Stem-loop Research Articles

A passive star polymer in a dense active bath: insights from computer simulations.

Using computer simulations in two dimensions (2D), we explore the structure and dynamics of a star polymer with three arms made of passive monomers immersed in a bath of active Brownian particles (ABPs). We analyze the conformational and dynamical changes of the polymer as a function of activity and packing fraction. We also study the process of motility induced phase separation (MIPS) in the presence of a star polymer, which acts as a mobile nucleation center. The presence of the polymer increases the growth rate of the clusters in comparison to a bath without the polymer. In particular, for low packing fraction, both nucleation and cluster growth are affected by the inclusion of the star polymer. Clusters grow in the vicinity of the star polymer, resulting in the star polymer experiencing a caged motion similar to a tagged ABP in the dense phase. Due to the topological constraints of the star polymers and clustering nearby, the conformational changes of the star polymer lead to interesting observations. Inter alia, we observe the shrinking of the arm with increasing activity along with a short-lived hairpin structure of one arm formed. We also see the transient pairing of two arms of the star polymer, while the third is largely separated at high activity. We hope our findings will help in understanding the behavior of active-passive mixtures, including biopolymers of complex topology in dense active suspensions.

Controlled Sequential Assembly of DNA Nanoparticles Inside Cells Enabling Mitochondrial Interference

AbstractDeveloping artificial material systems to rationally interfere with organelles is emerging as a promising route to regulate the behaviors and fate of cells, thus providing new therapeutic strategies. Herein, a DNA nanoparticles (DNPs)‐based material system is reported, which achieves controlled sequential assembly inside cells, and realizes specific interference toward mitochondria. Two types of DNPs are synthesized via radical polymerization, followed with cascade hybridization chain reaction of hairpin DNA with Cyanine5 (Cy5) for mitochondrial targeting, and with complementary sequences for base pairing, respectively. DNPs with Cy5 (Cy5‐DNPs) first entered cells to target mitochondria, and the other DNPs with complementary sequences entered cells after an interval to sequentially assemble with Cy5‐DNPs into aggregates via base pairing. Mitochondrial interference is achieved, including increased reactive oxygen species, decreased membrane potential, abnormal elevation of Ca2+ level, decreased adenosine triphosphate, and attenuated cellular migration rate. In particular, by regulating the intervals of two types of DNPs entering cells, the mitochondrial interference degree is controllably modulated. This work achieves regulated organelles interference via the precise controlled self‐assembly of DNA nanostructures inside cells, which is envisioned to have great potential in precision biomedicine.

Intelligent chemiluminescent system for wash-free and ultrasensitive assay of aflatoxin B1 in coix seed and decoction pieces

Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins. Convenient and reliable determination of trace AFB1 is highly desired. Herein, dual hemin labeled DNA hairpin was designed as a chemiluminescence (CL) switch and combined with aptamer of AFB1 and its partial complementary strand for homogenous assay of AFB1. Due to the judicious design of three DNA sequences and the help of nicking enzyme, the AFB1 recognition, signal transduction and recycling amplification can be implemented during one-step reaction. The whole protocol only involved the simple mixing of samples with homogeneous solution, avoiding the preparation of sophisticated solid-phase sensor and eliminating the multi-step washing and resulting errors, which provided a unique tool for on-site rapid testing. Importantly, when the operation process was simplified, the detection sensitivity can be enhanced facilely due to the enzyme recycling. Under optimal conditions, the intelligent CL system for sensing AFB1 exhibited a wide dynamic range (1.0 × 10-2 ∼ 100 ng mL−1) with a low detection limit (down to 1.5 pg mL−1), suitable for detecting trace AFB1 in complex matrix. The methodology was validated in coix seed and decoction pieces. The superiority including free of washing and separation, high sensitivity, acceptable accuracy, and greenness, showed good applicability of the proposed CL system in safety evaluation of food and medicine.

A new small molecule DoNA binding to CAG repeat RNA

We here report a new molecule DoNA binding to a CAG repeat RNA. DoNA is a dimer of the NA molecule that we previously reported. NA binds with high affinity to a CAG repeat DNA but not significantly to a CAG repeat RNA. Binding analyses using SPR and CSI-TOF MS indicated a significant increase in the affinity of DoNA to a single stranded CAG repeat RNA compared to NA. Systematic investigation of the RNA motifs bound by DoNA using hairpin RNA models revealed that DoNA binds to the CAG units at overhang and terminal positions, and notably, it binds to the structurally flexible internal and hairpin loop region.

Structural Dynamics Role of AGG Interruptions in Inhibition CGG Repeat Expansion Associated with Fragile X Syndrome.

Abnormal expansion of trinucleotide CGG repeats is responsible for Fragile X syndrome. AGG interruptions in CGG repeat tracts were found in most healthy individuals, suggesting a crucial role in preventing disease-prone repeat expansion. Previous biophysics studies emphasize a difference in the secondary structure affected by AGG interruptions. However, the mechanism of how AGG interruptions impede repeat expansion remains elusive. We utilized single-molecule fluorescence resonance energy transfer spectroscopy to investigate the structural dynamics of CGG repeats and their AGG-interrupted variants. Tandem CGG repeats fold into a stem-loop hairpin structure with the capability to undergo a conformational rearrangement to modulate the length of the overhang. However, this conformational rearrangement is much more retarded when two AGG interruptions are present. Considering the significance of hairpin slippage in repeat expansion, we present a molecular basis suggesting that the internal loop created by two AGG interruptions acts as a barrier, obstructing the hairpin slippage reconfiguration. This impediment potentially plays a crucial role in curbing abnormal expansion, thereby contributing to the genomic stability.

In Silico Evaluation of the PCR Performance of Different Tests for Detection of WSSV

In this study, the primers of different protocols for the detection of White Spot Syndrome Virus (WSSV) were analyzed in silico to evaluate their potential performance in PCR. As with any biological entity, this virus evolves constantly. Thus, this analysis showed that a few primers, including those recommended by the World Organization for Animal Health (WOAH), might mismatch with some isolates of WSSV, specially with isolates more recently sequenced. Furthermore, a set of primers recommended by WOAH, showed the potential to self-dimer and form hairpin loop structures, which could affect the efficiency of PCR, resulting in an inaccurate diagnostic result. From our perspective, and considering the evolutionary trajectory of this virus, it may be time for the WOAH to update the PCR protocols recommended for WSSV detection, which remains as a highly prevalent and lethal virus.

Using supramolecular polymerization of DNA to produce tandem repeat proteins

AbstractTandem repeat (TR) proteins are abundant in nature and show diverse biochemical and mechanical properties. However, these proteins can be difficult to synthesize in a precise way because of their repetitive nature. Using a supramolecular polymerization scheme known as hybridization chain reaction, we show how to generate the DNA sequences encoding such TR proteins. The polymerization mechanism allows for identical “monomers” to selectively create TR proteins of different molecular weight. We demonstrate this strategy with examples from elastin‐like polypeptides (ELPs) and mussel foot proteins (mfps). Moreover, the scheme can be adapted to create protein libraries through the use of multiple DNA hairpin “monomers.”

DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts.

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes.

Dual-signal SERS biosensor based on spindle-shaped gold array for sensitive and accurate detection of miRNA 21

MicroRNAs (miRNAs) play an important regulatory role in tumor progression and have been emerging as promising biomarkers for early diagnosis of breast cancer. Herein, we developed a dual-signal surface-enhanced Raman scattering (SERS) biosensor for the quantitative detection of miRNA 21 (miR-21) by utilizing a tightly ordered spindle-shaped gold (SAu) array with 5, 5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) molecule as an internal standard. Meanwhile, 6-carboxyl-Xrhodamine (ROX) labeled hairpin DNA H1 was linked to the SAu array by Au–S bonds generating another SERS signal. In the presence of miR-21, H1 was turned on with a decrease in SERS signal of ROX, while the SERS signal of DTNB remained stable. The SAu array as substrate provides a large area of hot spots, and the dual-signal can avoid background interference and achieve signal self-calibration. By the dual-signal SERS biosensor, target miR-21 could be detected over a broad linear range (0.1 −105 pM) with a low detection limit (0.046 pM) without signal amplification strategy. Thanks to the SAu array and dual-signal output, the sensor showed high sensitivity, good signal reproducibility, excellent specificity, and high detection accuracy. When this sensor was applied to the miR-21 assay in human serum samples, its performance was comparable to quantitative reverse transcription polymerase chain reaction (qRT-PCR), indicating promising applications of the developed sensor for cancer diagnostics.

LeishIF4E2 is a cap-binding protein that plays a role in Leishmania cell cycle progression.

Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

Hierarchical Mechanical Transduction of Precision-Engineered DNA Hydrogels with Sacrificial Bonds.

Engineering the response to external signals in mechanically switchable hydrogels is important to promote smart materials applications. However, comparably little attention has focused on embedded precision mechanisms for autonomous nonlinear response in mechanical profiles in hydrogels, and we lack understanding of how the behavior from the molecular scale transduces to the macroscale. Here, we design a nonlinear stress-strain response into hydrogels by engineering sacrificial DNA hairpin loops into model network hydrogels formed from star-shaped building blocks. We characterize the force-extension response of single DNA hairpins and are able to describe how the specific topology influences the nonlinear mechanical behavior at different length scales. For this purpose, we utilize force spectroscopy as well as microscopic and macroscopic deformation tests. This study contributes to a better understanding of designing nonlinear strain-adaptive features into hydrogel materials.

Visualizing a two-state conformational ensemble in stem-loop 3 of the transcriptional regulator 7SK RNA.

Structural plasticity is integral to RNA function; however, there are currently few methods to quantitatively resolve RNAs that have multiple structural states. NMR spectroscopy is a powerful approach for resolving conformational ensembles but is size-limited. Chemical probing is well-suited for large RNAs but provides limited structural and kinetics information. Here, we integrate the two approaches to visualize a two-state conformational ensemble for the central stem-loop 3 (SL3) of 7SK RNA, a critical element for 7SK RNA function in transcription regulation. We find that the SL3 distal end exchanges between two equally populated yet structurally distinct states in both isolated SL3 constructs and full-length 7SK RNA. We rationally designed constructs that lock SL3 into a single state and demonstrate that both chemical probing and NMR data fit to a linear combination of the two states. Comparison of vertebrate 7SK RNA sequences shows either or both states are highly conserved. These results provide new insights into 7SK RNA structural dynamics and demonstrate the utility of integrating chemical probing with NMR spectroscopy to gain quantitative insights into RNA conformational ensembles.

Bipedal DNA walker- CRISPR/Cas12a coupling with sulfur doped boron nitride–carbon nitride composite biosensing for the detection of miRNA-21

A novel photoelectrochemical (PEC) biosensor based on 2D–2D heterojunction S-BN/CN and dual-orbit three-dimensional (3D) bipedal DNA walker-synergized CRISPR/Cas12a signal amplification was fabricated for sensitive detection of miRNA-21. First of all, S-BN/CN, which sulfur was doped into boron nitride (BN) to construct heterojunction of 2D S-BN and 2D g-C3N4, was used as photosensitive materials to provide initial photocurrent, and combined methylene blue (MB) labeled hairpin DNA (MB-HP) as a signal probe. Secondly, after identifying the target miRNA-21, the two swing arms were simultaneously activated on two 3D orbits, cleaving along the orbit to output massive short single strand DNA (O-DNA) under the action of Nt.BsmAl endonuclease. Then trigger the trans-cleavage of CRISPR/Cas12a and cut off the hairpin DNA labeled with MB to achieve signal-off. The result showed that the proposed PEC biosensor had a good sensitivity for the detection of miRNA-21. It was a good linearity in the range of 10 fM - 100 nM, and the limit of detection was 3.98 fM (S/N = 3). The proposed PEC biosensor achieved sensitive detection with high specificity and low limit of detection for miRNA-21, while the rational design could be extended to achieve the detection of other targets.

Exploring the folding landscape of leptin: Insights into threading pathways

The discovery of new protein topologies with entanglements and loop-crossings have shown the impact of local amino acid arrangement and global three-dimensional structures. This phenomenon plays a crucial role in understanding how protein structure relates to folding and function, affecting the global stability, and biological activity. Protein entanglements encompassing knots and non-trivial topologies add complexity to their folding free energy landscapes. However, the initial native contacts driving the threading event for entangled proteins remains elusive. The Pierced Lasso Topology (PLT) represents an entangled topology where a covalent linker creates a loop in which the polypeptide backbone is threaded through. Compared to true knotted topologies, PLTs are simpler topologies where the covalent-loop persists in all conformations. In this work, the PLT protein leptin, is used to visualize and differentiate the preference for slipknotting over plugging transition pathways along the folding route. We utilize the Energy Landscape Visualization Method (ELViM), a multidimensional projection technique, to visualize and distinguish early threaded conformations that cannot be observed in an in vitro experiment. Critical contacts for the leptin threading mechanisms were identified where the competing pathways are determined by the formation of a hairpin loop in the unfolded basin. Thus, prohibiting the dominant slipknotting pathway. Furthermore, ELViM offers insights into distinct folding pathways associated with slipknotting and plugging providing a novel tool for de novo design and in vitro experiments with residue specific information of threading events in silico.

Detection of Hofmeister effects at a single-molecule level

Metal cations are essential for the function of nucleic acids. Given their efficiency for DNA overwinding and the DNA hairpin folding/unfolding process coupled with DNA twist, we systematically investigated the effects of cation identities and increasing concentrations of NaCl, KCl, and LiCl from 50 mM to 300 mM on the folding/unfolding process by manipulating a DNA hairpin construct with magnetic tweezers. The critical forces and free energy of the unfolding transition of the DNA hairpin at a fixed concentration of KCl, NaCl, and LiCl followed the same ranking as , and are reversed with the unfolding rate ranking of {}\ ext{Na}^{+}>{}\ ext{Li}^{+}$ ?> . This highlights that the addition of Li+ in solution obviously enhances the stability of the DNA hairpin when compared to Na+, and K+, and their efficiency on the stability of the DNA hairpin was ranked as .

G-U base-paired hpRNA confers potent inhibition of small RNA function in plants.

MicroRNA (miRNA) target mimicry technologies, utilizing naturally occurring miRNA decoy molecules, represent a potent tool for analyzing miRNA function. In this study, we present a highly efficient small RNA (sRNA) target mimicry design based on G-U base-paired hairpin RNA (hpG:U), which allows for the simultaneous targeting of multiple sRNAs. The hpG:U constructs consistently generate high amounts of intact, polyadenylated stem-loop (SL) RNA outside the nuclei, in contrast to traditional hairpin RNA designs with canonical base pairing (hpWT), which were predominantly processed resulting in a loop. By incorporating a 460-bp G-U base-paired double-stranded stem and a 312-576 nt loop carrying multiple miRNA target mimicry sites (GUMIC), the hpG:U construct displayed effective repression of three Arabidopsis miRNAs, namely miR165/166, miR157, and miR160, both individually and in combination. Additionally, a GUMIC construct targeting a prominent cluster of siRNAs derived from cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) effectively inhibited Y-Sat siRNA-directed silencing of the chlorophyll biosynthetic gene CHLI, thereby reducing the yellowing symptoms in infected Nicotiana plants. Therefore, the G-U base-paired hpRNA, characterized by differential processing compared to traditional hpRNA, acts as an efficient decoy for both miRNAs and siRNAs. This technology holds great potential for sRNA functional analysis and the management of sRNA-mediated diseases.

Dissecting the role of GGGGCC repeat RNA secondary structure in cellular toxicity associated with C9 FTD/ALS

AbstractBackgroundAberrant expansions of simple nucleotide repeat sequences can cause more than fifty human diseases. For example, the most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is the expansion of an intronic GGGGCC hexanucleotide repeat sequence in C9orf72 gene (C9 FTD/ALS). Transcribed GGGGCC repeat RNAs can form complex secondary structures, which facilitate unusual interactions with cellular RNA‐binding proteins (RBPs). Sequestration of crucial RBPs into nuclear foci can inhibit the normal functions of those proteins. In addition, expanded GGGGCC repeat RNAs can support the translation of highly toxic repetitive peptides through a non‐canonical process known as repeat associated non‐AUG translation (RAN translation), which may require some specialized factors. Here we sought to understand how RNA secondary structure influence GGGGCC RAN translation and repeat elicited toxicity in C9 FTD/ALS.MethodWe used chemical modification coupled with traditional high‐throughput sequencing and nano‐pore sequencing approaches to probe GGGGCC repeat RNA secondary structures inside cells. We further use in vitro and in vivo RAN translation assays to determine whether repeat RNA secondary structures influence RAN translation efficiency.ResultOur RNA structural studies revealed that GGGGCC repeat RNAs form primarily form hairpin structures in cells. With nanopore sequencing, we were also able to detect the possible presence of unfolded and/or g‐quadruplex folded GGGGCC repeat RNAs in cells. Folding GGGGCC RNAs into hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits RAN translation in both in vitro and in vivo assays. Our results suggest that RNA secondary structure can affect the RAN translation elongation efficiency and g‐quadruplex structures may lead to the termination of elongating ribosomes.ConclusionWe present a deep sequencing‐based chemical probing technique to evaluate the roles of repeat RNA secondary structures in RNA toxicity underlying dementia‐associated repeat expansion disorders. Repeat RNA structural information can be used to identify chemical compounds that can bind to the RNA and prevent cytotoxicity in cells.

4.5SH RNA counteracts deleterious exonization of SINE B1 in mice

4.5SH RNA is a highly abundant, small rodent-specific noncoding RNA that localizes to nuclear speckles enriched in pre-mRNA-splicing regulators. To investigate the physiological functions of 4.5SH RNA, we have created mutant mice that lack the expression of 4.5SH RNA. The mutant mice exhibited embryonic lethality, suggesting that 4.5SH RNA is an essential species-specific noncoding RNA in mice. RNA-sequencing analyses revealed that 4.5SH RNA protects the transcriptome from abnormal exonizations of the antisense insertions of the retrotransposon SINE B1 (asB1), which would otherwise introduce deleterious premature stop codons or frameshift mutations. Mechanistically, 4.5SH RNA base pairs with complementary asB1-containing exons via the target recognition region and recruits effector proteins including Hnrnpm via its 5' stem loop region. The modular organization of 4.5SH RNA allows us to engineer a programmable splicing regulator to induce the skipping of target exons of interest. Our results also suggest the general existence of splicing regulatory noncoding RNAs.

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Exonuclease-catalyzed recycling and annular four-footed DNA walking amplification-assisted “on-off-super on” signal transitions for photoelectrochemical biosensing of kanamycin

Photoelectrochemical (PEC) biosensors have exhibited a promising potential for assays of a large variety of analytes; however, how to realize their low background-based “super on” signal output is still a great challenge. Herein, we report a novel multiple nucleic acid amplification-assisted “on-off-super on” signal transition mechanism for the PEC biosensing of kanamycin antibiotics. The biosensing platform was constructed on a perylene-3,4,9,10-tetracarboxylic dianhydride-based photoelectrode, and its strong photocurrent could be well inhibited by an anchored ferrocene (Fc)-labeled hairpin DNA to produce a low background signal. Two target biorecognition-triggered exonuclease III-catalytic reactions were adopted to produce an annular four-footed DNA walker (AFW) and a methylene blue (MB)-labeled DNA strand. By using their synergistic effect to release Fc quenchers and simultaneously capture MB sensitizers, a “super on” signal output was realized. As a result, a very wide linear range from 10 fg mL−1 to 10 ng mL−1 and an ultra-low detection limit of 7.8 fg mL−1 were obtained. Meanwhile, the aptamer recognition-based homogeneous reaction and AFW-based multiple nucleic acid amplification effectively simplified the assay manipulation and well ensured the repeatability of the method. The satisfactory sample experiment results indicated its good reliability and accuracy for the antibiotic residue analysis application.