Patent Application
20180172678
The present invention generally relates to methods and devices for transcription factor-DNA interaction determination, and more particularly relates to methods and devices for using dynamic light scattering for high-throughput sequence-specific transcription factor-DNA interaction determination for drug and/or disease screening.
The investigation of important biomolecular events such as DNA mutation and gene transcription have been made possible with the advent of nanotechnology. Various nanosensing probes, such as metal nanoparticles, quantum dots, and silicon nanowires have been utilized to lend insight into the intertwining complexities between biomolecules, by transducing ‘invisible’ biological signals into measurable output. In particular, gold nanoparticles (AuNPs) have added a new dimension to the realm of biosensing through their exhibition of localized surface plasmon resonance (LSPR). The strong absorbance and scattering characteristics of AuNPs at the visible light region render them as ideal sensing probes for various bioassay developments based on different optical responses. For example, the interparticle-distance dependent plasmonic coupling of AuNPs has been utilized to design colorimetric assays for biomolecular detection. However, a high concentration of targets are needed in the colorimetric assay to aggregate the AuNP to elicit appreciable color changes to be visibly perceived, which results in less than ideal sensitivity. In addition, it is not suitable for use in coloured samples such as blood, which would interfere with the red to purple/blue transition in observations by the naked eye or using UV-vis spectroscopy.
Given that low sensitivity is one of the main limitations of gold nanoparticles-based colorimetric assays, methods such as biobarcode and silver staining amplifications have been carried out to address the problem, but such methods are still complicated and time consuming. As such, it has been shown that AuNP probes carrying recognition sequences for protein-protein binding and DNA-DNA hybridization would cluster and aggregate in the presence of their target binders. The increase in particle size led to a wholesale shift in the population distribution from tens of nm to the hundreds nm range. Given that larger AuNPs show greater scattering cross section, the overall size increase from the aggregation amplifies the readout, further enhancing the sensitivity and clarity of the readout.
If a sensitive platform for detecting AuNP-transduced biorecognition signals and biodiagnostic strategies involving increase in AuNP size could be addressed, such a platform could be used for cancer screening. Cancer is one of the prevalent causes of death worldwide and can take more than 200 diverse forms, including lung cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, hematologic cancer, colon cancer, or leukemia. Environmental factors as well as genetic factors have been linked with an increased threat in the development and progression of cancer. However, many developed cancer therapies are specific only to a certain kind of cancer. Among cancer treatments, chemotherapy is a more ‘general’ anti-cancer method but is very invasive and non-targeting. Chemotherapy drugs kill both cancer cells and normal cells, thereby bringing severe side effects to patients.
The p53 protein is a general tumor suppressor which governs cell fates, thus it has been called “the guardian of the genome”. As a typical transcription factor, p53 binds to specific DNA response elements (REs) which regulate the expression of target genes. Approximately half of all cancers have been found to result from mutations in p53, thereby making p53 pathway a prime target for cancer therapy development. Of the dozens of p53 drugs currently in development, the vast majority simply try to boost levels of healthy p53. When p53 proteins are mutated, they lose their ability to bind to specific DNA promoter sequences containing DNA REs and, thus, are unable to trigger processes that safeguard a normal cell such as cell cycle arrest, DNA repair or apoptosis.
The discovery of a drug that is able to restore mutant p53's DNA binding ability is of high clinical importance and promises to change the landscape for cancer treatment and for treatment of other diseases that involve misfolded proteins such as Alzheimer's disease. More generally, if the tumor suppressor functions of p53 could be activated by an anticancer drug, it would greatly improve the drug efficacy. It is envisioned that a p53 activation or reactivation drug will present a general strategy to treat many kinds of cancer with just a few drugs. Unfortunately, there is a lack of a simple, fast, sensitive and high-throughput drug screening assay to target p53 activation in a complex biological setting. In addition, estrogen receptor (ER) is a protein biomarker that has significant implication in breast cancer prognosis and treatment. Thus, a sensitive and selective method for detection of binding interactions of ER with its consensus DNA containing estrogen response element (ERE) in a fast and simple manner is highly desirable.
Conventional methods that have been used to ascertain p53-DNA binding include gel shift assay, DNA footprinting, fluorescence anisotropy, Chromatin Immunoprecipitation (ChIP), Surface Plasmon Resonance (SPR) and enzyme-linked immunosorbent assay (ELISA). These methods are mostly heterogeneous-phase assays which involve multiple surface treatments, a high level of technical expertise, and require expensive reagents and sophisticated instrumentation. Thus, they are not suitable for a high-throughput drug screening targeting p53 pathway. In addition, these methods have high background noise and are mainly used to detect purified protein samples. More recently, two new approaches, multiplex in vitro binding assay and microsphere assay for protein-DNA binding (MAPD), have been designed to detect p53-DNA binding in in vitro transcription/translation (IVT) samples or nuclear extracts in a semi-quantitative manner. These assays still suffer from tedious procedures and, more critically, reliance on multiple expensive reagents such as antibodies, primers and beads for signal readout.
Dynamic light scattering (DLS) can also detect 100 nm AuNPs at as low as fM level without added processing or amplification, which makes DLS a sensitive platform for detecting AuNP-transduced biorecognition signals, and also biodiagnostic strategies involving increase in AuNP size. However, as in most aggregation-based systems, particle aggregation is an uncontrolled process, with the biomolecular targets causing the AuNPs to aggregate and grow extensively, leading to large variations in AuNP aggregate size and complex DLS readouts that are complicated to analyze especially for more subtle size changes. Greater control over the probe-analyte interaction process is necessary to leverage the size growth of AuNPs, detected by DLS machine.
Thus, what is urgently needed is a simple, fast, sensitive, label-free and high-throughput assay platform to identify and evaluate binding interactions directly in live cells, cell lysates and/or other biological protein samples. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.
According to at least one embodiment of the present invention, a method for identifying sequence-specific transcription factor DNA interactions is provided. The method includes homogeneously mixing plasmonic metal nanoparticle probes with protein samples as an assay in multi-well plates. The plasmonic metal nanoparticle probes comprise a plurality of plasmonic metal nanoparticles and a specific response element and the protein samples bind with the specific response elements to form an assembly of the plasmonic metal nanoparticle probes. The method further includes measuring particle size distribution of the assembly of the plasmonic metal nanoparticle probes in the solution by dynamic light scattering and determining one or more sequence-specific transcription factor DNA interactions from a curve of the particle size distribution determined from light scattered by the dynamic light scattering.
According to another embodiment of the present invention, a nanoprobe is provided. The nanoprobe includes a plurality of plasmonic metal nanoparticles and a DNA linker. The DNA linker forms a link between the two plasmonic metal nanoparticles, the DNA linker including a double stranded region encoding a specific response element.
According to a further embodiment of the present invention a system for drug screening is provided. A system includes an assay, multi-well plates for combining the assay with a biological sample, and a measurement device. The assay includes a plurality of specific transcription factor DNA response elements having large light scattering dimensions when binded to drug-activated or -reactivated transcription factor proteins. The biological sample includes drug-activated or -reactivated transcription factor proteins. And the measurement device determines binding affinity of the drug-activated or -reactivated transcription factor proteins with the plurality of specific transcription factor DNA response elements by measuring particle size dimensions in the multi-well plates by dynamic light scattering.
And according to yet a further embodiment of the present invention a system for disease screening is provided. The system includes an assay, multi-well plates and a measurement device. The assay includes a plurality of specific DNA response elements having large light scattering dimensions when binded to receptor elements. The assay is combined with a disease screening biological sample in the multi-well plates. And the measurement device determines binding of the specific DNA response elements with the receptor elements in the disease screening biological sample by measuring particle size dimensions in the multi-well plates by dynamic light scattering.
The accompanying figures, where like reference numerals refer to identical or functionally similar elements throughout the separate views and which together with the detailed description below are incorporated in and form part of the specification, serve to illustrate various embodiments and to explain various principles and advantages in accordance with a present embodiment.
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale.
The following detailed description is merely exemplary in nature and is not intended to limit the invention or the application and uses of the invention. Furthermore, there is no intention to be bound by any theory presented in the preceding background of the invention or the following detailed description. It is the intent of the present embodiment to present a unique DNA-assembled gold nanoparticle (AuNP) probe for dynamic light scattering (DLS) sensing of transcription factors. A specific response element sequence is incorporated into DNA linkers used to bridge the AuNPs in the AuNP probe. Coupled with the DLS measurement, this AuNP probe-based DLS detection system provides specific readouts in the presence of target molecule. This unique optical signature enables the nanostructures to be used in conjunction with a DLS platform to study transcription factor-DNA interactions. In addition, the AuNP nanoprobes could also suppress the light-scattering signal from unbound proteins and other interfering factors (e.g., buffer background), and provide highly sensitive detection of target proteins in complex biological samples such as cell lysates. Thus, the AuNP probe coupled with DLS measurement is a simple (mix and test), rapid (readout in ˜5 min) and sensitive (low nM levels) platform to detect sequence-specific protein-DNA binding event.
In addition, a new dynamic light scattering (DLS) based high-throughput anticancer drug screening nanoplatform targeting the p53 pathway. The nanoplatform in accordance with a present embodiment is capable of quantitatively measuring p53-DNA binding in real-time, determining whether a drug can reactivate mutant p53 protein to restore its sequence-specific binding ability to p53 reaction elements (REs) or increase wildtype p53 activity, and evaluating sequence specificity for drug validation. The nanoplatform capitalizes on the large scattering dimension of gold nanoprobes (AuNPs) (approximately 106-fold larger than fluorescent probes) due to the localized surface plasmon resonance (LSPR) effect. The use of AuNPs coupled with DLS leads to excellent sensitivity with an ultralow detection limit of 0.06 pM, a marked improvement over conventional techniques. The assay can be carried out in a ‘mix-and-measure’ manner that is faster, simpler, and cheaper than the conventional methods where, in general, multiple incubation with multiple labeled reagents and repeated washing steps are required. This assay is highly specific due to the fact that the p53 REs (DNA) are conjugated onto the AuNPs, thereby suppressing any signals arising from non-binding substances and allowing drug screening in complex mediums such as cell lysate or blood serum.
The DLS equipment is a common characterization equipment used by pharmaceutical companies, providing an easy adaptation pathway for this assay to be adapted by the pharmaceutical industry to screen and validate p53 activation or reactivation drugs. In 2014, the global market value for cancer drugs reached $100 billion per annum, and is expected to increase to $147 billion by 2018. This large market promotes increased investment in cancer drug research and development evidencing an urgent need for anticancer drug screening assays targeting the p53 pathway for the development of general anticancer therapeutics.
Dynamic light scattering is a well-known analytical technique capable of analyzing particle size distribution down to the nanometer range. The particle size is determined by monitoring fluctuations in scattered light intensity caused by Brownian motion of particles in a solution. DLS has been used to measure the hydrodynamic radius or size of purified proteins and DNAs at very high concentrations. However, since all biomolecules scatter light to a similar extent, it is almost impossible to detect the binding interactions, especially at low concentrations in a complex biological sample. Therefore, the drug screening nanoplatform in accordance with the present embodiment includes a DLS probe designed with to include a gold nanoparticle (AuNP) with high scattering dimensions to enhance the DLS signal and at the same time suppress any background noise, conjugate/link with one p53 response element (RE) to allow sequence specific binding, and provide a passivated probe surface with oligonucleotides to prevent non-specific adsorption of irrelevant biomolecules.
Referring to
Utilizing the above-mentioned criteria, the p53 RE linked AuNP (RE-AuNP) DLS probe 102 is prepared. Briefly, two 5′-thiolated single-stranded DNAs (ssDNAs) 104, 106 hereby termed A 104 and B 106, are conjugated onto AuNPs 108, 110 to form AuNP-A 112 and AuNP-B 114, respectively. After passivation with polyT oligonucleotides, AuNP-A 112 and AuNP-B 114 are hybridized to a target ssDNA AB 116 which is complementary in sequence to both probes. A p53 reaction element (RE) 118 (e.g. a PUMA sequence where p53 unregulated modulator of apoptosis (PUMA) is a pro-apoptotic protein, a member of the Bcl-2 protein family) is on the side with the AuNP-B 114. The RE-AuNPs 108, 110 are linked by one single strand of double-stranded DNA (dsDNA) between the two AuNPs 108, 110, looking like a dumbbell. The nano-dumbbell AuNP probes 102 exhibit a distinct size that is about two times larger than individual AuNPs 108, 110 as measured by DLS.
When the nano-dumbbell AuNP probe 102 is incubated 120 with wildtype p53 (wtp53) proteins 122, the proteins 122 will bind to the p53 RE 102 as a multimer 124 such as a tetramer, leading to assembling 126 or stacking of multiple nano-dumbbell probes 102. The resulting increase in hydrodynamic radius of the assembly of nano-dumbbell AuNPs probes 126 gives a significant change in a position 128 of the DLS signal 130. A similar aggregation phenomenon was observed previously with RE conjugated microbeads (1 μm) via fluorescence imaging.
In contrast, mutant p53 (mutp53) proteins 132 are unable to bind to the nano-dumbbell probes 102 and, thus, there is no change in a position 134 of the DLS signal 136 since this assay only detects proteins that specifically bind to nano-dumbbell probes 102 but receives no interference from the solution background. When a drug successfully reactivates mutp53 to restore its RE binding ability, the drug-reactivated p53 proteins will bind to the nano-dumbbell probes 102 and the extent of reactivation (i.e., effectiveness of the anticancer drug) can be evaluated by measuring a position 140 of the resultant DLS signal 142. Based on the novel sensing principle in accordance with the present embodiment, a versatile DLS-based AuNP probe assay has been designed that can advantageously be applied to quantitatively measure sequence-specific p53-DNA binding with high sensitivity and selectivity, allow high-throughput screening of p53 activation or mutant reactivation drugs in cell lysates, and evaluate the binding affinities of various DNA promoter sequences to wildtype p53.
Fast and Quantitative Measurement of p53 Protein-DNA Binding with High Sensitivity and Specificity in Real Time.
Thus it can be seen that the assay in accordance with the present embodiment demonstrates quantitative measurement of wildtype p53 interacting with the PUMA RE sequence with excellent sensitivity and ultralow detection limit. The linear range of the wildtype p53 protein concentration is from 0 to 12 pM with an ultra-low detection limit of 0.06 pM (S/N=3) achievable in accordance with the present embodiment. Furthermore, the calibration plot 256 in the graph 250 advantageously enables determination of wildtype p53 protein concentration in an unknown sample.
Since p53-DNA binding is a time-dependent event, the ability to real-time monitor the binding interaction in a homogeneous solution is of great importance. Surface plasmon resonance (SPR) is typically considered the standard method for real-time monitoring of protein-DNA or protein-protein binding, but SPR has limitations. For instance, SPR requires surface functionalization of a probe and then detects the binding interaction at a solid-liquid interface which may introduce steric hindrance for the binding event and also does not accurately represent the physiological situation. However, accuracy is especially critical in the case of p53-DNA binding since p53 proteins tend to aggregate upon binding. Unlike SPR, the system in accordance with the present embodiment provides a direct, single-step, real-time monitoring of wildtype p53 protein binding to its RE in a homogeneous solution, without requiring surface immobilization and advantageously having the ability to better mimic actual physiological conditions.
Referring to
Referring to the graph 350, time is plotted on the x-axis 352 and particle size is plotted on the y-axis 354. The statistical plot 356 shows that a size shift can be observed after one minute and it increases with incubation time reaching a plateau at around fifteen minutes. The distinct size change observable after just one minute of incubation evidences that DLS promptly detects the specific binding interaction between wtp53 and RE. Thus, the present embodiment advantageously provides reduced assay time and allows faster detection.
High Sequence Specificity Wildtype p53-DNA Binding.
The DNA sequence selectivity in accordance with the present embodiment is demonstrated in
Excellent Differentiation of Mutant Proteins Vs. Wildtype Proteins.
The capability to differentiate wildtype p53 from mutant p53 is critical towards the success of a drug screening assay. When p53 proteins are mutated, they lose their capability of binding to specific DNA promoter sequences containing the REs. The specially designed PUMA-AuNP nano-dumbbell probe in accordance with the present embodiment easily distinguishes the binding wildtype p53 protein from the non-binding mutant p53 proteins. Referring to
The major approaches to correct the dysfunctional p53 regulatory pathway are to inhibit the p53-MDM2 (ubiquitin protein ligase that targets p53 for degradation) interactions or to restore the functions of mutant p53.
Screen for a Drug that Increases p53 Activity.
Unlike most current techniques that require purified protein samples, the assay in accordance with the present embodiment is able to directly detect the protein in cell lysates, allowing more clinically-relevant data to be obtained. Although cell lysates typically contain many other substances such as proteins and DNAs which will also scatter light, the intensity is significantly lower than that exhibited by the AuNPs due to the large scattering cross-section of AuNPs. The fact that p53 REs are conjugated onto the AuNPs probe also ensures that the detection of p53-DNA binding is highly specific and any signals arising from the non-binding substances are significantly suppressed, thereby allowing testing for native p53 protein in cell lysates.
To investigate the applicability of the assay in accordance with the present embodiment to cell lysates, the H1299 Ecdysone-Inducible (EI) system for controllable and constitutive expression of p53 in H1299 cells induced by ponasterone A (PonA) was used. Referring to
Referring to
Referring to
The ability to clearly distinguish wildtype p53 from mutant p53 in cell lysates provides the potential of the assay in accordance with the present embodiment for screening drugs that can effectively reactivate mutant p53 directly in cells and recovered the biological samples from cell lysates for detection.
Referring back to Step 1, drugs to be tested are first incubated 912 with live cancer cells expressing mutp53 proteins. Drugs are then uptaken 914 by the cells and interact with the mutp53 proteins. The drug treated cancer cells are subsequently lysed at Step 2 (920) to obtain crude cell lysates. These cell lysates are incubated with RE-AuNP probes and DLS is finally employed to evaluate the efficiency of mutp53 reactivation 932. If the reactivation is successful 934, the reactivated p53 protein will have a restored binding toward the RE, thus leading to increase in the diameter of the p53 RE-AuNP probes as measured 936 by DLS. In contrast, failure in reactivation 938 will not restore the binding of mutp53 protein to the RE sequence on the nano-dumbbell probes, thus no change in DLS signal is measured 940. Thus, the bioassay in accordance with the present embodiment is able to evaluate the extent of mutant reactivation by comparing the DLS signal obtained with the wtp53 binding to RE. The effective concentration of drugs for reactivation can also be determined by performing a series of concentration-dependent experiments during cell reactivation
There are very limited numbers of p53 reactivation drugs available currently, very possibly due to the lack of an efficient and high-throughput drug screening method. The methodology in accordance with the present embodiment provides a DLS based drug screening assay with huge potential for high-throughput screening of p53 reactivation drugs.
To demonstrate the feasibility and reliability of the assay in accordance with the present embodiment, three p53 reactivation drugs including COTI-2 owned by a private company, PRIMA-1met undergoing clinical trial, and mutant specific NSC3198726 were tested in PonA induced H1299 cells and the results are shown in
Referring to the bar graph 1050, successful reactivation of mutant p53 (R273H) by COTI-2 at high concentrations (++) 1060 but not at low concentrations (+) 1062 and successful reactivation of mutant p53 (R273H) by Prima-1met at high concentrations (++) 1064 but not at low concentrations (+) 1066 is shown. However, the NSC drug has no effect at either low concentrations 1068 or high concentrations 1070 due to its specificity only to R175H mutant.
Thus, in accordance with the present embodiment, higher concentrations of COTI-2 (1 μM) 1012, 1060 and Prima-1met (20 μM) 1016, 1064 can successfully reactivate both p53 mutants tested (R175H and R273H) to bind with PUMA-AuNPs. In contrast, NSC reactivates R175H at both low concentrations (0.3 μM) 1018 and high concentrations (3 μM) 1020, but has no effect on the R273H mutant 1068, 1070. This is explained by the NSC's specific action on the R175H mutant. Notably, since the assay in accordance with the present embodiment is performed in a 384 multi-well plates, it is amenable for high-throughput homogeneous drug screening.
As aforestated, the p53 protein is a transcription factor which will bind specifically to DNA that contains RE sequences. In nature, wildtype p53 protein will bind to many promoter sequences and subsequently activate a wide range of genes for DNA repair, cell cycle arrest, apoptosis and its own degradation. As shown hereinabove, the bioassay in accordance with the present embodiment can be used for DNA selectivity study by conjugating different RE sequences to the AuNPs. However, it would be technically tedious to carry out conjugation for each DNA sequence and then screen for the binding affinity of a large number of promoter sequences. Therefore, a convenient competition assay which requires only one set of p53 RE-linked AuNPs has been designed in accordance with the present embodiment to evaluate the binding affinity of p53 protein to various promoter sequences listed in Table 1. This competition assay further allows the identification of the specific downstream pathway that is triggered upon p53 activation or reactivation, providing crucial information on the drug validation and outcome of the drug action.
Referring to
The dissociation constant (KD) is defined as the concentration of p53 for 50% of the DNA to be bound. The lower the KD value, the stronger the binding affinity between wtp53 and the tested free DNA sequence. Thus, a smaller change in the size of the complex bound probes is expected due to the competitive binding of wtp53 between the free DNAs and conjugated RE sequences on AuNPs. Referring to
Referring to
A 100 mer DNA linker 1220 containing two 50-mer complementary sequence to seq A and seq B bridges the two sets of AuNP-ssDNA conjugates (i.e., AuNP-seq A 1202 and AuNP-seq B 1204) and forms a double-stranded DNA (dsDNA) bridged dumbbell nanostructure construct 1222. The AuNP dumbbell probe 1222 contains a consensus wildtype ERE sequence 1224 (GGTCAnnnTGACC) located at seq B 1204 where an ER 1226 can recognize and specifically bind to it. The ERE-containing AuNP dumbbell probe 1222 is then purified on agarose gel and characterized by a DLS measurement showing, in a graph 1230, a ˜10 nm rightward peak shift 1232 relative to that of the individual conjugate peak 1208 at 20 nm. The formation of the AuNP dumbbell probe construct 1222 is confirmed by a TEM image 1240. The as-formed 30 nm ERE-containing AuNP dumbbell probes 1250 can then be used as a highly specific sensing probe 1250 to detect DNA-ER binding interactions in a homogenous solution. The DLS readout in a graph 1252 shows the appearance of a ‘complex peak’ 1254 in the 200-300 nm region, which was accompanied by a decrease in the DLS signal intensity of the original dumbbell probe peak 1256 shifted to 30 nm. It is conjectured that these distinctive, two population optical signature is believed to be the result of the sequence-specific binding of ER onto the ERE-containing AuNP dumbbell probes as shown in a TEM image 1260 evidencing that the AuNP dumbbell probe nanostructures in accordance with the present embodiment can advantageously be used in conjunction with a DLS platform to study transcription factor-DNA interactions.
To better establish the phenomenon of ER and ERE-containing AuNP dumbbell probe interaction, DLS analysis of ER interaction with different AuNP nanostructures, namely unmodified citrate-anion capped AuNPs, OEG passivated AuNP, and AuNPs bearing one strand of ssDNA was conducted.
Referring to
The graph 1330 of
The graph 1360 (
The ER-ERE interaction could not be studied on DLS without the transduction of the signal readout by the AuNP probes. The readouts of the ER-only, and ER-bound ERE samples (all without AuNPs) showed no significant difference from that of the buffer only. In addition, the graph 1330 depicting the DLS results of the OEG-passivated AuNPs evidences that the presence of AuNP could suppress the light-scattering signal from unbound proteins, buffers and other background noises. These factors indicate that the DLS nanoplatform in accordance with the present embodiment provides a highly sensitive and specific DLS readout with the biorecognition transduced by the unique ERE-containing AuNP dumbbell probes for the detection of target transcription factor in complicated biological samples such as blood or cell lysates that have less distinct light scattering cross sections.
For bioassay development, it is important to quantify the amount of analytes at low detection limits, as well as to establish the rapidity of the technique. Referring to
Referring to
Referring to
To establish the system specificity for the target proteins, the ERE-containing AuNP dumbbell probes 1250 were queried with bovine serum albumin (BSA) 1552. At comparable concentrations of protein, the graph 1550 of the DLS readout indicates that the size of the system was essentially unchanged, and that no complex peaks were observed. Since changes in the transcription factor levels in cells are the subject of much scientific study, such as the reprogramming of stem cells and study of oncogenic pathways, any system querying the cell extract has to be minimally affected by the presence of many different proteins in a sample and not give any non-specific readouts. In accordance with the present embodiment, the graph 1550 indicates that an unrelated protein (BSA) 1552 was unable to elicit any aggregation in ERE-containing AuNP probes. While proteins are known to induce AuNP aggregation through charge interaction, the OEG passivation of the AuNP probes prevents this from happening, thereby maintaining the specificity of the DNA-bridged dumbbell probes for the ER target.
AuNPs and DLS are two highly complementary platforms as the large scattering cross section of the AuNPs facilitates a clear and distinct DLS readout. The DLS nanoplatform system in accordance with the present embodiment was designed such that ERE-containing AuNP dumbbell probes could interact with the ER 1302 through specific binding of the protein that eventually presented as a unique DLS readout wherein the localization of positively-charged ER on the ERE negated the negative charge of the AuNPs and the reduction of the electrostatic repulsion provided a driving force for their clustering. In addition, as ERβ binds to ERE as a tetramer, a few ER-bound AuNP dumbbell probes would cluster as their respective ERs are assembled or interacted non-specifically through the protein side chains. All of these factors lead to the increase in the overall size of the system, which also amplifies the intensity of the DLS readout. Typically, AuNPs aggregate when they lose their colloidal stability, which can be attributed mainly to electrostatic and steric factors, and environmental conditions as the presence of ions like Na+ and Cl− can negate the AuNP surface charges, and such screening effects lead to increased clustering and aggregation. Generally, it is desirable to maintain the Coulombic repulsion and ensure the colloidal stability of the system until the target is introduced. The unique design with ERE localized in the dumbbell probes imparts a level of control such that only specific dumbbell probe-protein interaction can induce a change in the colloidal stability of the system, and the AuNPs will cluster to certain extent of particle stability instead of aggregating uncontrollably. This stability also ensures that readout changes, if any, must be due to the presence of the protein target. Moreover, the ER binding results in a distortion of the response element with bending towards its major groove, which inadvertently causes the AuNPs in the dumbbell probe construct to come into closer proximity. Such plasmonic coupling would also contribute to the red shift and increase in light scattering signature, which is translated as a unique DLS readout, enhancing the signal readout.
The use of nanostructures with a large scattering cross section also reduced the amount of AuNP probes required to bring about meaningful signal changes. In fact, the AuNPs samples that showed no appreciable signal on the UV-vis spectroscopy would still present a clear signal on DLS measurement, which is an additional advantage of using DLS for bimolecular detection over the conventional spectroscopic techniques. Thus, unlike conventional AuNP detection systems where the AuNP probes are used excessively, the amount of probes used here could be purposefully kept low such that even if ER is at low concentrations, their interaction with ERE could still elicit an appreciable positive readout. Further, the system in accordance with the present embodiment presents a label-free detection method where the ER is detected in its native form, as desired in biomarker sensing in general.
Thus, it can be seen that the present embodiment provides a novel ERE-containing AuNP dumbbell probe that is used for the detection of ER protein, via the signature readout on DLS. Complex peaks are observed only in the presence of ERE and ER, thus indicating both sequence specificity and protein selectivity. The quantification potential of the system has been evidenced through protein concentration dependent DLS signal outputs. Moreover, the system the system in accordance with the present embodiment can provide a DLS readout in as quickly as five minutes, thereby providing a high-throughput advantageous bioassay system for the study of not just transcription factors, but also other valuable biomarkers. The assay in accordance with the present embodiment provides a low nM level sensitivity more favorable than other AuNP-based aggregation assays that measure bulk-phase changes of particle size under UV-vis spectroscopy. In addition, the system in accordance with the present embodiment is not just limited to ER protein detection, the single-tube ‘mix and test’ AuNP dumbbell probe DLS-based bioassays in accordance with the present embodiment offer flexibility for detecting other DNA binding molecules by simply changing the conjugated DNA sequence, making the nanoprobes in accordance with the present embodiment versatile probes for use in biomedical research and diagnostic applications.
It can also be seen that the present embodiment provides a novel DLS-based anticancer drug screening assay targeting the druggable p53 pathways which include the wildtype activation and mutant reactivation. A first key aspect provided by the present embodiment is convenience. In accordance with the present embodiment, a convenient homogeneous-phase assay is provided in a single-tube format; label-free detection can be achieved with no chemical modification of the p53 proteins or the drug molecules; and a single-step “mix-and-measure” assay protocol is provided without multiple washing steps as required by conventional protocols. A second key aspect provided by the present embodiment is sensitivity and specificity. In accordance with the present embodiment, an ultralow detection limit of 0.6 pM is provided due to the strong light-scattering property of the AuNP nanoprobes. Also, the high specificity allows sequence specific detection of wildtype p53 binding using p53 RE linked AuNPs nano-dumbbell probe with negligible background interference. And reliable drug screening is provided in accordance with the present embodiment to detect drug-reactivated p53-DNA binding complexes.
A third key aspect provided by the present embodiment is efficiency. In accordance with the present embodiment, fast detection occurs within one minute. An efficient protein-DNA binding in a physiological solution is provided and the bioassay can be performed using multiwell plates with high amenability to high-throughput screening. A fourth key aspect provided by the present embodiment is cost-effectiveness. In accordance with the present embodiment, small sample volumes (1-5 μL), low p53 RE-AuNP nano-dumbbell probe concentrations (0.3 nM), and simple instrumentation (just need DLS which is available in most pharmaceutical companies) provides significant cost reduction for both drug and disease screening.
A fifth key aspect provided by the present embodiment is physiologically relevant results. In accordance with the present embodiment, the ability to test directly on cell lysates offers the following benefits: (a) provides more clinically-relevant data than using purified proteins, (b) accounts for all possible interactions between drugs and cellular molecules, (c) distinguishes differences in reactivation efficacy of various compounds, and (d) allows study of small molecule structure-activity relationships.
While exemplary embodiments have been presented in the foregoing detailed description of the invention, it should be appreciated that a vast number of variations exist. It should further be appreciated that the exemplary embodiments are only examples, and are not intended to limit the scope, applicability, operation, or configuration of the invention in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment of the invention, it being understood that various changes may be made in the function and arrangement of steps and method of operation described in the exemplary embodiment without departing from the scope of the invention as set forth in the appended claims.
