electrochemical detections have been recently developed to tackle some of the challenges in high-throughput screening technology for large scale analysis, which is especially useful for drug development and mass screening for pandemic outbreak such as SARS or COVID-19.
Deoxyribonucleic acid (DNA) is a polymeric chain comprising of repeating units called nucleotides, which carries important genetic instructions that play vital roles in regulating biological activities in living cells. Mutations in DNA have always been correlated with various diseases including cancer. DNA typically exists as two strands of oligonucleotides wrapped around each other to form a double helix [1, 2] . The double helix structure of DNA is characterized by two grooves which allow binding with a large variety of proteins or drug molecules [3] . Proteins such as transcription factors, nucleases or enzymes bind to DNA and regulate the biological functions of DNA, usually gene expression. On the other hand, DNA is the main target of many drug molecules that interacts with the DNA in various modes to manipulate its function [4] . Today, DNA is strategically set as a target for ligands in treating many human diseases especially for cancer therapeutics [5, 6] . Therefore, in-depth understanding of protein-DNA and drug-DNA binding interactions is the key to understand disease mechanisms which helps in the drug design in order to successfully tackle a range of diseases [7] [8] [9] .
Besides performing critical cellular functions, there is an artificially selected group of short, single-stranded DNA/RNA called aptamers that can bind to a diverse range of analytes including metal ions, small molecules, proteins and even pathogens with high affinity and selectivity [10] [11] [12] . Although aptamers are frequently considered as the nucleic acid equivalent of antibodies, they offer many unique advantages due to their relatively small size, versatility in binding to any target of choice (especially small molecular analytes of J o u r n a l P r e -p r o o f <1700kDa), higher target selectivity, better stability under non-physiological conditions, prolonged shelf life, ease of chemical modification and cost-effectiveness for large quantity synthesis, which are all beneficial in developing nucleic acid nanosensors [10, 12] . Cai et al. have reviewed the recent advances in studying the interactions of aptamers with their binding targets, which offers great insights into the range of binding targets as well as the binding constants and binding forces between the aptamer and their targets [13] . These insights are especially useful in the design of aptamer based nanosensors for detecting these specific binding interactions.
The advancement of nanotechnology has contributed significantly in developing novel nucleic acid-based biosensors involving the use of different types of nanostructures.
Plasmonic nanomaterials such as gold and silver nanoparticles, fluorescent nanomaterials like quantum dots and carbon dots, electrochemically active materials including carbon nanotubes and graphene are frequently used as the key components in these nanosensors [14] [15] [16] . The interaction of DNA with nanomaterials for biosensing applications has been reviewed by He et al. [17] . In this review, we focus on the design of nucleic acid nanosensors for detecting a broader range of biological analytes, which include ions, protein, bacteria, etc. According to the signal generation mechanism, nanosensors can be detected in different sensing modes such as optical or electrochemical detection [14] . The uniqueness of a nanosensor lies in the similar length scale of the nanostructures to the biological targets to facilitate better binding interactions mimicking natural interactions, which may further allow in vivo applications. In addition, large surface-to-volume ratios provided by nanostructures enable the binding of a large number of bio-recognition elements to capture the target analyte, thus greatly improving the detection sensitivity.
Due to the importance of understanding protein-DNA and drug-DNA interactions and the prevalent applications of aptamers in various fields, it is not surprising to observe synergistic J o u r n a l P r e -p r o o f combination of nucleic acids as the recognition element and nanostructures as the signalling element for the advancement of nucleic acid nanosensors to detect sequence specific binding interactions. Many of these nucleic acid nanosensors incorporate natural DNA sequences or in vitro selected aptamers as the bio-recognition element to investigate a wide range of targets from metal ions, drug binding, proteinaceous targets such as antibodies or enzymes to the entire cells such as viruses or bacteria. Both optical and electrochemical sensing principles are widely employed in these nucleic acid nanosensors to study the binding interactions between nucleic acids and targets of interest.
As mentioned earlier, DNA is a polymer comprising of repeating units of four nucleotides [18] . The nucleotides typically constitute a five-carbon sugar called deoxyribose and a nitrogen containing base, which are connected through phosphodiester linkages within a strand. The bases include thymine (T), cytosine (C), guanine (G) and adenine (A) [19] . The secondary structure of DNA is a double helix with two antiparallel polynucleotide chains firmly held together by hydrogen bonds, where A forms two hydrogen bonds with T while C forms three hydrogen bonds with G. In the double helix, the sequences of the strands are complementary to each other. The interactions of biomolecules or drugs with DNA double helix are varied. Typically, proteins interact with DNA through specific base-pair recognition, multi-specific binding or non-specific binding independent of DNA sequences [20] , whereas, drug molecules can interact in covalent or non-covalent interactions [19, 21, 22] .
Proteins that possess DNA-binding domains can bind to DNA through non-specific, specific or multi-specific interactions [3] . The most extensively studied DNA-binding protein is called J o u r n a l P r e -p r o o f transcription factor (TF), which regulates gene transcription such as the famous tumour suppressor protein, p53 [23] . When the amino acid residues of a TF interact with DNA, they recognize a specific sequence stringently to form a stable complex, although there is still an underlying difference in the extent of stability depending on the particular family of TFs. On the other hand, non-specific DNA-binding proteins prefer to bind in the minor grooves of double helix, without a good discrimination between the bases. As for the multi-specific binding, this group of DNA-binding proteins recognize target sequences less stringently, even though some bases are frequently mutated [20, 24] .
For the sequence-specific protein-DNA binding interactions, TFs bind to a specific DNA sequence called the response elements (RE) through hydrogen bonding and Van der Waals forces [3, 25] . Generally, TFs interact with the major groove of DNA double helix as the bases are exposed for ease of identification and interaction [26] . There are approximately 3,000 TFs with sequence-specific DNA binding characteristics [27, 28] , and they are classified based on their DNA-binding domains [29] which may predict their unique functions. Among the various binding domains existed in TFs (Figure 1) , the three main groups of TFs include 1) C 2 H 2 zinc fingers, 2) homeodomain (HD) and 3) helix-loop-helix (HLH).
J o u r n a l P r e -p r o o f HTH stands for helix-turn-helix domain. bHLH stands for basic helix-loop-helix motif. HD and HMG stand for homeodomain and high-mobility group box domain, respectively. Adapted from ref. [30] Detailed investigations have discovered that TFs can bind their RE as homo or hetero dimers.
Homodimeric TFs clamp the DNA on both strands. In addition, higher oligomerization has also been observed, for example, heat shock factors act as trimers and p53 proteins bind as In principle, full-length p53 proteins dimerize through C-terminal oligomerization domain and then the RE half-sites bind to the p53 core domain in a symmetric interface. Finally, the dimers tetramerize with the second half-site. This example illustrates how complicated TF-DNA binding can be, thus, smart and sensitive detection methods need to be developed to gain an in-depth understanding of varied protein-DNA binding interactions including binding affinity evaluation, the mode of binding, the effect of protein mutation, etc, which will be discussed in details in Section 3.
DNA, especially in its double-stranded form, is able to complex with a large number of natural or synthetic molecules, therefore DNA has become the pharmacological target of many drugs. For example, chemotherapeutic drugs bind to DNA and tend to alter the properties of DNA which then influence critical cellular processes [31] . Elucidating the detailed interactions between drug molecules and DNA has always been a hot research topic which aids drug design and drug screening.
In general, drug-DNA interactions can be classified as covalent or non-covalent [31] . The covalent interaction is an irreversible reaction which inhibits the functions of DNA and consequently causes cell apoptosis. A popular example is a platinum based anticancer drug, cisplatin, which is frequently used in cancer treatment due to their strong interactions with DNAs ( Figure 2a) . This drug typically forms bifunctional intra-and inter-strand DNA crosslinks through covalent bonds with purine nucleobases, which subsequently disrupts DNA replication, inhibits transcription and consequently kills the cancer cells. Unfortunately, the platinum-based drugs are limited by side effects and poor activity in certain types of cancer because their detailed mechanisms of action are still not clearly understood [32] .
Another example is alkylator which interacts covalently with the nucleophilic group of DNA through a strong electrostatic interaction to form an irreversible adduct (Figure 3b ). Typical On the other hand, the three major modes of non-covalent drug-DNA interactions include (1) groove binding, (2) intercalative binding, (3) external binding [31] . Groove binders possess free rotating heterocyclic or aromatic hydrocarbon ring groups that allow them to fit into the major or minor groove of DNA double helix. For example, netropsin and distamycin interact with DNA minor groove through hydrogen bonding and hydrophobic interactions ( by exponential enrichment (SELEX) [37] . The SELEX process is used to screen oligonucleotides from large libraries of RNA or single-stranded DNA. This in vitro selection process is iteratively performed and subsequently the selected sequences are amplified by polymerase chain reaction (PCR). Aptamers bind to their targets via the complementary shape interactions and three-dimensional folding with high affinity. The most common secondary or tertiary structural motifs for aptamer include G-quadraplexes, stem-loops, pseudoknots, kissing complexes, three-way junctions, and hairpins [12] . Compared to the most commonly used biorecognition element-antibodies that mainly targets proteins, aptamers can be generated against a myriad of targets ranging from metal ions (e.g. Cu 2+ , Hg 2+ ), small molecules (e.g. ATP, cocaine, aflatoxin B1) to proteins (e.g. thrombin, streptavidin) and even whole cells (e.g. viruses, bacteria, cancer cells) [12, 13, [38] [39] [40] [41] . This gives great versatility to design nucleic acid sensors to target any analyte of interest.
To detect DNA binding interactions, such as protein-DNA binding, drug-DNA binding, aptamer-target binding, researchers have developed various sensing methods. Among which, the most promising one is the nanosensor design, which uses either the plasmonic nanostructures for direct optical sensing or employ various type of nanomaterials such as graphene, metal oxides, rare-earth metal, magnetic nanoparticles, etc. to achieve signal amplification or enhancement in sensing performance [42] . Particularly, optical and electrochemical detection principles have been widely applied in developing nanosensors for various analytes, which will be discussed in details with specific examples highlighted in the following subsections.
J o u r n a l P r e -p r o o f
Colorimetric sensors are simple, low cost due to their easy read out, which is even possible by naked eyes detection [43] . Noble metal nanoparticles such as gold, silver are suitable colorimetric indicators due to their excellent extinction coefficients and also unique distancedependent optical properties [44] . For example, Zhang's group reported a sensitive approach to detect transcription factor NF-kB p50 by converting them into reporter oligonucleotides through protein-DNA interaction, exonuclease (III) digestion and isothermal exponential amplification [45] . If NF-kB is present, its binding to DNA protects the DNA from nuclease digestion, the DNAs are then amplified to cause aggregation of the DNA-modified gold nanoparticle (AuNP) probes leading to precipitation of AuNPs. In the absence of NF-kB,
DNAs are digested and no sequences can be amplified, therefore the AuNP probes remain well suspended and the solution is still red in color. This sensing method is very sensitive, achieving a limit of detection of 3.8 pM. However, the procedure of this assay is quite time consuming and tedious, requiring many steps of heating and cooling to allow DNA digestion and signal amplification.
Our lab has been developing a series of easy-to-perform gold nanoparticles (AuNP)-enabled colorimetric sensors to quickly detect protein-DNA binding with various sensing strategies (Figure 3a) . Moreover, this assay offers high sequence specificity which distinguishes single-base variation in the ERE [46, 52] . A similar detection strategy is later applied to monitor the binding of single strand binding protein (SSB) to a single-stranded DNA (ssDNA) of various base sequences [48] . It was successfully revealed that the minimum DNA length required to form a stable SSB-ssDNA complex is more than J o u r n a l P r e -p r o o f 10-mer, and the SSB proteins binds to homopyrimidines with a higher affinity than the homopurines of similar length (Figure 3b ). In these two nanosensor designs, noncrosslinking AuNPs aggregation strategy has been employed successfully to detect not only the sequence-specific protein-DNA interactions without any tedious surface modification steps, but also provide useful insights into their intrinsic binding mechanisms in homogenous solution similar to that in the biological systems.
As the citrate-capped AuNPs may be prone to aggregation induced by environmental factors, our group has further developed a new sensing strategy based on the use of segmented DNAmodified AuNPs and non-crosslinking mechanism to detect sequence-specific ER (protein)-ERE (DNA) interactions. Specifically, two sets of double-stranded DNA (dsDNA) modified
AuNPs, each carrying a half site segment of the ERE was designed to contain a short complementary sticky end for base pairing to cause particle aggregation and change in solution colour from red to purple (Figure 3c) . In the presence of ER, the sequence-specific protein binding is able to stabilize the transient nanostructures formed by the two sets of segmented ERE-linked AuNPs upon mixing due to steric forces, thus the particle solution remains red [47] . In this work, silver nanoparticles (AgNPs) has been demonstrated to give a more sensitive detection limit with a different visual color change from yellow to brown. Tan and co-workers later carried out a systematic investigation on the decorating DNA design to gain better insight into the non-crosslinking AuNPs aggregation using DNA-conjugated
AuNPs for more efficient assay development [49] . It was found that (1) the dsDNA spacer of 11-base pairs (bp) or longer are more effective than the ssDNA spacer and (2) the symmetrically spaced sticky ends could facilitate the dsDNA-AuNPs aggregation better than the asymmetrical combination. More recently, our lab developed a new "mix-and-measure"
protein-DNA binding assay utilizing only one set of dsDNA-modified AuNPs, which contains the p53 response element (RE-AuNP) for rapid detection of wildtype p53 proteins J o u r n a l P r e -p r o o f from the complex cell lysates [50] . As shown in Figure 3d , the wildtype p53 proteins bind to RE-AuNPs in tetrameric forms causing particle aggregations and thus solution color change from red to purple. As no tetramerization occurs in the presence of mutant p53 proteins, the RE-AuNPs are still dispersed and the solution remains red. The colorimetric difference in this assay is very distinct which can be easily observed by naked eyes and quantified by the UVvisible spectroscopy. This protein tetramerization-induced RE-AuNPs aggregation sensing principle has been further employed for small molecular drug screening and cancer diagnostics in the reported studies [50] , [53] .
Other than utilizing natural DNA sequences in designing colorimetric nucleic acid nanosensors, aptamer represents a popular choice due to its wide-ranging targets. 
SERS is one of the most promising technique for molecular detection which merges the benefits of molecular fingerprint specificity with single molecule sensitivity [59] . Simulation studies have shown that metallic nanostructures of higher orders (e.g., trimer and tetramer) and different configurations can lead to better SERS enhancement effects [60, 61] . For instance, SERS have been employed to study protein-DNA or drug-DNA interactions, achieving enhanced signal and therefore improved detection sensitivity [62] . Bonham et al.
designed a short DNA duplex to have a core sequence to which the protein binds specifically and two short single-strand overhangs that hybridize with a pair of DNA-modified gold nanoparticles. DNA-bridged nanoparticle assemblies were formed and then silver plated to detect sequence-dependent protein-DNA interactions using confocal Raman microprobe [62] .
In addition, the Raman intensity correlates with the protein concentration in a sequence specific manner leading to the estimation of the apparent dissociation constant (Figure 5a ) [62] .
SERS is also an excellent technique to study drug-DNA interaction. Barhoumi To study protein-DNA interactions, protein or oligonucleotides need to be fluorescently labelled. So far, most of the FRET assays for detecting DNA-binding proteins work in a "turn off" mode by monitoring fluorescence quenched upon binding, which tend to give false positive results due to non-specific quenching by environmental factors. Recently, a FRET assay utilizing cationic conjugated polymer (CCP) nanoparticles, hairpin DNA and an intercalative dye is developed to detect nuclear factor-kappa B (NF-kB) (Figure 7 ) [67] . If NF-kB is present, it binds to the hairpin DNA probe and protect it from digestion by J o u r n a l P r e -p r o o f exonuclease III, thus resulting in efficient FRET between the intercalative dye and the CCP nanoparticles. As low as 1 pg/mL of NF-kB in HeLa nuclear extracts were successfully detected, which is 10,000-fold more sensitive than the previously reported methods.
Fluorescent nanosensors are promising tools in studying both drug-DNA and protein-DNA interaction with high sensitivity. In addition to fluorescent dyes, semiconductor quantum dots, fluorescent conjugated polymers, novel fluorescent nanomaterials such as bioinspired carbon dots [68] [69] [70] [71] [72] , metal nanoclusters [73] [74] [75] [76] [77] and aggregation induced emission dyes [78, 79] can also be used in developing sensitive assays to monitoring biomolecular interactions including protein-DNA and drug-DNA binding.
J o u r n a l P r e -p r o o f leading to a reduction in electrochemical signal [85] . Electrochemical sensors are usually very sensitive and fast in detecting protein-DNA binding. Gold nanoparticles (AuNPs) and
AuNPs-enhanced silver deposition strategies have been used to further improve the electrochemical signal to achieve even lower detection limits [86] .
In addition to act as the electrochemical signal enhancer, gold nanoparticles can be synthesized to exhibit peroxidase like activities. With the addition of an electrochemically active species, this approach enables rapid detection of P. aeruginosa with a detection limit of 60 cfu/mL [87] .
Other than metal nanoparticles, chemically modified graphene is also excellent electroactive indicators. Hernandez et al. created an electrochemical nanosensor by non-covalently conjugating S. aureus specific aptamer to graphene via π-π interactions. With the addition of S. aureus, the aptamer prefers to bind to the bacteria and thus separate its negatively ionized phosphodiester groups from the graphene surface at pH 7.4. Since the graphene sheet act as asymmetric capacitors, this charge separation leads to a change of the recorded potential.
This nanosensor is highly sensitive, achieving an ultralow detection limit of 1 cfu/mL [88] . However, when P. aeruginosa is added, the Fc-GO is displaced from the electrode surface causing electrochemical signal reduction due to their higher affinity binding with the aptamer modified ZIF-8. This particular nanosensor design also achieves an excellent detection limit of 12 cfu/mL and can be used to directly diagnose P. aeruginosa infection in urine [89] . We believe that these aptamer-based nanosensor designs offers a new angle to develop novel sensors to detect SARS-COV-2 in a rapid and sensitive manner. infections, the recent discovery of specific aptamer sequence that targets the SARS-CoV-2 spike protein [90] will allow the research community to quickly design aptamer based nanosensors for rapid and accurate detection of COVID-19 infections. Furthermore, nanoDLS assay also presents a feasible way to mass screen drug candidate targeting the virus. It is expected that these nucleic acid nanosensors would play very critical roles in both drug development and mass screening for pandemic outbreak such as SARS or COVID-19. 
Understanding and detecting DNA binding interactions, specifically protein-DNA binding and drug-DNA binding are very crucial in revealing disease mechanisms and mode of actions for drugs which will then guide diagnosis and drug development. With the recent development of various nanosensors to detect various types of DNA binding interactions, we are expecting to see promising progresses with in-depth understanding of these interactions in various aspects, including binding kinetics, binding affinities, stoichoimetry, multiple component interactions and many more, which will certainly provide better guidance on the J o u r n a l P r e -p r o o f development of disease diagnosis and help to inform drug design. Furthermore, the advancement in aptamer design and synthesis has helped to greatly expand the range of analytes detectable by nucleic acid nanosensors. The development of many easy-to-use nanosensors that only requires one-step mix-and-measure protocol to operate are similarly important, which will help to greatly improve the throughput of screening platform for largescale analysis and mass screening for infectious diseases containment applications.