Stem-loop Research Articles

Synthesis of fluorescent AuNCs with RNA as template

Although nucleic acids have been widely used as templates for the synthesis of nanomaterials, the synthesis of RNA-templated gold nanoclusters (AuNCs) has not been explored. In this work, we developed a simple strategy for synthesis of RNA-templated fluorescent AuNCs. We first evaluated the adsorption of different nucleoside monophosphates (NMP) on gold atoms. Our density function theory simulation and isothermal titration calorimetry measurements demonstrated that adenosine monophosphate (AMP) is a superior gold binder than other NMPs or deoxyadenosine monophosphate. Afterwards, NMP-templated synthesis of AuNCs was conducted in various pH environments, and our results indicated that bright green light-emitting AMP-templated AuNCs can be obtained at pH ∼6.0. In order to study the synthesis mechanism of AuNCs, we investigated the effects of reducing agent type and addition time, and the negative charge carried by template nucleotides on the fluorescence of AuNCs. Finally, we extended the template AMP into RNA hairpin structure, the fluorescence intensity was the highest when the cyclic bases were poly 16 A. This study opens new routes to synthesize fluorescent AuNCs using RNA templates.

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion.

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1×1 nucleotide UU loops (5'CUG/3'GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5'CUG/3'GUC motifs in complex with three different small molecules, a di-guandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5'CUG/3'GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

Metal ion-complexed DNA probe coupled with CRISPR/Cas12a amplification and AuNPs for sensitive colorimetric assay of metallothionein in fish

The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg2+ and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion-induced water bio-pollution. The target MT molecules bind Hg2+ in the Hg2+- complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence-containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.

Reynolds number effect on the Lagrangian evolution of hairpin vortices in turbulent channel flow

The evolution and generation of hairpins are studied in a Lagrangian perspective in turbulent channel flows. Direct numerical simulation database of turbulent channel flow at friction Reynolds numbers R e τ = 590, 390, and 180 is employed to perform tracking and interpolation algorithms of fluid particles on hairpins. These particles are carefully selected using a vortex line identification technique developed to extract the hairpin's core line. Interesting results are observed regarding the Reynolds number impact on the evolution and regeneration of hairpins. As the Reynolds number increases, the head and the vortical tongues are compressed and aligned in the main flow direction and the hairpin structures tend to orient more horizontally. The deformation shape of hairpins using the strain state parameter, which is correlated with the dissipation rate, is analysed using the probability density function. Most phenomena related to the generation of secondary hairpins, including the existence of vortical tongues, occur in the range 35 ≲ y + ≲ 50 . The results reveal that the Lagrangian perspective is a proper technique to address the extension of vortical tongues, generation of the secondary hairpin, and stretch of hairpin legs.

Evaluation of RNA Secondary Stem-Loop Structures in the UTRs of Mouse Hepatitis Virus as New Therapeutic Targets.

MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an alternative animal infection model for SARS-CoV and SARS-CoV-2, aiding the development of new antiviral drugs. In this study, the MHV-A59 model was employed to investigate the potential of SARS-CoV-2 UTRs as new targets for antiviral drugs. Optimal targets within the MHV-A59 UTRs were identified using a shRNA and siRNA design tool, focusing on RNA secondary stem-loop (SL) structures in the UTRs. We then examined whether the designed RNAi constructs could inhibit MHV-A59 replication. In the 5'UTR, the stem-loop 1 (SL1) was identified as the most effective target, while in the 3'UTR, the minimal element for the initiation of negative-strand RNA synthesis (MIN) proved to be the most effective. Importantly, siRNAs targeting SL1 and MIN structures significantly reduced total RNA synthesis, negative-strand genomic RNA synthesis, subgenomic (sg) RNA synthesis, viral titer, and the plaque size of MHV-A59 compared to the control. Although not statistically significant, the combination of siSL1 and siMIN had a stronger effect on inhibiting MHV-A59 replication than either siRNA monotherapy. Interestingly, while the SL1 structure is present in both MHV and SARS-CoV-2, the MIN structure is unique to MHV. Thus, the SL1 of SARS-CoV-2 may represent a novel and promising target for RNAi-based antiviral drugs.

A novel dual-signal output strategy for POCT of CEA based on asmartphone electrochemical aptasensing platform.

A smartphone-based electrochemical aptasensing platform was developed for the point-of-care testing (POCT) of carcinoembryonic antigen (CEA) based on the ferrocene (Fc) and PdPt@PCN-224 dual-signal labeled strategy. The prepared PdPt@PCN-224 nanocomposite showed a strong catalytic property for the reduction of H2O2. Phosphate group-labeled aptamer could capture PdPt@PCN-224 by Zr-O-P bonds to form PdPt@PCN-224-P-Apt. Therefore, a dual signal labeled probe was formed by the hybridization between Fc-DNA and PdPt@PCN-224-P-Apt. The presence of CEA forced PdPt@PCN-224-P-Apt to leave the electrode surface due to the specific affinity, leading to the decrease of the reduction current of H2O2. At the same time, the Fc-DNA strand changed to hairpin structure, which made Fc closer to the electrode and resulted in the increase of the oxidation current of Fc. Thus, CEA can be accurately determined through both signals: the decrease of H2O2 reduction current and the increase of Fc oxidation current, which could avoid the false positive signal. Under the optimal conditions, the prepared aptasensor exhibited a wide linear range from 1pg·mL-1 to 100ng·mL-1 and low detection limits of 0.98pg·mL-1 and 0.27pg·mL-1 with Fc and PdPt@PCN-224 as signal labels, respectively. The aptasensor developed in this study has successfully demonstrated its capability to detect CEA in real human serum samples. These findings suggest that the proposed sensing platform will hold great potential for clinical tumor diagnosis and monitoring.

An Effective Model for Capturing the Role of Excitonic Interactions in the Wave-Packet Dynamics of DNA Nucleobases

Investigating exciton dynamics within DNA nucleobases is essential for comprehensively understanding how inherent photostability mechanisms function at the molecular level, particularly in the context of life’s resilience to solar radiation. In this paper, we introduce a mathematical model that effectively simulates the photoexcitation and deactivation dynamics of nucleobases within an ultrafast timeframe, particularly focusing on wave-packet dynamics under conditions of strong nonadiabatic coupling. Employing the hierarchy equation of motion, we simulate two-dimensional electronic spectra (2DES) and calibrate our model by comparing it with experimentally obtained spectra. This study also explores the effects of base stacking on the photo-deactivation dynamics in DNA. Our results demonstrate that, while strong excitonic interactions between nucleobases are present, they have a minimal impact on the deactivation dynamics of the wave packet in the electronic excited states. We further observe that the longevity of electronic excited states increases with additional base stacking and pairing, a phenomenon accurately depicted by our excitonic model. This model enables a detailed examination of the wave packet’s motion on electronic excited states and its rapid transition to the ground state. Additionally, using this model, we studied base stacks in DNA hairpins to effectively capture the primary exciton dynamics at a reasonable computational scale. Overall, this work provides a valuable framework for studying exciton dynamics from single nucleobases to complex structures such as DNA hairpins.

Encoding signal propagation on topology-programmed DNA origami.

Biological systems often rely on topological transformation to reconfigure connectivity between nodes to guide the flux of molecular information. Here we develop a topology-programmed DNA origami system that encodes signal propagation at the nanoscale, analogous to topologically efficient information processing in cellular systems. We present a systematic molecular implementation of topological operations involving 'glue-cut' processes that can prompt global conformational change of DNA origami structures, with demonstrated major topological properties including genus, number of boundary components and orientability. By spatially arranging reactive DNA hairpins, we demonstrate signal propagation across transmission paths of varying lengths and orientations, and curvatures on the curved surfaces of three-dimensional origamis. These DNA origamis can also form dynamic scaffolds for regulating the spatial and temporal signal propagations whereby topological transformations spontaneously alter the location of nodes and boundary of signal propagation network. We anticipate that our strategy for topological operations will provide a general route to manufacture dynamic DNA origami nanostructures capable of performing global structural transformations under programmable control.

Conformational transformation of activator strand directly regulates CRISPR/Cas12a system activity for live cell sensing of multiple biomolecules

In recent years, biosensors designed based on the CRISPR/Cas12a system have opened up a new era in biosensing. However, the activator strand in the existing CRISPR/Cas12a-based sensing strategies are usually designed with 20 bases complementary to crRNA, it is difficult to completely shut down the trans-cleavage activity of the CRISPR/Cas12a system. To address this issue and simultaneously construct a biosensor with an “on-off-on” mode, we shortened the activator strand complementary to crRNA to 16 bases and embedded it into a hairpin structure depriving its ability to activate the Cas12a. We all know that the binding of aptamers with small molecules involves folding into a complex structure that is complementary to the surface of small molecules, thus forming a stable complex. To further expand the application of the CRISPR/Cas12a system in the field of biosensors, we selected small-molecule ATP and nucleolin as targets to confirm that the formation of the complex three-dimensional structure at the 3′- end of activator strand does not affect the activity of CRISPR/Cas12a system. Therefore, by designing only one allosteric activator strand, it is possible to regulate CRISPR/Cas12a system’s activity. This design not only reduces the number of DNA strands used in the sensing system, but also eliminates the need for complex concentration optimization operations. Furthermore, the stability of the intramolecular hairpin structure can minimize fluctuations in the background signal, ensuring stable and low-background signals in live cell applications, this further expands the toolbox of applications for the CRISPR/Cas12a system.

Exploration of new ways for CRISPR/Cas12a activation: DNA hairpins without PAM and toehold and single strands containing DNA and RNA bases

The CRISPR/Cas12a system is emerging as a promising candidate for next-generation diagnostic biosensing platforms, with the discovery of new activation modes greatly expanding its applications. Here, we have identified two novel CRISPR/Cas12a system activation modes: PAM- and toehold-free DNA hairpins, and DNA-RNA hybrid strands. Utilizing a well-established real-time fluorescence method, we have demonstrated a strong correlation between DNA hairpin structures and Cas12a activation. Compared with previously reported activation modes involving single-stranded DNA and PAM-contained double-stranded DNA, the DNA hairpin activation way exhibits similar specificity and generality. Moreover, our findings indicate that increasing the number of RNA bases in DNA-RNA hybrid strands can decelerate the kinetics of Cas12a-triggered trans-cleavage of reporter probes. These newly discovered CRISPR/Cas12a activation ways hold significant potential for the development of high-performance biosensing strategies.

Sensitive aptasensing of ATP based on a PAM site-regulated CRISPR/Cas12a activation.

The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25-750 nM, and the whole workflow of the assay was approximately 60min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity.

Detection of miRNA-378 based on a catalytic hairpin self-assembly reaction combined with gold nanoparticle colorimetry

Recent studies have shown that abnormal miRNA-378 expression is a rule, rather than an exception, in cervical cancer and can be used as a diagnostic and prognostic biomarker to assess tumor initiation. In this study, we developed a general, sensitive strategy for detecting miRNA-378 using catalytic hairpin self-assembly (CHA) combined with gold nanoparticles (AuNP) colorimetry. The presence of miRNA-378 triggers the repeated self-assembly of two designed hairpin DNAs (H1 and H2) into dsDNA polymers, which leads to changes in the surface plasmon resonance absorption band and the macroscopic color of the AuNP colloids due to the formation of nanoparticle-DNA conjugates. This experimental phenomenon can be observed by ultraviolet-visible spectrometry or even with the naked eye. Using this method, miRNA-378 could be quantitatively detected at the picomolar level (as low as 20.7 pM). Compared with traditional methods, such as quantitative polymerase chain reaction and RNA blotting, this strategy has a simple operation, low cost, and high sensitivity and selectivity, and thus, exhibits significant potential for miRNA detection.

Dynamic stem–loop extension by Pol θ and templated insertion during DNA repair

Theta-mediated end-joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend single-stranded DNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both single-stranded DNA and RNA substrates by unimolecular stem loop synthesis initiated by only two 3ʹ terminal base-pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem loop synthesis competes with bimolecular end-joining, even when a longer terminal microhomology for end-joining is available. Both reactions are partially suppressed by the ssDNA binding protein RPA. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare amongst human DNA polymerases but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.

Hyphal editing of the conserved premature stop codon in CHE1 is stimulated by oxidative stress in Fusarium graminearum

Although genome-wide A-to-I editing mediated by adenosine-deaminase-acting-on-tRNA (ADAT) occurs during sexual reproduction in the presence of stage-specific cofactors, RNA editing is not known to occur during vegetative growth in filamentous fungi. Here we identified 33 A-to-I RNA editing events in vegetative hyphae of Fusarium graminearum and functionally characterized one conserved hyphal-editing site. Similar to ADAT-mediated editing during sexual reproduction, majority of hyphal-editing sites are in coding sequences and nonsynonymous, and have strong preference for U at -1 position and hairpin loops. Editing at TA437G, one of the hyphal-specific editing sites, is a premature stop codon correction (PSC) event that enables CHE1 gene to encode a full-length zinc fingertranscription factor. Manual annotations showed that this PSC site is conserved in CHE1 orthologs from closely-related Fusarium species. Whereas the che1 deletion and CHE1TAA (G438 to A) mutants had no detectable phenotype, the CHE1TGG (A437 to G) mutant was defective in hyphal growth, conidiation, sexual reproduction, and plant infection. However, the CHE1TGG mutant was increased in tolerance against oxidative stress and editing of TA437G in CHE1 was stimulated by H2O2 treatment in F. graminearum. These results indicate that fixation of the premature stop codon in CHE1 has a fitness cost on normal hyphal growth and reproduction but provides a benefit to tolerance against oxidative stress. Taken together, A-to-I editing events, although rare (not genome-wide), occur during vegetative growth and editing in CHE1 plays a role in response to oxidative stress in F. graminearum and likely in other fungal pathogens.

High-Frequency Quartz Crystal Microbalance and Dual-Signaling Electrochemical Ratiometric Assays of PTP1B Activity Based on COF@Au@Fc Hybrids.

The abnormal expression of protein tyrosine phosphatase 1B (PTP1B) is highly related to several serious human diseases. Therefore, an accurate PTP1B activity assay is beneficial to the diagnosis and treatment of these diseases. In this study, a dual-mode biosensing platform that enabled the sensitive and accurate assay of PTP1B activity was constructed based on the high-frequency (100 MHz) quartz crystal microbalance (QCM) and dual-signaling electrochemical (EC) ratiometric strategy. Covalent-organic framework@gold nanoparticles@ferrocene@single-strand DNA (COF@Au@Fc-S0) was introduced onto the QCM Au chip via the chelation between Zr4+ and phosphate groups (phosphate group of the phosphopeptide (P-peptide) on the QCM Au chip and the phosphate group of thiol-labeled single-stranded DNA (S0) on COF@Au@Fc-S0) and used as a signal reporter. When PTP1B was present, the dephosphorylation of the P-peptide led to the release of COF@Au@Fc-S0 from the QCM Au chip, resulting in an increase in the frequency of the QCM. Meanwhile, the released COF@Au@Fc-S0 hybridized with thiol/methylene blue (MB)-labeled hairpin DNA (S1-MB) on the Au NPs-modified indium-tin oxide (ITO) electrode. This caused MB to be far away from the electrode surface and Fc to be close to the electrode, leading to a decrease in the oxidation peak current of MB and an increase in the oxidation peak current of Fc. Thus, PTP1B-induced dephosphorylation of the P-peptide was monitored in real time by QCM, and PTP1B activity was detected sensitively and reliably using this innovative QCM-EC dual-mode sensing platform with an ultralow detection limit. This platform is anticipated to serve as a robust tool for the analysis of protein phosphatase activity and the discovery of drugs targeting protein phosphatase.

Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing thiC Riboswitch in Escherichia coli.

Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.

Deciphering the Binding of 5' Stem Loop RNA to the La Domain of Human LARP6.

La-related protein 6 regulates the highly organized biosynthesis of type I procollagen polypeptides and affects proper assembly of procollagen peptides into heterotrimers of type I procollagen. LARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic instability of the La domain of HsLARP6 hampers studies probing the molecular basis of biologically- and disease-relevant structure-function relationship, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effect of the construct length as well as RNA binding on protein stability. N- and C-terminal extensions greatly impact stability based on interactions with the core domain and modulation of the pI. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50x longer than the unbound state, under identical conditions, likely due to a global change in conformational plasticity upon RNA binding. These results provide a foundation for further studies of the molecular recognition of 5'SL by HsLARP6 as well as a platform for refining potential antifibrotic therapeutics.

DNAzyme and controllable cholesterol stacking DNA machine integrates dual-target recognition CTCs enable homogeneous liquid biopsy of breast cancer

Although circulating tumor cells (CTCs) have demonstrated considerable importance in liquid biopsy, their detection is limited by low concentrations and complex sample components. Herein, we developed a homogeneous, simple, and high-sensitivity strategy targeting breast cancer cells. This method was based on a non-immunological stepwise centrifugation preprocessing approach to isolate CTCs from whole blood. Precise quantification is achieved through the specific binding of aptamers to the overexpressed mucin 1 (MUC1) and human epidermal growth factor receptor 2 (HER2) proteins of breast cancer cells. Subsequently, DNAzyme cleavage and parallel catalytic hairpin assembly (CHA) reactions on the cholesterol-stacking DNA machine were initiated, which opened the hairpin structures T-Hg2+-T and C–Ag+-C, enabling multiple amplifications. This leads to the fluorescence signal reduction from Hg2+-specific carbon dots (CDs) and CdTe quantum dots (QDs) by released ions. This strategy demonstrated a detection performance with a limit of detection (LOD) of 3 cells/mL and a linear range of 5–100 cells/mL. 42 clinical samples have been validated, confirming their consistency with clinical imaging, pathology findings and the folate receptor (FR)-PCR kit results, exhibiting desirable specificity of 100% and sensitivity of 80.6%. These results highlight the promising applicability of our method for diagnosing and monitoring breast cancer.

Determinants of the interaction between the 5′-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3′ end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5′ leader (5′L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5′L responsible for LysRS binding, how the conformations and various hairpin structures of 5′L regulate 5′L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5′L significantly influences its binding affinity with LysRS. The 5′L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5′L conformation that is not selected for packaging. Additionally, dimerization of 5′L impairs LysRS-5′L interaction. Furthermore, among various regions of 5′L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5′L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.

CRISPR/Cas12a-mediated DNA-AgNC label-free logical gate for multiple microRNAs' assay.

CRISPR/Cas system has been widely applied in the assay of disease-related nucleic acids. However, it is still challenging to use CRISPR/Cas system to detect multiple nucleic acids at the same time. Herein, we combined the preponderance of DNA logic circuit, label-free, and CRISPR/Cas technology to construct a label-free "AND" logical gate for multiple microRNAs detectionwith high specificity and sensitivity. With the simultaneous input of miRNA-155 and miRNA-141, the logic gate starts, and the activation chain of Cas12a is destroyed; thus, the activity is inhibited and the fluorescence of the signal probe ssDNA-AgNCs is turned on. The detection limit of this method for simultaneous quantitative detection of double target is 84 fmol/L (S/N = 3). In this "AND" logic gate, it is only necessary for the design of a simple DNA hairpin probe, which is inexpensive and easy, and sincethis method involves only one signal output, the data processing is very simple. What is more important, in this strategy two types of microRNAs can be monitored simultaneously by only using CRISPR/Cas12a and a type of crRNA, which offers a new design concept for the exploitation of single CRISPR/Cas system for multiple nucleic acid assays.