Allosteric Signal Research Articles

PIM-1L Kinase Binds to and Inactivates SRPK1: A Biochemical and Molecular Dynamics Study.

SR/RS dipeptide repeats vary in both length and position, and are phosphorylated by SR protein kinases (SRPKs). PIM-1L, the long isoform of PIM-1 kinase, the splicing of which has been implicated in acute myeloid leukemia, contains a domain that consists largely of repeating SR/RS and SH/HS dipeptides (SR/SH-rich). In order to extend our knowledge on the specificity and cellular functions of SRPK1, here we investigate whether PIM-1L could act as substrate of SRPK1 by a combination of biochemical and computational approaches. Our biochemical data showed that the SR/SH-rich domain of PIM-1L was able to associate with SRPK1, yet it could not act as a substrate but, instead, inactivated the kinase. In line with our biochemical data, molecular modeling followed by a microsecond-scale all-atom molecular dynamics (MD) simulation suggests that the SR/SH-rich domain acts as a pseudo-docking peptide that binds to the same acidic docking-groove used in other SRPK1 interactions and induces inactive SRPK1 conformations. Comparative community network analysis of the MD trajectories, unraveled the dynamic architecture of apo SRPK1 and notable alterations of allosteric communications upon PIM-1L peptide binding. This analysis also allowed us to identify key SRPK1 residues, including unique ones, with a pivotal role in mediating allosteric signal propagation within the kinase core. Interestingly, most of the identified amino acids correspond to cancer-associated amino acid changes, validating our results. In total, this work provides insights not only on the details of SRPK1 inhibition by the PIM-1L SR/SH-domain, but also contributes to an in-depth understanding of SRPK1 regulation.

Cooperative Substructure and Energetics of Allosteric Regulation of the Catalytic Core of the E3 Ubiquitin Ligase Parkin by Phosphorylated Ubiquitin.

Mutations in the parkin gene product Parkin give rise to autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase that is a critical participant in the process of mitophagy. Parkin has a complex structure that integrates several allosteric signals to maintain precise control of its catalytic activity. Though its allosterically controlled structural reorganization has been extensively characterized by crystallography, the energetics and mechanisms of allosteric regulation of Parkin are much less well understood. Allostery is fundamentally linked to the energetics of the cooperative (sub)structure of the protein. Herein, we examine the mechanism of allosteric activation by phosphorylated ubiquitin binding to the enzymatic core of Parkin, which lacks the antagonistic Ubl domain. In this way, the allosteric effects of the agonist phosphorylated ubiquitin can be isolated. Using native-state hydrogen exchange monitored by mass spectrometry, we find that the five structural domains of the core of Parkin are energetically distinct. Nevertheless, association of phosphorylated ubiquitin destabilizes structural elements that bind the ubiquitin-like domain antagonist while promoting the dissociation of the catalytic domain and energetically poises the protein for transition to the fully activated structure.

6813 Des-Ciclesonide: A Novel Selective Glucocorticoid Receptor Modulator With Therapeutic Potential for Bronchopulmonary Dysplasia, a Chronic Lung Disease of Prematurity

Abstract Disclosure: N. El Khoury: None. J. Jaumotte: None. C. Min: None. C. Madigan: None. J. Wang: None. U. Chandran: None. V. Sampath: None. T.J. Cole: None. P. Monaghan-Nichols: None. R. Houtman: None. K.W. Nettles: None. D.B. DeFranco: None. Infants born before 30 weeks gestation are at risk of developing bronchopulmonary dysplasia (BPD), a prevalent chronic lung disease of prematurity. Glucocorticoids (GCs) such as dexamethasone (DEX) have been a mainstay of BPD prevention as they reduce lung inflammation and injury. However, their use is encumbered with several adverse effects including hyperglycemia, impaired somatic and brain growth and cerebral palsy. These major concerns have resulted in the AAP recommending caution in GC use in preterm infants.Ciclesonide (CIC), an inhaled GC prodrug approved for the treatment of asthma and allergic rhinitis in children, is converted to the potent glucocorticoid receptor (GR) agonist des-ciclesonide (DES) by enzymes enriched in the airways. We previously reported that s.c. delivery of CIC in neonatal rats activated GR transcriptional responses in lung but did not trigger the adverse effects on somatic growth, brain weight or cerebral white matter loss caused by DEX leading us to propose CIC as a potential new pharmacotherapy for BPD. To determine whether limited systemic metabolism of CIC in neonatal rats was solely responsible for its enhanced safety profile, we assessed various outcomes in neonatal rats followed s.c. delivery of its active metabolite DES. Consistent with previous studies, five daily s.c. injections of DEX led to a reduction in body and brain weight as well as reduced serum levels of insulin-like growth factor-1 and chronic hyperglycemia. Surprisingly, analogous s.c. injection of neonatal rats with DES, which has a higher binding affinity for GR than DEX, did not trigger any of these adverse effects. However, DES is as effective as DEX in reducing the expression of various proinflammatory cytokine mRNAs in neonatal rat lung induced by an 11-day systemic treatment with bleomycin. Bulk RNAseq analyses revealed transcriptional responses of neonatal lung and liver to DES, although less robust than that observed to DEX (i.e., 25% and 13% in lung and liver, respectively). Nonetheless, a NAPing assay for coregulator peptide binding showed increased binding of all the DES responsive peptide interactions relative to DEX and cortisol. All-atom molecular dynamics simulations revealed site-specific H-bonding changes within the GR ligand binding domain (LBD) caused by the bulky C16/C17substitution within DES, which was translated into broader changes in LBD structure, specifically revealed as differences in RMSF in helix 7/8 and the surfaces interacting with the NCOA2 peptide. Furthermore, network pathway analysis illustrated that DES mobility at helix 7/8 uniquely generated a meaningful allosteric signaling pathway between Q642 and NCOA2 peptide. In conclusion, the pharmacokinetic properties of the GR prodrug CIC coupled with the tissue-selective pharmacodynamic properties of its active metabolite DES make this FDA approved drug a potential new therapeutic agent for BPD. Presentation: 6/3/2024

Crystal structures of cables formed by the acetylated and unacetylated forms of the Schizosaccharomyces pombe tropomyosin orthologue TpmCdc8

Cables formed by head-to-tail polymerization of tropomyosin, localized along the length of sarcomeric and cytoskeletal actin filaments, play a key role in regulating a wide range of motile and contractile processes. The stability of tropomyosin cables, their interaction with actin filaments and the functional properties of the resulting co-filaments are thought to be affected by N-terminal acetylation of tropomyosin. Here, we present high–resolution structures of cables formed by acetylated and unacetylated Schizosaccharomyces pombe tropomyosin orthologue TpmCdc8. The crystal structures represent different types of cables, each consisting of TpmCdc8 homodimers in a different conformation. The structures show how the interactions of the residues in the overlap junction contribute to cable formation and how local structural perturbations affect the conformational dynamics of the protein and its ability to transmit allosteric signals. In particular, N-terminal acetylation increases the helicity of the adjacent region, which leads to a local reduction in conformational dynamics and consequently to less fraying of the N-terminal region. This creates a more consistent complementary surface facilitating the formation of specific interactions across the overlap junction.

SecM leader peptide as an allosteric translation inhibitor: a molecular dynamics study

The SecM leader peptide regulates translation of the SecA protein, being a part of the Sec translocase, that reversibly arrests the ribosome. In the present study the structure of the SecM complex with the E. coli A/A,P/P–ribosome was obtained by means of docking and molecular dynamics simulation methods. It has been established that binding of the SecM leader peptide in the nascent peptide exit tunnel leads to a turn of the aminoacylating proline residue away from the C–terminal SecM glycine residue, which is adverse to the peptidyltransferase reaction. Besides, the SecM binding leads to a disturbance of the A–tRNA contacts with the tip of the H38 helix of the 23S rRNA (the A–site finger, ASF) and ribosomal protein uL16. Allosteric interrelation between these events has been proved by a construction of networks of concerted changes in non–covalent interactions throughout the whole ribosome, whereupon the A1614 and A751 residues of the 23S rRNA in the exit tunnel that formed stacking interactions with the SecM residues during the MD simulations, were found to be the principal triggers, inducing crucial alterations in the A–tRNA binding. The allosteric signal from the SecM peptide to the ASF, according to our model, is transmitted through ribosomal protein uL22, and there is reason to believe that this sensor is used not only by the SecM leader peptide, but also by other peptides that cause translation arrest.

Toward the Design of Allosteric Effectors: Gaining Comprehensive Control of Drug Properties and Actions.

While the therapeutic potential of allosteric drugs is increasingly realized, the discovery of effectors is largely incidental. The rational design of allosteric effectors requires new state-of-the-art approaches to account for the distinct characteristics of allosteric ligands and their modes of action. We present a broadly applicable computational framework for obtaining allosteric site-effector pairs, providing targeted, highly specific, and tunable regulation to any functional site. We validated the framework using the main protease from SARS-CoV-2 and the K-RasG12D oncoprotein. High-throughput per-residue quantification of the energetics of allosteric signaling and effector binding revealed known drugs capable of inducing the required modulation upon binding. Starting from fragments of known well-characterized drugs, allosteric effectors and binding sites were designed and optimized simultaneously to achieve targeted and specific signaling to distinct functional sites, such as, for example, the switch regions of K-RasG12D. The generic framework proposed in this work will be instrumental in developing allosteric therapies aligned with a precision medicine approach.

Revealing the graded activation mechanism of neurotensin receptor 1

Graded activation contributes to the precise regulation of GPCR activity, presenting new opportunities for drug design. In this work, a total of 10 μs enhanced-sampling simulations are performed to provide molecular insights into the binding dynamics differences of the neurotensin receptor 1 (NTSR1) to the full agonist SRI-9829, partial agonist RTI-3a and inverse agonist SR48692. The possible graded activation mechanism of NTSR1 is revealed by an integrated analysis utilizing the reweighted potential of mean force (PMF), deep learning (DL) and transfer entropy (TE). Specifically, the orthosteric pocket is observed to undergo expansion and contraction, with the G-protein-binding site experiencing interconversions among the inactive, intermediate and active-like states. Detailed structural comparisons capture subtle conformational differences arising from ligand binding in allosteric signaling, which can well explain the graded activation. Critical microswitches that contribute to graded activation are efficiently identified with the DL model. TE calculations enable the visualization of allosteric communication networks within the receptor, elucidating the driver-responder relationships associated with signal transduction. Fortunately, the dissociation of the full agonist from the orthosteric pocket is observed. The current findings systematically reveal the mechanism of NTSR1 graded activation, and also provide implications for structure-based drug design.

GPCR Signaling: A Study of the Interplay Between Structure, Energy, and Function.

G protein-coupled receptors (GPCRs) exemplify sophisticated allosteric communication, transducing extracellular signals through ligand-induced structural rearrangements that resonate through the molecular scaffold. Despite extensive study, the biophysical underpinnings of how conformational changes spread remain unclear. This work employs a novel physics-based framework to characterize the role of energy dissipation in directing intramolecular signaling pathways. By modeling each residue as a network of coupled oscillators, we generate a localization landscape depicting the vibrational energy distribution throughout the protein scaffold. Quantifying directional energy flux between residues reveals distinct pathways for energy and information transfer, illuminating sequences of allosteric communication. Our analysis of CB1 and CCR5 crystal structures unveils an anisotropic pattern of energy dissipation aligning with key functional dynamics, such as activation-related conformational changes. These anisotropic patterns of vibrational energy flow constitute pre-configured channels for allosteric signaling. Elucidating the relationship between structural topology and energy dissipation patterns provides key insights into the thermodynamic drivers of conformational signaling. This methodology significantly advances our mechanistic understanding of allostery in GPCRs and presents a broadly applicable approach for rationally dissecting allosteric communication pathways, with potential implications for structure-based drug design targeting these critical receptors.

Insights into Allosteric Inhibition of the AcrB Efflux Pump: Role of Distinct Binding Pockets, Protomer Preferences, and Crosstalk Disruption.

AcrB, a key component in bacterial efflux processes, exhibits distinct binding pockets that influence inhibitor interactions. In addition to the well-known distal binding pocket within the periplasmic domain, a noteworthy pocket amidst the transmembrane (TM) helices serves as an alternate binding site for inhibitors. The bacterial efflux mechanism involves a pivotal functional rotation of the TM protein, inducing conformational changes in each protomer and propelling drugs toward the outer membrane domain. Surprisingly, inhibitors binding to the TM domain display a preference for L protomers over T protomers. Metadynamics simulations elucidate that Lys940 in the TM domain of AcrB can adopt two conformations in L protomers, whereas the energy barrier for such transitions is higher in T protomers. This phenomenon results in stable inhibitor binding in l protomers. Upon a detailed analysis of unbinding pathways using random accelerated molecular dynamics and umbrella sampling, we have identified three distinct routes for ligand exit from the allosteric site, specifically involving regions within the TM domains─TM4, TM5, and TM10. To explore allosteric crosstalk, we focused on the following key residues: Val452 from the TM domain and Ala831 from the porter domain. Surprisingly, our findings reveal that inhibitor binding disrupts this communication. The shortest path connecting Val452 and Ala831 increases upon inhibitor binding, suggesting sabotage of the natural interdomain communication dynamics. This result highlights the intricate interplay between inhibitor binding and allosteric signaling within our studied system.

COMPARATIVE STUDY OF THE DYNAMIC BEHAVIOR OF BACTERIAL PHOTOREGULATED ADENYLATE CYCLASES BPAC AND OAPAC

Photoregulated adenylate cyclases bPAC and OaPAC are compared using classical molecular dynamics simulations in both states, before and after blue light irradiation. The pathways of signal transduction from the photoreceptor to the catalytic domain are determined. The more pronounced acceleration of the catalytic reaction rate as a result of light irradiation in the case of bPAC is explained both by a shift in equilibrium towards a closed conformation and by a larger number of suboptimal pathways of allosteric signal transduction from photoreceptor to catalytic domain

Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

SARS-CoV-2 3CLPro Dihedral Angles Reveal Allosteric Signaling.

In allosteric proteins, identifying the pathways that signals take from allosteric ligand-binding sites to enzyme active sites or binding pockets and interfaces remains challenging. This avenue of research is motivated by the goals of understanding particular macromolecular systems of interest and creating general methods for their study. An especially important protein that is the subject of many investigations in allostery is the SARS-CoV-2 main protease (Mpro), which is necessary for coronaviral replication. It is both an attractive drug target and, due to intense interest in it for the development of pharmaceutical compounds, a gauge of the state-of-the-art approaches in studying protein inhibition. Here we develop a computational method for characterizing protein allostery and use it to study Mpro. We propose a role of the protein's C-terminal tail in allosteric modulation and warn of unintuitive traps that can plague studies of the role of protein dihedrals angles in transmitting allosteric signals.

Target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive and direct Pseudomonas aeruginosa detection

Pseudomonas aeruginosa (P. aeruginosa), a kind of gram-negative pathogenic bacteria, are causative agents of severe infections, such as lower respiratory tract infections in children and cancers. Detecting low levels of P. aeruginosa in clinical samples in an easy-to-operate manner is highly desired but still poses a problem. Herein, we established a target recognition-initiated allosteric probe-based multiple signal amplification strategy for sensitive detection of P. aeruginosa in a wash-free way. This approach involves the allosteric probe’s accurate recognition and binding to target P. aeruginosa, leading to subsequent multiple-cycle amplification. Afterward, the amplified products were translated to induce the aggregation of gold nanoparticles (AuNPs), resulting in color variations. The utilization of the allosteric probe, which is integrated with the aptamer sequences, enables wash-free detection of P. aeruginosa. Taking the merit of multiple signal amplification process, the suggested method showed a strong linear response to the extracted P. aeruginosa within a concentration range of 10–105 cfu/mL, with a low limit of detection for individual P. aeruginosa detection. The proposed technique has considerable clinical promise for early disease diagnosis because to its high sensitivity and wash-free simplicity.

Deciphering S100B Allosteric Signaling: The Role of a Peptide Target, TRTK-12, as an Ensemble Modulator.

Allostery is an essential biological phenomenon in which perturbation at one site in a biomolecule elicits a functional response at a distal location(s). It is integral to biological processes, such as cellular signaling, metabolism, and transcription regulation. Understanding allostery is also crucial for rational drug discovery. In this work, we focus on an allosteric S100B protein that belongs to the S100 class of EF-hand Ca2+-binding proteins. The Ca2+-binding affinity of S100B is modulated allosterically by TRTK-12 peptide binding 25 Å away from the Ca2+-binding site. We investigated S100B allostery by carrying out nuclear magnetic resonance (NMR) measurements along with microsecond-long molecular dynamics (MD) simulations on S100B/Ca2+ with/without TRTK-12 at different NaCl salt concentrations. NMR HSQC results show that TRTK-12 reorganizes how S100B/Ca2+ responds to different salt concentrations at both orthosteric and allosteric sites. The MD data suggest that TRTK-12 breaks the dynamic aromatic and hydrogen-bond interactions (not observed in X-ray crystallographic structures) between the hinge/helix and Ca2+-binding EF-hand loop of the two subunits in the homodimeric protein. This triggers rearrangement in the protein network architectures and leads to allosteric communication. Finally, computational studies of S100B at distinct ionic strengths suggest that ligand-bound species are more robust to the changing environment relative to the S100B/Ca2+ complex.

β-sheets mediate the conformational change and allosteric signal transmission between the AsLOV2 termini.

Avena sativa phototropin 1 light-oxygen-voltage 2 domain (AsLOV2) is a model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change begins with the unfolding of the N-terminal A'α helix in the dark state followed by the unfolding of the C-terminal Jα helix. The light state is characterized by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, β-sheets are identified as crucial components for mediating allosteric signal transmission between the two termini. Through combined experimental and computational investigations, the Hβ and Iβ strands are recognized as the most critical and influential β-sheets in AsLOV2's allosteric mechanism. To elucidate the role of these β-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive molecular dynamics simulations. In-depth hydrogen bond analyses emphasized the role of two hydrogen bonds, Asn482-Leu453 and Gln479-Val520, in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. This illustrates the role of β-sheets in the transmission of the allosteric signal upon the photoactivation of the light state.

Allosteric pathways of SARS and SARS-CoV-2 spike protein identified by neural relational inference.

The receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein must undergo a crucial conformational transition to invade human cells. It is intriguing that this transition is accompanied by a synchronized movement of the entire spike protein. Therefore, it is possible to design allosteric regulators targeting non-RBD but hindering the conformational transition of RBD. To understand the allosteric mechanism in detail, we establish a computational framework by integrating coarse-grained molecular dynamic simulations and a state-of-the-art neural network model called neural relational inference. Leveraging this framework, we have elucidated the allosteric pathway of the SARS-CoV-2 spike protein at the residue level and identified the molecular mechanisms involved in the transmission of allosteric signals. The movement of D614 is coupled with that of Q321. This interaction subsequently influences the movement of K528/K529, ultimately coupling with the movement of RBD during conformational changes. Mutations that weaken the interactions within this pathway naturally block the allosteric signal transmission, thereby modulating the conformational transitions. This observation also offers a rationale for the distinct allosteric patterns observed in the SARS-CoV spike protein. Our result provides a useful method for analyzing the dynamics of potential viral variants in the future.

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

Deciphering S100B allosteric signaling: The role of TRTK as an ensemble modulator

Deciphering S100B allosteric signaling: The role of TRTK as an ensemble modulator

Allosteric Signal within the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein Mediated by a Class 3 Monoclonal Antibody Revealed through Molecular Dynamics Simulations and Protein Residue Networks.

This study investigated the allosteric action within the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein caused by class 3 monoclonal antibody (mAb) binding. As the emergence of SARS-CoV-2 variants has raised concerns about the effectiveness of treatments by antibodies, targeting the highly conserved class 3 epitopes has become an alternative strategy of antibody design. Simulations of explicitly solvated RBD of the BA.2.75 omicron subvariants were carried out both in the presence and in the absence of bebtelovimab, as a model example of class 3 monoclonal antibodies against the RBD of the SARS-CoV-2 spike protein. The comparative analysis showed that bebtelovimab's binding on two α helices at the epitope region disrupted the nearby interaction network, which triggered a denser interaction network formation on the opposite side of the receptor-binding motif (RBM) region and resulted in a "close" conformation that could prevent the ACE2 binding. A better understanding of this allosteric action could lead to the development of alternative mAbs for further variants of concern. In terms of computational techniques, the communicability matrix could serve as a tool to visualize the effects of allostery, as the pairs of amino acids or secondary structures with high communicability could pinpoint the possible sites to transfer the allosteric signal. Additionally, the communicability gain/loss matrix could help elucidate the consequences of allosteric actions, which could be employed along with other allostery quantification techniques in some previous studies.

A label-free fluorescent magnetic dual-aptasensor based on aptamer allosteric regulation of β-lactoglobulin

We presented a label-free fluorescent biosensor based on magnetic dual-aptamer allosteric regulation of β-lactoglobulin (β-LG) detection. The bovine serum albumin (BSA) acted as the bridge to connect amino-modified magnetic beads and aptamer, which synthesized pyramid-type probes (MBAP) with high capture and reduced nonspecific adsorption. Moreover, the original aptamer was tailored and then designed as a bivalent aptamer to fabricate allosteric signal probes (ASP). The ASP can both specifically capture β-LG and output the fluorescence signal. The detection mechanism is as follows. The combination of the dual-aptamer and β-LG triggered the allosteric change, resulting in the release of SYBR Green (SG I) from the allosteric signal probe and change signals. This method exhibits a broad linear detection range from 10 ng/mL to 1 mg/mL and the limit of detection reaches as low as 8.06 ng/mL. This study provides a highly generalizable strategy for protein biomolecular detection via replacing different target aptamers.