotactic bacteria, as well as by some non-magnetotactic bacteria.^[127] Bacterial magnetosomes are synthesized under strict genetic control with uniform shape, size, dispersion, and chemical composition.^[128] The size of a magnetosome is normally between 35–120 nm (Figure 3E).^[129] In general, magnetosomes are arranged in single or multiple chains or dispersed in the microorganism.^[130] They allow a cell to passively align along magnetic field lines—a behavior known as magnetotaxis. The various crystal morphologies of magnetosomes include cubo-octahedral, hexagonal prism, bullet-shaped, and rectangular morphologies.^[131] The magnetosome membrane contains proteins involved in the synthesis, shape, size, and location of magnetosomes. Some research demonstrates that magnetosomes possess intrinsic peroxidase-like activity, linking the peroxidase-like activity of magnetosomes to the proteins on the magnetosome membrane.^[132] Based on this principle, FNA-coupled magnetosomes can be widely used in the development of liquid-phase or paper-based visual biosensors. Further, there are also many −NH₂ groups on the magnetosome surface, making it easy to bioconjugate FNAs by simple carboxyamidation. In 2015, Borg et al. prepared a ZnO/SiO₂-encapsulated magnetosome core–shell heterojunction nanostructure (Figure 9A).^[133] The novel nanostructure possesses advantages over the component materials: the magnetosome nanosize and the modification ability of silica. This made it possible to product FNA-magnetosome composite nanomaterials by new construction methods.

Figure 9.

Magnetic compound nanomaterials and their applications. A) Schematic illustration of SiO₂/ZnO-magnetosome magnetic hybrid nanoparticle.^[133] B) Route for DNA adsorption onto MSN.^[9]

3.5.4. Magnetic Composite Nanomaterials

Bare magnetic nanoparticles are likely to form large aggregates, have fewer activating groups, and are easily oxidized or dissolved in an acidic medium, which seriously restricts their potential applications as functional materials. To overcome these limitations, a suitable coating on the surface of the bare magnetic nanoparticles is required. In addition to polymeric coatings, silica is very promising as a coating material. Recent explorations of silica have been conducted because magnetite’s surface has a strong affinity towards the material, and no primer is required to promote its deposition and adhesion.^[134] Furthermore, the silica shell stabilizes the magnetic nanoparticles in two different ways. First, it shields the magnetic dipole interaction. Secondly, it attracts negative charges onto the surface of the shells, which enhances the coulomb repulsion of the magnetic particles.^[135] The silica shell is easily modified with −NH₂, −Cl, and −N₃ functionalities, so it is easy to attach FNAs to the surface of the silica/magnetic core–shell structure. The novel FNA magnetic nanomaterial possesses the function of a magnetic nucleic acid probe and can also be used for magnetic hyperthermia or magnetic resonance imaging (MRI) of tumors or other diseases through the targeting function of FNAs (Figure 9B).

Au can also be used as the shell of a core–shell composite nanostructure, due to the superparamagnetic properties of the magnetic nanoparticles andthe easy functionalization and fluorescence quenching ability of the Au shell.^[136] Furthermore, AgNP decoration on the surface of magnetic nanoparticles leads to an important composite magnetic nanomaterial that can enhance SERS sensitivity and simplify the operation process.^[137]

3.6. Quantum Dots (QDs)

QDs are semiconductor nanoparticles that are so small that quantum confinement occurs in all three dimensions. The first systematic dynamic investigations of interfacial electron and hole processes, as well as reports on the size-dependent optical properties of QDs in solution, date back to the early 1980s.^[138] QDs can be divided into several types, including metal-iondoped inorganic semiconductor QDs, metal nanoparticles and nanoclusters, silicon QDs, carbon QDs, and graphene QDs.^[139] QDs have received much attention owing to their unique, highly sensitive optical and electronic properties.

FNAs can be easily introduced onto the surface of QDs based on their abundant surface amino and carboxyl groups (Figure 10A,B).^[140] Based on the FRET effect and the tunable, narrow-band emission and broad excitation spectral features of QDs, fluorescence emission and quenching occur quickly between QDs and the fluorescence groups attached to the FNAs, such that the fluorescent FNA-QD nanomaterials can be applied in bioimaging and biosensing.^[141] As shown in Figure 10C,D, the exponential amplification reaction (EXPAR) and rolling-circle amplification (RCA) were separately combined with QDs for the detection of microRNA by utilizing the FRET effect between QDs and fluorescence groups.^[142]

Figure 10.

Surface functional groups of composite nanomaterials and their applications with FNA. A) QD with amino groups on surface.^[140b] B) QD with carboxyl groups on surface.^[140a] C) Schematic illustration of a detection method based on QDs combined with EXPAR.^[149] D) Schematic illustration of a detection method based on QDs combined with RCA.^[142a] E) MOF with azido groups on surface.^[150] F) MOF with amino groups on surface.^[150] G) Schematic illustration and characterization of the encapsulation and unblocking process of a dye/drug loaded MOF.^[72] H) Principle behind the preparation of the i-motif gated MOF and its pH-stimulated switchable release ability.^[146] I) Principle behind the preparation of the triplex DNA assembly gated MOF and its pH-stimulated switchable release ability.^[146]

3.7. Composite Nanomaterials

MOFs are a widely used nanomaterial self-assembled from metal ions and organic linkers under relatively gentle conditions.^[143] Amines, carboxylic acids, nitrates, sulfonates, and phosphates are the most commonly used organic ligands, and there are abundant corresponding chemical groups on MOF surfaces. It is easy to transform these groups and attach biomacromolecules to them (Figure 10E,F). The specific features of MOFs include ultrahigh porosity, excellent structural adjustability, large internal surface area, diversity in structure, high chemical stability, and strong thermal stability.^[144] MOF structures can be 1D, 2D (Figure 3I), or 3D (Figure 3J).^[145] In terms of interaction with nucleic acids, many MOFs show obvious fluorescence quenching ability for dye-labeled single-strand DNA (ssDNA).^[143b]

Combined with FNAs, the most commonly used property of MOFs is their excellent loading capability, so FNAs are often used as a logic switch to give MOFs a stimuli-response encapsulation and release ability. As shown in Figure 10G–I, C-rich DNA strands and triplex DNA structures were used as logic switches to control the stimuli-response release. The flexibility of FNAs thus give MOFs intelligent functions.^[146]

The combinations of composite nanomaterials are abundant, and the core construction principle is in exerting all the nanomaterials’ unique advantages. For example, Jurado-Sanchez and colleagues invented a Janus micromotor, which consists of polycaprolactone (PCL) semiconducting polymers, graphene quantum dots (GQDs), and Pt/Fe₃O₄ NPs.^[147] This Janus micromotor efficiently is propelled in the presence of H₂O₂ or magnetic actuation without the addition of other chemical fuels. The construction of Janus particles is inspiring: by conjugation with FNAs, a micromotor probe with both autonomous movement and capture capability can be prepared and utilized in environmental and life sciences.

Furthermore, some researchers also reported some remarkable synergistic catalysis effects of FNA-peptide composite nanomaterials. For example, in 2017, Liu et al. designed a hybrid system that assembled G-rich DNA strands and histidine-rich peptides into a sticky composite nanomaterial, which possesses enzyme-like hierarchical active sites for synergistic catalytic activity.^[148]

4. Interactions between FNAs and Nanomaterials

FNA nanomaterials are novel composite nanomaterials formed by the combination of various FNAs (including DNAzymes, aptamers, triplex DNA assemblies, and DNA tiles and origami) and nanomaterials (such as metal nanomaterials, carbon and silicon-based nanomaterials, bionanomaterials, and composite nanomaterials) or controllable and programmatic self-assembly of FNAs. FNA and nanomaterials can be prepared in a myriad of ways, including bioconjugation, adsorption interaction, FNA-templated assembly, FNA–nanomaterials heterojunction, and FNA self-assembly. Among them, the heterojunction of FNA and nanomaterials, and the FNA self-assembled nanostructures are especially noteworthy. There are a number of traditional conjugation methods, including bioconjugation and adsorption interactions. For example, the click reaction hemistry has been widely used and is known for its efficiency and reliability. The required groups, including azido and alkynyl functions, are easily modifiable on the surface or at the 5’ and 3’ nucleic acid ends, making this approach an attractive bioconjugation method. With respect to the adsorption interaction, A-rich sequences adsorbed on the surface of Au nanomaterials, which result from the affinity between adenine and Au, further command researcher interest. In Section 4, we separately elaborate the mechanism and advantages of the five methods for the preparation of FNA nanomaterials.

4.1. Bioconjugation

Although many methods for the preparation of FNA nanomaterials, bioconjugation is the most common and widely used. As described in Section 3.1.1, formation of a Au–S covalent bond is a well-known and highly efficient bioconjugation method. It can be used to conjugate FNAs on the surface of AuNPs. In the conjugation process, through the gradually increase of the ionic strength, the negatively charged phosphate backbone of nucleic acids strands could be effectively screened, and the Au–S bond facilitates the formation of extremely dense monolayers of oligonucleotides on gold nanoparticle surface.^[151] Most AuNP-DNA nano-motors are prepared by this method.^[152] Based on the same principle, Ag–S or Pt–S covalent bonds can be used to fix FNAs on the surface of noble metal NPs.^[153] In conclusion, the metal–S covalent bond is stable and is formed quickly. The activated nucleic acids, labeled with a thiol group, need only to be incubated with metal NPs and no other operations are necessary.

Functional groups on FNAs can also be linked to the surface of carbon-based nanomaterials. Generally, the bioconjugation of FNAs on carbon-based nanomaterials is based on the interaction between amino and carboxyl groups. This reaction is easy to achieve utilizing a well-known carbodiimide-mediated wet-chemistry approach.^[45] The binding of nucleic acid strands may result in significant changes in the electrochemical signal. This mechanism has been used to sense DNA or RNA with high sensitivity and single-base specificity. In conclusion, FNA-carbon-based nanomaterials possess not only the targeting ability of FNAs, but also the ion-to-electron transduction ability of single-walled CNTs or GO.^[45]

The bioconjugation between FNAs and MSNs differs somewhat. Although ssDNA can be fixed on the MSN surface via covalent bonds such as amide bonds,^[108a,154] the Li group indicated three main driving forces of DNA–MSN combination when they studied the mechanism of DNA adsorption, including electrostatic shielding effect, dehydration, and the intermolecular hydrogen bonding.^[101] As mentioned before, FNA-MSNs are novel nanomaterials for automatic encapsulation and delivery.

The click reaction is an important bioconjugation method. Owing to its stability, efficiency, and moderate reaction conditions, the click reaction can be carried out over a wide pH value range and under various buffer conditions. Nucleic acid strands labeled with click groups can be firmly fixed on the nanomaterial surface, or with other nucleic acid strands, within 2 h at room temperature.^[106,155] The catalyzed click reaction is simple and inexpensive; the reaction proceeds smoothly in the presence of Cu²⁺ and sodium ascorbate.

4.2. Adsorption Interactions

Other promising methods for the preparation of FNA nanomaterials are important include adsorption mediated by electrostatic interactions and van der Waals interactions (π–π stacking interactions).^[95] For metal-based nanomaterials, their surface charges determine the adsorption properties. For AuNPs, the negatively charged surface favors the attractive binding of positively charged nucleobases hanging from ssDNA,^[88] while the binding between gold nanomaterials and dsDNA is weak due to the occultation of nucleobases in the phosphor-deoxyribose backbone of double-stranded DNA (dsDNA). Similarly, carbon-based nanomaterials also possess FNA adsorption ability: bases of FNA interact with the surface of carbon-based nanomaterials through electrostatic interactions and π–π stacking interactions and solvation energy provided by the solvent molecules. Graphene’s large 2D aromatic surface structure makes it an ideal substrate for the adsorption of nucleic acids and other biomolecules. For 1D or 2D carbon-based nanomaterials such as single-walled CNTs or GO, the π–π stacking interaction often occurs between their surface and the nitrogen bases of nucleic acid strands. Since the nitrogen bases of ssDNA are exposed, there is a significant difference in the strength of adsorption interactions between carbon-based nanomaterials and single- or double-stranded nucleic acids. Utilizing this property, researchers have developed many FNA-carbon-based nanomaterial probes for quick detection of genetically modified products or microRNA. In addition, some researchers even constructed stable GO-FNA hydrogels based on the π–π stacking interaction, which utilize natural nucleic acids from animal sources.^[156]

Many studies demonstrate that affinity of the poly-A nucleic acids with metal nanomaterials is much stronger than that of poly-G, poly-C, and poly-T nucleic acid strands.^[157] Therefore, the poly-A strands can be used as ideal binding elements for the preparation of FNA nanomaterials. The use of poly-A strands is highly efficient; some researchers reported that the immobilization of FNA probes by poly-A strands can be accomplished in just 10 min.^[158]

4.3. FNA-Templated Assembly

As described in Section 3.1.2, AgNCs and CuNCs are formed by FNA-templated assembly and well-defined organization of metal atoms. The FNA-templated assembly of AgNCs brings results in some important fluorescence features: i) AgNCs with different emission colors, ranging from blue to near- infrared (NIR), with high quantum yield (QY) could be obtained by simply changing the sequence of DNA (Figure 11A);^[159] ii) the same AgNCs might emit different colors in different environments; iii) the emitted fluorescence of a few AgNCs might be enhanced upon hybridizing with a DNA overhang containing different base sequences; iv) G-rich overhang sequences have excellent fluorescence enhancement ability (Figure 11B).^[159]

Figure 11.

FNA-templated metal NCs and their emission sequences. A) A few DNA sequences with their templated, different color fluorescent AgNCs.^[159] B) Design principle behind the AgNCs beacon G-rich DNA probe design.^[163] C) Methods for the construction of DNA-templated AgNCs.^[159] D) FNA-AgNCs biosensors based on nucleic acid hybridization related to changing the local environment of the AgNCs.^[159]

In the assemby of metal NCs, FNAs should not only be considered as a self-assembly template, but also an important molecular recognition element of probes. Compared with traditional protein-based biosensors, the FNA-templated metal NCs are more flexible and possess more advanced signal transmission, amplification, and output potentials: i) the easy-to-prepare FNA-templated metal NCs are potential alternatives for complicated fluorescence labels and modifications. ii) Some specific nucleic acid sequences were needed for generation or to enhance the fluorescence signal of NCs in some cases, so it is possible to achieve signal amplification based on a designed FNA probe with special sequences and “turn-on” secondary structures. For example, AgNCs were used as signal output element to detect specific nucleic acid sequences (Figure 11C) and thrombin (Figure 11D).^[160] iii) Certain species or chemicals, such as heavy metal ions or thiolated compounds, can modulate the fluorescence color and intensity of metal NCs. Based on this breakthrough, the applications of FNA-templated metal NCs can be broadened beyond nucleic acids. Furthermore, AgNCs and CuNCs have their own unique features in their generation. The generation time of AgNCs is much longer than that of CuNCs and the formation of CuNCs has two different steps. At low ion concentration, copper ions prefer to interact with the backbone phosphate negative groups through nonspecific electrostatic attraction.^[161] With increasing concentration, the Cu²⁺ ions begin to bind to DNA bases, with much higher affinity than to the phosphate groups, and are further reduced by ascorbic acids to form fluorescent CuNCs.^[162] In addition, the generation of CuNCs depends largely on the poly-T nucleic acid sequences and indicates a clear relationship to the length of the nucleic acid sequence.

4.4. FNA-Nanomaterial Heterojunctions

The FNA-nanomaterial heterojunction nanostructures are a new type of composite nanostructure that is prepared by producing nucleic acid hydrogels with uniform thickness on the surface of various inorganic nanomaterials, such as MSNs and MOFs.^[164] The most commonly observed feature of these FNA-nanomaterial heterojunction nanostructures is their drug delivery capability, in which the nanomaterial serves as the carriers and FNA serves as the encapsulation gate. As shown in Figure 12, in 2018, Willner and colleagues prepared nucleic acid based polyacrylamide hydrogel-coated MOF nanoparticles for controlling drug encapsulation and release.^[164] In this heterojunction nanostructure, the formation of the hydrogel is stimulated by crosslinking two polyacrylamide chains, functionalized with nucleic acid hairpins. Meanwhile, the resulting duplex-bridged hydrogel, which included an anti-ATP aptamer sequence in a caged configuration, encapsulated drugs in the pores of the MOF. Overall, the construction of FNA-nanomaterial heterojunction promotes the drug loading ability of porous nanomaterials and overcomes the nonspecific leakage of drugs. Further, the formation and disintegration of FNA hydrogels is controllable, thus providing drug target delivery and sustained release. A magnetic FNA hydrogel-silica heterojunction nanostructure was also prepared by the Yang group. Similar to the FNA hydrogel-MOF nanomaterial, the DNA hydrogel was prepared on the surface of magnetic silica composite nanoparticles.^[165]

Figure 12.

Typical formation process of FNA-nanomaterial heterojunction and electron microscope images.^[150]

4.5. FNA Self-Assembly

Utilizing the “bottom-up” fabrication strategies, self-assembly-based DNA nanotechnology provides a purist’s approach to create various 2D and 3D structures in different sizes or shapes, such as tetrahedra, triangular prisms, octahedra, icosahedra, and other complicated DNA origami structures.^[6b,165] These designed self-assembled FNA nanostructures inherit the advantages of nucleic acids, particularly their small size, high programmability, capacity for multiple functionalizations with high addressability, and excellent biocompatibility.

The development of self-assembled FNA nanomaterials started with Seeman’s construction of artificial, branched DNA nanostructures. The four sequence-specific oligomeric nucleic acid strands with extended sticky ends at each arm of the ssDNA strands self-assemble into an immobile four-way junction.^[167] However, branched DNA junctions have low rigidity, and DNA branched junction-based assembly does not often yield a regular structure. Therefore, to address these issues, researchers established modified branched DNA junctions to generate rigid and defined nanostructures, such as DX and TX techniques.^[168] These approaches were hugely successful in the fabrication of DNA nanostructures. In 2004, Rothemund first introduced the strong flexibility and simplicity of DNA origami technology to the nanomaterial field. As shown in Figure 13A, the square, rectangle, pentagram, and smiley face patterns were all prepared by DNA origami technology.^[6c] At the beginning of the self-assembly process, the staple strands bind to and crossover onto the scaffold strand, thus strengthening the scaffold strand into an addressable shape that can display desired patterns on its surface.^[6c,166a] Once the staple strands are prepared, the DNA origami nanostructure develops when all of the staple strands and the scaffold are simply mixed in a buffered solution and allowed to slowly cool. Staple strands can be modified with a stem-loop or a dumbbell hairpin to provide pixels for surface patterning of origami structures with local height differences detectable by AFM.

Figure 13.

Self-assembled 2D and 3D FNA nanostructures. A) Various kinds of 2D DNA origami shapes.^[6c] B) DNA nanostructures with complex 3D curvatures.^[76] C) Schematic illustration of the controlled opening of the 3D DNA box lid.^[6b] D) Schematic illustration of the construction of thrombin-loaded nanorobot by DNA origami, and its reconfiguration into a rectangular DNA sheet in response to nucleolin binding.^[165] E) Strand-level diagram of a square DNA origami Mona Lisa pattern.^[166a]

After the discovery that each DNA unit had the ability to bind four neighboring DNA units, 3D DNA assembly technology boomed. Various 3D DNA nanostructures were designed and constructed. Among these nanostructures, the DNA tetrahedron is a classic three-dimensional framework.^[6e] Turberfield and colleagues developed a rapid, single-step synthesis method of this platonic solid, in which only an annealing process was needed. To form the tetrahedron, four well-designed oligonucleotides are mixed in equimolar amounts in a salt-containing buffer solution. When the reaction mixture is heated to 95°C for several minutes and then quickly cooled to room temperature, a regular tetrahedron with six edges and four vertexes can be successfully assembled with high yield.^[6e] In addition to the DNA tetrahedron, other 3D DNA nanostructures were also prepared and characterized. As shown in Figure 13B, a self-assembled DNA nanostructure with complex curvature was constructed using the DNA origami folding technology.^[76] Double-helical DNA is bent to follow the rounded contours of the target object, and potential strand crossovers are subsequently identified. Concentric rings of DNA are used to generate in-plane curvature, constrained to two dimensions by rationally designed geometries and crossover networks. A series of DNA nanostructures with high curvature and the ellipsoidal shells shown in Figure 13B is one example.

In 2009, Andersen and colleagues created an addressable DNA box that can be opened in the presence of externally supplied DNA “keys” drawn from the principle of complementary base pairing (Figure 13C).^[6b] Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications. Meanwhile, the close and open mechanism of the DNA box inspired researchers to construct iterations of complicated and multifunctional 3D nanocargos and nanorobots. To give an example, the nucleolin-targeting aptamer serves as a targeting domain and as a molecular trigger for the mechanical opening of the DNA nanorobot (Figure 13 D). The thrombin inside is thus exposed and activates coagulation at the tumor site. Apart from these, DNA origami technology also can be used to create DNA arrays of larger sizes, as well as those with arbitrary patterns. Tikhomirov et al. drew various patterns and images, including the Mona Lisa, a rooster, and bacteria by producing DNA origami arrays with sizes up to 0.5 μm² and with up to 8704 pixels (Figure 13E).^[166a] These DNA nanostructures could also facilitate the study of molecular interactions in chemical and biological systems through the construction of spatially organized molecular networks which could be used as molecular devices with information-processing capability that are more complex than previously possible,^[169] or lead to the construction of more sophisticated structural components in DNA robots and localized DNA circuits.

5. Applications of FNA-Nanomaterial Composites

FNA nanomaterials primarily make use of the targeting, signal conversion, and amplification ability of FNAs as well as the stability and multifunctionality of nanomaterials. The foremost applications of FNA nanomaterials fall into several main categories, including i) bioimaging, which precisely image microRNAs, metal ions, gene damage, etc.; ii) biosensing, which utilizes FNAs for signal conversion and amplification and nanomaterials as a sensing platform to detect abundant types of targets; iii) biomedical, which includes early disease diagnosis and drug delivery; iv) information storage, which represents the most advanced application of FNAs.^[170]

5.1. Bioimaging

So far, the bioimaging function of FNA nanomaterials has greatly promoted the development of early disease diagnosis and lesion imaging. Targeting ability and fluorescence restoration are the key points of FNA-nanomaterial bioimaging. The basic principle of bioimaging focuses on the specific affinity between targets (including tumor cells, proteins, metal ions, or specific DNA sequences) and probes, as well as effective delivery and restoration of fluorescence substances. Therefore, FNA nanomaterials perfectly meet the requirements of bioimaging. Nanomaterials prevent FNA from degrading by the action of various nucleases in vivo, and FNAs provide targeting ability, fluorescence signal generation, and amplification ability for nanomaterials. Some nanomaterials, such as MNPs, also possess photothermal effects that further activate the release of fluorescence substances.

Metal NPs, such as AuNPs, are selected as a basis for immobilization and cleavage of DNAzymes. By attaching fluorescence groups at the 5′-end of the DNAzyme’s substrate strands, we can explore the positioning of metal ions. Meanwhile, the AuNPs’ size also allows them to be transported into cells by endocytosis. This is the theoretical basis of AuNP-FNA bioimaging in cells. As shown in Figure 14A, in 2017, Peng and colleagues reported a DNAzyme motor that operates in living cells in response to a specific intracellular target.^[171] The intracellular interaction of a target molecule with the motor system initiates the autonomous walking of the motor on the AuNP. By utilizing a fuel-strand-triggered cleavage of substrate strands, this AuNP-DNAzyme motor, which is responsive to a specific microRNA, realized the amplified detection and bioimaging of the specific microRNA in individual cancer cells. In the combination of metal nanoparticles with DNAzymes, nanoparticles provide a stable reaction platform, but meanwhile, the degree of dispersion of nanoparticles in cells and the efficiency of the DNAzyme cleavage on AuNP surfaces still need further research.

Figure 14.

Bioimaging applications of FNA-nanomaterials. (A) Intracellular operation of a DNAzyme motor initiated by a specific miRNA.^[171] (B) Bioassisted, sequence-independent self-assembly of multicolor FRET bioimaging DNA nanoflowers.^[172] (C) Schematic illustration of the G-based fluorescence enhanced bioimaging DNA templated AgNCs.^[173] (D) Schematic illustration of the preparation of FNA-upconversion nanoprobes and application in monitoring microRNA-21 in living cells.^[174]

Self-assembled FNA nanomaterials are ideal bioimaging elements due to their excellent biocompatibility and self-assembly ability. In 2014, Hu et al. designed DNA nanoflowers containing an aptamer sequence and three types of fluorescent groups which exhibited multifluorescence emissions through RCA.^[172] The assembly of nanoflowers was independent of template sequences, avoiding the otherwise complicated design of DNA building blocks assembled into nanostructures by base-pairing (Figure 14 B). Furthermore, the nanoflowers were uniform and exhibited high fluorescence intensity and excellent photostability.

FNA-templated metal NCs can also be used to realize fluorescence imaging due to their fluorescence characteristics. As shown in Figure 14C, the Wang group studied the G-quadruplex-enhanced fluorescence of DNA-templated AgNCs and reached several significant conclusions. By combining the AS1411 aptamer with G-rich DNA strands, they designed AgNC-modified nucleic acid probes for cancer cell bioimaging.^[173] Of course, the stability of DNA probes determines the accuracy and efficiency of bioimaging, and the use of AgNCs will increase the stability of DNA probes in vivo.

We also note the bioimaging applications of upconversion nanomaterials. As shown in Figure 14D, some types of FNA probes were combined with upconversion nanoparticles (UCNPs).^[174] The non-enzymatic reactions between FNA probes resulted in the lighting up of UCNPs with a high detection signal gain. The large anti-Stokes shift, low background fluorescence, minimal photodamage, negligible biological toxicity, and high photostability of UCNPs make them an effective element for preparing FNA-nanomaterial bioimaging probes.

5.2. Biosensing

FNA nanomaterials appear most often in biosensing applications. Biosensing occurs in four steps: signal sensing, signal conversion, signal amplification, and signal output. Each step involves FNAs. In the signal sensing step, the targeting ability and cleavage capability ensure the precise capture of targets. In the signal conversion step, various chemical groups are labeled, utilizing fluorescence features of some FNAs such as is found in the fluorescent aptamers Mango and Spinach, or as found with other photometrical sources. The fluorescent and luminous properties of various nanomaterials and FNA nanomaterials convert abstract molecule signals into realistic visual, fluorescent, or electrochemical signals. As a result, various types of biosensors were constructed. The cleavage and extension of FNA strands help biosensors realize the signal amplification process. In the signal output step, various FNA-nanomaterial biosensors are classified as visual, fluorescent, or electrochemical biosensors to denote their detection principle, sensing process, and sensitivity. Similarly, nanomaterials also play an important role in the construction of biosensors. For example, nano-plasmonics with surface plasmon resonance can concentrate light fields into the deep sub-wavelength scale owing to the strong interaction between light fields and the collective oscillation and resonance of electrons at the surface of metallic nanostructures.^[175] Other examples show macroscopic phenomena such as fluorescence quenching and color change, which provide excellent platforms for signal conversion and signal amplification for the construction of biosensors.

5.2.1. FNA-Nanomaterial Visual Biosensors

It is necessary to review visual biosensors based on a colorimetric approach while analyzing their ideal characteristics. Current technology based on colorimetry focuses on miniaturization, cost, use in situ, and not using additional instruments. For FNA-nanomaterial visual biosensors, nanomaterials such as Au, Ag, and Cu are significant because of their surface plasmon resonance (SPR). Generally, the detection mechanism of FNA-nanomaterial visual biosensors is based on molecular interactions on the nanomaterial surface, which is modified, functionalized, or coated by FNAs or certain functional groups.

AuNPs are typical optical materials that display distance-dependent surface plasmon properties, resulting in strong color changes that rival, or even exceed, the color changes of organic dyes. Single-stranded and double-stranded DNA can mediate the dispersion and aggregation of AuNPs. As shown in Figure 15A, by utilizing the difference of ssDNA and dsDNA to mediate the degree of AuNP aggregation, the Xu group designed a double target detection method.^[176] However, we note that the degree of aggregation of AuNPs is affected by many factors, such as the photothermal effect, ionic strength, and nucleic acid strand concentration. Therefore, in order to ensure the accuracy of this detection method, it is extremely important to control these other factors. Meanwhile, this also inspired another new type of biosensor based on the fact that some nanomaterials or FNAs, which possess HRP-like catalytic activity, also could be utilized as biosensor signal conversion moieties and amplification elements, and their catalytic property excludes interference from other factors. As shown in Figure 15B, the Xu group designed Au nanorod based visual biosensors for the detection of telomerase activity, which utilized the catalytic ability of FNAs.^[177] In this visual biosensor, they used the catalytic activity of G-quadruplexes to induce the color change of Au nanorods.

Figure 15.

FNA-nanomaterial visual biosensors. A) Construction method of the AuNP-based two-way visual biosensing system.^[176] B) Principle behind the multicolor visual detection of telomerase based on catalytic hairpin assembly and etching of Au nanorods.^[177] C) Fabrication of the visual RCA DNA origami biosensor.^[78]

By transferring detection mechanisms from the liquid phase to paper-based, point-of-care FNA nanomaterials, visual strip biosensors, can be designed to detect a specific analyte in a given sample, just like traditional lateral flow immunoassay strips. Just like liquid-phase detection mechanisms, various nanoparticles (such as metal nanoparticles, carbon-based nanoparticles, quantum dots, lanthanides, and up-converting phosphors) can be used to functionalize FNAs. Lateral flow technology was applied for fast and low-cost detection of various targets. FNA-nanomaterial visual strip biosensors are defined by the movement of the liquid sample containing the analyte of interest along a strip. Whereas the basic capture principle of the immunoassay involves the specific binding reaction between antigens and antibodies, the FNA strips utilize the interaction between FNAs and nanomaterials, or FNA linkage and cleavage ability. Compared to antibodies, FNAs are more flexible and even possess the ability of signal conversion and output. By combining various FNAs, such as aptamers and G-quadruplexes, with nanomaterials (especially nanozymes, which possess high catalytic ability), we have constructed various lateral flow FNA strips for the quick detection of genetically modified products, microRNA, metal ions, and pathogens.^[178]

Self-assembled FNA nanostructures can also be used as a biosensor detection platform. As shown in Figure 15C, Yan and colleagues exploited rolling-circle amplification (RCA) and DNA origami to design a sensitive biosensor suitable for the detection of prostate-specific antigen (PSA). In this design, they first fabricated DNA nanostructures by using RCA. After amplification with phi29 polymerase, single-stranded DNA containing 96-base periodic units could be seen. Next, the long, RCA-based ssDNA scaffold was folded into DNA belts with staple strands. Biotin recognition sites were designed to be incorporated into the DNA belts in the folding process for signal amplification via the avidin–HRP conjugation. Finally, DNA belt nanostructures were employed in novel signal amplification, then combined with the MB-based ELISA strategy for improved PSA detection. Based on the stability and the ease of functionalization of DNA origami, it is an excellent platform for the HRP catalytic reaction.

5.2.2. FNA-Nanomaterial Fluorescent Biosensors

Fluorescence signals are the most common signal output of FNA-nanomaterial biosensors. Based on the specific properties of FNAs and nanomaterials, fluorescent or quenching groups can be attached to the ends of nucleic acids, and fluorescent substances can be encapsulated or loaded by the interaction between FNAs and nanomaterials. As shown in Figure 16A, the Xu group prepared AuNP-DNAzyme molecular motor fluorescent biosensors for the detection of microRNA. In this fluorescent biosensor, the click reaction triggered by miRNA-155 converted the intermolecular hybridization of a Mg²⁺-dependent DNAzyme into an intramolecular hybridization on the surface of AuNPs. The signal amplification came from the circular cleavage of the Mg²⁺-dependent DNAzyme and the retrieval of fluorescence from the free fluorescent group labeled DNA strands.^[53b] This method greatly elevated the stability of AuNP-DNAzyme molecular motors based on conventional strand-triggered DNAzyme circular cleavage. Further, some metal NPs even possess fluorescence quenching ability, while QDs can excite the fluorescence of fluorescent groups by the FRET effect. Compared to other signal output methods, the fluorescence signal is more flexible, diverse, and sensitive. Meanwhile, the combination of metal NPs and QDs could realize two kinds of optical signal detection.

Figure 16.

FNA-nanomaterial fluorescent biosensors. A) Schematic illustration of the AuNP-DNAzyme molecular motor fluorescent biosensor.^[53b] B) Schematic illustration of the proposed fluorescent DNA-templated CuNC biosensor.^[179] C) Fluorescent detection principle of FNA-MOFs.^[181] D) Preparation of FNA-composite nanomaterial fluorescent biosensors.^[183] E) Schematic illustration of the dynamic light–matter AuNP-DNA origami interaction system.^[184]

FNA-templated metal NCs are also excellent elements for the construction of fluorescent biosensors based on their flexibility, simplicity, and high fluorescence intensity. As shown in Figure 16B, nuclease-assisted FNA “turn-on” ATP biosensors were prepared based on CuNCs fluorescence.^[179] Comparing the applications of AgNCs and CuNCs, we can conclude that the color variability of AgNCs make them easy to be used in complex nucleic acid target detection through the design of different DNA probes, while the very short formation time of CuNCs make them suitable for the rapid detection of single targets.

Based on the porous feature of MSNs, the base pairing, cleavage, and structural transformation of FNAs have been utilized as logic gates to control the encapsulation and release of fluorescent agents within the pores of MSNs. Chen et al. utilized the click reaction to conjugate a thrombin aptamer with MSNs, such that the binding of thrombin with its aptamer controlled the release of FITC.^[106] They linked thrombin-containing dsDNA to the surface of MSNs in order to control the encapsulation and release of FITC.^[106]

This method provided a basic platform for sensors based on the combination of MSNs and FNAs. Afterwards, various FNAs, including DNAzymes, aptamers, and even DNA origami, have been used as “goalkeeper” of MSNs.

Since most MOFs have an inherent fluorescence quenching ability, they can quench the fluorescent groups labeled on DNA probes.^[180] Researchers used MOFs and functional nucleic acids to detect DNA, RNA, and other biomolecules or metal ions with fluorescent biosensors. The basic fluorescence quenching and restoration principle is shown in Figure 16C.^[181] In 2013, Zhu et al. proposed the use of MOFs as a sensing platform for the detection of biomolecules. They constructed 2D Cu²⁺-core MOFs for immobilizing DNA fluorescent probes by hydrophobic and π–π interactions between nitrogen bases and MOFs,^[182] utilizing the fluorescence quenching ability of the MOF for the quantitative detection of HIV viral DNA. In addition to MOFs, other composite nanomaterials have also been used in the preparation of fluorescent biosensors. As shown in Figure 16D, a fluorescent biosensor for the sensitive detection of intracellular telomerase activity was prepared by hybridization chain reaction (HCR) and Au@carbon nanospheres.^[183] In an intracellular confocal microscopy study, the Cao group demonstrated that the FNA-composite nanomaterial biosensor can enter into cancer cells such as A549 cells and lead to an increase in luminescence. Furthermore, the Au@carbon composite nanomaterial increased the delivery efficiency of DNA.

Furthermore, as shown in Figure 16E, the Liu group constructed a dynamic light–matter interaction nanosystem enabled by the DNA origami technology. A single fluorophore molecule can autonomously and unidirectionally walk into the hotspot of a plasmonic nanoantenna along a designated origami track. Successive fluorescence intensity increase and lifetime reduction are in situ monitored using single-molecule fluorescence spectroscopy, while the fluorophore walker gradually approaches and eventually enters the plasmonic hotspot.^[184] The dynamic DNA origami platform is a great fluorescent biosensor platform.

5.2.3. FNA-Nanomaterial Electrochemical Biosensors

Electrochemical biosensors are devices that allow information to be obtained with minimal manipulation of the system in situ, generating results that can be analyzed and correlated with other environmental parameters. Electrochemical biosensors are promising candidates for quantitatively detecting various targets because of their high sensitivity, low cost, and the speed of the test.^[185] By utilizing FNAs, selectivity has been further improved. Compared to traditional electrochemical immunoassay biosensors, the FNA-nanomaterial electrochemical biosensors have notable advantages, like higher affinity, lower price, easier fabrication, higher sensitivity, and wider sensing application of analytes. FNAs have shown more stable binding properties in terms of their interaction with electrodes. For the sensitive and selective detection of proteins, DNA, small molecules, cells, metal ions, and tumor biomarkers, FNA-nanomaterial electrochemical biosensors play increasingly important roles.^[186]

In the application of FNA-MSN electrochemical biosensors, there is some variance in detection methods, but many researchers also use MB as a signal molecule to monitor the change of concentration of targets. As shown in Figure 17A, the DNAzyme was used as a lock to control the encapsulation and release of MB. The content of released signal molecules can be converted to the corresponding current signal which positively correlates with the concentration of UO₂²⁺. This method has the advantage of high selectivity, convenience, low cost, and quick completion timeline. Meanwhile, Au-SiO₂ was used in combination with ssDNA and antibodies for the detection of microcystin-LR (Figure 17 B).^[187]

Figure 17.

FNA-nanomaterial electrochemical biosensors. A) Synthesis, MB loading, and dsDNA binding of the MSNs, as well as the release of MB from MSN in the presence of UO₂²⁺.^[191] B) Preparation of FNA-composite nanomaterials and the construction of electrochemical biosensors.^[187] C) Schematic illustration of an electrochemical sensing platform for PSA by coupling with “Z-Scheme” double photosystems and a 3D DNA walker amplification strategy.^[188] D) Schematic illustration of the electrochemical FNA-nanomaterial biosensor.^[189]

Some DNA molecular motor walkers also have been combined with nanomaterials to realize precise electrochemical detection of some targets. The signal amplification function of DNA walkers further improved the sensitivity of electrochemical biosensors. As shown in Figure 17C, a new electrochemical biosensing platform is designed for the ultrasensitive detection of PSA by coupling with 3D DNA walkers. The amplification strategy is based on interactions of hairpin DNA1 conjugated onto the AuNPs, hairpin DNA2 labeled with CdS QDs, and the DNA walker complementary strand with the PSA aptamer attached to a magnetic bead. Upon addition of target, the DNA walker strand is displaced from DNA walker/Apt-MB to open hairpin DNA1 on AuNP@BiVO₄.^[188] Other composite nanomaterials, such as CuO/Au nanomaterials (Figure 17 D), also have been used in FNA-nanomaterial electrochemical biosensors to detect targets with the aid of FNAs for signal amplification.^[189] The combination of multiple nanomaterials provides abundant signal amplification strategies for a stable non-enzymatic FNA reaction platform.

Utilizing DNA origami as the fixed platform for a DNA probe, Liu and colleagues realized the precise detection of lung cancer specific microRNA. The sensing part of the unique 3D DNA probe consisted of a stem-loop structure constructed on the top of a three-dimensional DNA tetrahedron, bearing three thiol groups as anchors on a gold surface.^[190] The other terminal of the stem-loop portion was labeled with a ferrocene tag, giving rise to the electrochemical signal during the detection process. Compared with traditional ssDNA probes, the addition of the DNA tetrahedron gives the electrochemical biosensors more stability owing to the greater number of Au–S bonds on the electrode surface.

5.3. Biomedicine

The molecular recognition and drug delivery ability of FNA nanomaterials can be widely utilized in biomedical fields, which mainly include disease diagnosis and therapy.^[192] Nanomaterials, such as AuNPs, AuNRs, and magnetic NPs, possess well-known advantages and optical properties in synthesis, bioconjugation, lesion imaging, and photothermal therapy that could prove immensely beneficial in biomedical applications. Self-assembled 3D FNA nanostructures, such as DNA origami and DNA hydrogels, possess great biocompatibility, stability, flexibility, and precise programmability, as well as switchable properties and facile synthesis and modification.^[193] These materials are also promising for use in areas like diseases analysis and drug delivery.

5.3.1. Disease Diagnostics

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor the enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In 2017, Qing and colleagues reported a new strategy for label-free monitoring of PNK and ligase activity by using dumbbell-shaped DNA-templated CuNCs.^[194] In the presence of PNK and ligase, the dumbbell-shaped DNA probe was locked and could resist the digestion of exonucleases, and then serve as an efficient template for synthesizing fluorescent CuNCs. However, in the absence of ligase or PNK, the nicked DNA probe was digested by exonucleases and failed to template fluorescent CuNCs. The fluorescent changes of CuNCs could be used to evaluate these enzymes’ activity.

Utilizing the combination of aptamers and G-quadruplexes, the Ye group developed a microfluidic exosome detection platform, which integrated on-chip isolation and in situ electrochemical analysis of exosomes from serum (Figure 18A).^[195] The liver cancer patients could be well discriminated from the healthy controls by this FNA microfluidic chip, and it may serve as a comprehensive exosome analysis tool and potential noninvasive diagnostic platform.

Figure 18.

Disease diagnosis applications of FNA-nanomaterials. A) Integrated exosome isolation and analysis platform.^[195] B) Fabrication procedure for a cancer cell monitoring platform.^[196] C) Use of the double-signal amplification mechanism-based SERS-microfluidics platform for the measurement of miRNA-21.^[197]

As shown in Figure 18B, the Ge group also constructed a rapid, facile, portable, disposable, and low-cost diagnostic method for multiplexed monitoring of cancer cells using aptamer-GO nanomaterials in microfluidic paper-based analytical devices. This method combined the exceptional quenching capability of GO with the high specificity and affinity of aptamers employed as the molecular recognition element. The QD-coated MSN labeled with aptamers could be adsorbed on the surface of GO, and the fluorescence was quenched via the FRET effect, with subsequent recovery of the fluorescence upon addition of target cells.^[196]

Based on the HCR of FNAs, enzyme-free target-strand HCR was also combined with SERS microfluidic techniques for the detection of microRNA in disease diagnosis (Figure 18C).^[197] In this diagnosis method, many DNA double strands were produced via an enzyme-free target strand displacement recycling reaction initiated by the target microRNA, which resulted in the generation of an enhanced SERS signal. Compared with traditional methods, the method enabled the sensitive and rapid detection of microRNA and overcame the shortcomings due to complex operations.

5.3.2. Drug Delivery

FNA nanomaterials are excellent nanocarriers for drug delivery. Guo et al. conjugated MNPs with DNA to form DNA spheres which were integrated with disulfides or aptamers for drug delivery.^[198] MSNs are also effective drug delivery nanocarriers for the targeted treatment of cancer. The targeted drug transport of mesoporous silica-functional nucleic acid is also an important topic of research. Wang et al. fixed ssDNA on the MSN surface, which was treated with folic acid (FA) as a door control by electrostatic attraction, and sealed the anticancer drug DOX in the mesoporous material.^[199] The DNA/MSN/FA/DOX system can be recognized by the FA receptor on the surface of cell membranes to allow for DNA control of the release of DOX.

Self-assembled FNA nanomaterials, including DNA nanoflowers,^[200] DNA origami,^[201] and DNA hydrogels,^[202] also can be used as carriers to deliver medicine, such as nucleic acid therapeutics, small drug molecules, anthracyclines, proteins, and peptides. Further, the morphology of nanostructures such as size and shape also has a great influence on their localization to specific organs, cellular uptake behaviors, and therapeutic efficiency.^[203] DNA nanostructures with a polyanionic nature have been especially shown to internalize into different cell lines without the help of transfection agents through specific endocytic pathways. Compared to single-stranded DNA or DNA duplexes, the self-assembled compact structure of DNA nanostructures can significantly enhance their stability.^[38] The Tan group engineered stability-tunable DNA micelle flares using photo-controllable dissociation of intermolecular G-quadruplexes, which confers DNA micelle flares with robust structural stability against disruption by serum albumin. Once exposed to light, the G-quadruplex formation is blocked by strand hybridization, resulting in a loss of stability in the presence of serum albumin, allowing for subsequent cellular uptake. This programmable regulation to stabilize lipid-based micelles in the presence of fatty-acid-binding serum albumin further improved the biocompatibility of DNA micelles for in vivo drug delivery applications.^[200] This method realized precise regulation for the basic properties of DNA micelle flares by the simple conformational change of G-quadruplexes. The Yan group also presented a novel drug nanocarrier system, spatially addressable DNA origami nanostructures, based on DNA self-assembly.^[201] DOX was noncovalently attached to DNA origami nanostructures through inter-calation. A high level of drug loading efficiency was achieved, and the complex exhibited prominent cytotoxicity to both regular human breast cells and adenocarcinoma cancer cells.

5.4. Data Storage

Currently, the amount of data is increasing at exponential rates. Like-wise, the demand for better storage solutions continues to increase. Owing to its demonstrated information density (petabytes of data per gram), high durability, and evolutionarily optimized ability to faithfully replicate information, DNA is an excellent medium for data storage. Recently, a series of proof-of-principle experiments has demonstrated the feasibility and value of DNA as a storage medium.^{[170, 204]}

In 2016, Erlich and colleagues designed a DNA Fountain strategy for DNA data storage. They carefully adapted the power of fountain codes (a type of computer code) to overcome both oligo dropouts and the biochemical constraints of DNA storage. They enabled virtually unlimited data retrieval and high physical density when using DNA as data storage element.^[170] In a review Panda and co-workers critically analyzed the emergence of the concept of DNA as storage media, its historical perspective, feasibility, recent breakthroughs, and challenges to be overcome in order to for it to become a marketable data storage media. They concluded that storing astronomical amounts of data in nucleic acids is no longer the stuff of science fiction.^[204]

5.5. Other Applications

New therapeutic approaches are strongly needed to face the threat of resistant infections. The antibacterial effect and stability of FNAs, with proven in vivo activity when combined with nanomaterials or biomacromolecules, has good potential for the development of antibacterial FNA nanomaterials. In 2017, Mamusa and colleagues reformulated the 12-bis-THA/TFD nanoplexes in a liposomal carrier, which protects the therapeutic property of nucleic acids from degradation, preserving and delivering it across the bacterial cell wall and exerting the antibacterial effect of the FNA.^[205]

6. Summary and Outlook

The construction and application of FNA nanomaterials are the key process and goal of FNA nanotechnology. As extensively discussed in this Review, the appropriate combination of FNA and nanomaterials enables their respective advantages to be fully utilized. Remarkably, FNA nanomaterials have found particularly widespread application in bioimaging, biosensing, biomedicine, and other fields. Most references in this Review provide excellent examples or perspectives on the applications of FNA nanotechnology.

The benefits of FNA nanomaterials are manifold and evident: literally nanometer-precise manipulation of nanomaterials is paired with flexible FNAs, which allow for the production of large quantities of custom-made novel materials. This enables different nanomaterials and FNAs to readily combine and present their own unique advantages. In addition, considering the nanoscale positioning accuracy of FNAs, the well-organized operation of a large number of nucleic acids will greatly affect the arrangement and change some original attributes of nanomaterials. Furthermore, the FNA self-assembly technology and pure nucleic acid 2D and 3D nanostructures possess great potential for further breakthroughs to address the restrictions of other nanomaterials to create more refined and controllable nanoelements.

Meanwhile, several emerging trends and challenges in the FNA-nanomaterial field can be clearly discerned: i) researchers search for more kinds of FNAs and interpret reaction mechanisms of some existing FNAs, such as selection for multiple metal-ion-dependent DNAzymes and simulation of the binding mechanism between an aptamer and its target; ii) preparation of more flexible, uniform, controllable, and efficient FNA nanomaterials, which is determined by exploration of more advanced combination methods and instruments; iii) FNAs as ligands to directly involve the synthesis of nanomaterials without any additional modifications, which would help further simplify the preparation and control the morphology of nanomaterials; and iv) increase of the targeting efficiency of FNA nanomaterials when they are used in drug delivery systems. Based on the promising drug delivery abilities of FNAs, researchers are paying more attention to the targeting and stability of FNA nanomaterials, especially under the complex conditions of blood, cells, and the nucleus.

These advantages and challenges provide new opportunities for further research and development of improved interaction methods and applications of FNA nanomaterials. Furthermore, if we can learn the reaction mechanisms and overcome some stability problems of FNA nanomaterials when facing enzyme resistance and multifactor interference in vivo, FNA nanomaterials could be strong tools in understanding life processes, detecting various biomarkers, and treating diseases at a high level. Of course, the construction of FNA nanomaterials has been driven primarily by comprehension and utilization of advanced interactions between FNA sand nanomaterials, with applications being demonstrated only after their high-yielding construction as stable materials. Consequently, enormous focus is still needed on the development of bonds between FNAs and nanomaterials that are versatile and powerful for programming the construction of FNA nanomaterials. Sharp tools make good works: an understanding of the nature of the interaction of FNAs and nanomaterials and the ability to utilize them will allow researchers to create more and more accurate and efficient FNA nanomaterials.

Biographies

Wentao Xu studied at China Agricultural University (BS 2001, PhD 2006) and conducted postdoctoral research there before joining the faculty. He is currently an associate professor in the College of Food Science and Nutritional Engineering at China Agricultural University. His research interest is functional nucleic acid biosensors and nanomaterials.

Wanchong He obtained his BS degree from Huazhong Agricultural University in 2017. He is now a PhD candidate in the College of Food Science and Nutritional Engineering at China Agricultural University. His research interest is functional nucleic acid biosensors.

Yi Lu is the Jay and Ann Schenck Professor in the Department of Chemistry at the University of Illinois at Urbana-Champaign (UIUC). He received his BS degree from Peking University in 1986 and his PhD degree from UCLA in 1992 under Dr. Joan S. Valentine. After postdoctoral research with Dr. Harry B. Gray at Caltech, he started his own independent career at UIUC in 1994. His research interests include the design of functional metalloproteins as environmentally benign catalysts in renewable energy generation and DNA nanomaterials.

Yunbo Luo obtained his BS degree from Southwest Agricultural University in 1982 and his PhD degree from University of Bath in 1987. After postdoctoral research at the University of Bath and Beijing Agricultural University, he joined the faculty at Beijing Agricultural University. He is currently a professor in the College of Food Science and Nutritional Engineering at China Agricultural University. His research interests include food biotechnology and food safety.