Fluorescent 2-aminopurine Research Articles

An enzyme-free three-dimensional DNA walker powered by catalytic hairpin assembly for H5N1 DNA ratiometric detection

The reliable and quantitative detection of virus DNA is highly desirable for the early diagnosis and clinical treatment of diseases. In this work, a three-dimensional (3D) DNA walker based on a non-enzyme-mediated nucleic acid cascade amplification reaction was constructed for the ratiometric detection of H5N1 DNA. When the DNA walker was developed by the surface modification of aminated silica microsphere (SiO2-NH2) with H1 hairpin structures were activated in the presence of target DNA, the fluorescence of the H1-conjugated Thioflavin T (ThT) fluorophore (495 nm) was turned on, while the fluorescence of 2-aminopurine (2-AP) (363 nm) conjugated to the H2 hairpin remained unchanged. This strategy combines DNA walker powered by catalyzed hairpin assembly (CHA) reaction with proportional fluorescence signal output methods, which can effectively reduce the risk of generating false-positive signals. The developed DNA walker demonstrated excellent analytical sensitivity and specificity, with a detection limit of 60 pM. The DNA walker was also capable of selectively detecting H5N1 DNA in clinical blood serum samples, which demonstrated its potential for use in various clinical applications.

(Invited) Split G-quadruplex-based Strategy for Sensitive Detection of Pathogenic Bacteria

In this talk, I will introduce a simple, sequence-specific DNA detection method, utilizing a fluorescent 2-aminopurine (2-AP) nucleobase analogue-containing split G-quadruplex as the key detection component. In the sensor, the 2-AP-containing G-quadruplex is split into two segments and linked to a target-specific overhang sequence. The separate G-quadruplex sequences form an active G-quadruplex structure only in the presence of a complementary target DNA, which leads to a significant increase in the intensity of fluorescence from the 2-AP fluorophore. This simple, one-step, homogenous assay was successfully employed to detect target DNA with a high selectivity. In addition, the practical applicability of the detection method was demonstrated by its use in analyzing target DNAs in human serum. To the best of our knowledge, this is the first time that an investigation was carried out in which a fluorescent nucleobase analogue was incorporated into a split G-quadruplex structure and this structure was utilized as the foundation for a specific DNA sensor.

Dynamic and static quenching of 2-aminopurine fluorescence by the natural DNA nucleotides in solution

2-aminopurine (2AP) is a responsive fluorescent base analogue that is used widely as a probe of the local molecular environment in DNA. The ability of 2AP to report changes in local conformation and base-stacking interactions arises from the efficient quenching of its fluorescence by the natural DNA bases. However, the mechanism of this inter-base quenching remains imperfectly understood. Two previous studies of the collisional quenching of 2AP by the natural bases, in different buffer solutions, showed that dynamic quenching efficiency depends on the identity of the natural base, but disagreed on the relative quenching efficiencies of the bases. We report a comprehensive investigation of inter-base quenching of 2AP by the natural nucleoside monophosphates (NMPs), replicating the buffer conditions used in the previous studies. Using time-resolved fluorescence measurements to distinguish between dynamic and static quenching, we find that the dynamic quenching rate constants of the different bases show a consistent trend across both buffers, and this is in line with a charge-transfer mechanism. Time-resolved measurements also provide insight into static quenching, revealing formation of 2AP-NMP ground-state complexes in which 2AP displays a very short fluorescence lifetime, comparable to that seen in oligonucleotides. In these complexes, the dependence of the rate of quenching on the partner base also supports a charge-transfer mechanism.

Coordination Nanoparticles Formed by Fluorescent 2-Aminopurine and Au3+: Stability and Nanozyme Activities

Nucleobase–metal coordination polymers (CPs) have been the target of much recent research owing their interesting properties and ease of preparation. Natural nucleobases are nonfluorescent, and 2-aminopurine (2AP) is a strongly fluorescent analog of adenine. In this work, we compared the CPs formed by adenine, 2AP and adenosine with various metals. Only Au3+ produced CP nanoparticles with both adenine and 2AP. Both products were similar in terms of size, suggesting that the position of the exocyclic amine is unimportant for coordination with gold. Since 2AP is fluorescent, the dissolution of the nanoparticles in the presence of various chemicals including KCN, GSH, iodide, bromide and chloride, was studied and compared to fluorescent adenosine–Au nanoparticles. It appears that the 2AP CP was more robust than the adenosine one since even chloride can quench the fluorescence of the adenosine/Au complex. Finally, the adenine and 2AP nanoparticles exhibited weak oxidase-like activity for oxidation of TMB possibly by the Au3+ exposed on the surface.

A quencher-free DNAzyme beacon for fluorescently sensing uranyl ions via embedding 2-aminopurine

DNAzyme-based fluorescent probes have provided valuable protocols for detecting uranium, one of the most common radioactive contaminants in the environment, with ultra-high selectivity and sensitivity. Designing novel DNAzyme beacons to update the mode of fluorescence reporting and/or quenching will continuously enhance “turn-on” sensing performance as well as promote actual application of the biological probes. In this work, we developed a novel quencher-free DNAzyme beacon by embedding fluorescent 2-aminopurine for rapid detection of uranyl ion. 2-aminopurine is able to substitute adenine and keep strong fluorescence in single-stranded DNA whereas being quenched in the hybridized double-stranded DNA by the base-stacking interaction. The combination of such trait of 2-aminopurine and cleavage reaction of DNAzyme in the presence of target co-factors possesses two main advantages for ion sensing: simplicity for avoidance of extra quencher groups and high performance because of superiority of DNAzyme essence. The experimental conditions including embedding site, pH and salt concentration of buffer solutions, and the amount ratio of enzyme strand to substrate strand used to form DNAzymes were systematically optimized to inspire the highest performance of the biological beacon. Thus, a detection limit of 9.6 nM, a wide linear range from 5 nM to 400 nM (R2 = 0.997), and selectivity of more than 400 000-fold over other metal ions were achieved by the novel DNAzyme probes. The highly sensitive, selective and quencher-free DNAzyme probes accommodated a simple and cost-efficient alternative to current fluorescent counterparts, holding a great potential for further application in practical ion assay.

Ultrasensitive detection of telomerase activity in lung cancer cells with quencher-free molecular beacon-assisted quadratic signal amplification

Telomerase is an important biomarker for cancer diagnosis and a valuable target for cancer therapy. Most of the reported telomerase assays suffer from the repeating thermal cycles, laborious protocols, expensive instruments and the limited sensitivity. To solve these issues, we develop a new telomerase assay based on telomerization-driven release of fluorescent 2-aminopurine. We designed a 2-aminopurine molecular beacon in which the fluorescence of 2-aminopurine is quenched due to stacking interaction effect without the use of extra quenchers. The presence of target telomerase can open the 2-aminopurine molecular beacon, triggering a quadratic signal amplification reaction to digest abundant 2-aminopurine molecular beacons, releasing large amounts of free 2-aminopurine molecules with strong fluorescence emission. Due to the inherent low background signal of 2-aminopurine molecular beacon and the highly efficient telomerization-driven quadratic signal amplification, this method exhibits high sensitivity with a limit of detection equivalent to 1 human lung cancer A549 cell and a large dynamic range of 4 orders of magnitude from 1 to 10000 cells. Moreover, this method can be further applied for telomerase inhibition assay and the discrimination of cancer cells from normal cells, holding great potential in telomerase-related clinical diagnosis and drug discovery.

The use of a 2-aminopurine-containing split G-quadruplex for sequence-specific DNA detection

A simple, sequence-specific DNA detection method, utilizing a fluorescent 2-aminopurine (2-AP) nucleobase analogue-containing split G-quadruplex as the key detection component, is described. In the sensor, the 2-AP-containing G-quadruplex is split into two segments and linked to a target-specific overhang sequence. The separate G-quadruplex sequences form an active G-quadruplex structure only in the presence of a complementary target DNA, which leads to a significant increase in the intensity of fluorescence from the 2-AP fluorophore. This simple, one-step, homogenous assay was successfully employed to detect target DNA with a high selectivity. In addition, the practical applicability of the detection method was demonstrated by its use in analyzing target DNAs in human serum. To the best of our knowledge, this is the first time that an investigation was carried out in which a fluorescent nucleobase analogue was incorporated into a split G-quadruplex structure and this structure was utilized as the foundation for a specific DNA sensor.

Misfolding of a DNAzyme for ultrahigh sodium selectivity over potassium

Herein, the excellent Na+ selectivity of a few RNA-cleaving DNAzymes was exploited, where Na+ can be around 3000-fold more effective than K+ for promoting catalysis. By using a double mutant based on the Ce13d DNAzyme, and by lowering the temperature, increased 2-aminopurine (2AP) fluorescence was observed with addition of both Na+ and K+. The fluorescence increase was similar for these two metals at below 10 mM, after which K+ took a different pathway. Since 2AP probes its local base stacking environment, K+ can be considered to induce misfolding. Binding of both Na+ and K+ was specific, since single base mutations could fully inhibit 2AP fluorescence for both metals. The binding thermodynamics was measured by temperature-dependent experiments revealing enthalpy-driven binding for both metals and less coordination sites compared to G-quadruplex DNA. Cleavage activity assays indicated a moderate cleavage activity with 10 mM K+, while further increase of K+ inhibited the activity, also supporting its misfolding of the DNAzyme. For comparison, a G-quadruplex DNA was also studied using the same system, where Na+ and K+ led to the same final state with only around 8-fold difference in Kd. This study provides interesting insights into strategies for discriminating Na+ and K+.

Different Cations Change the Rates of an RNA Folding Pathway

RNA tertiary structure often depends on divalent ions for electrostatic shielding or binding that stabilize the folded structure. We employed machine learning to analyze 2-aminopurine stopped-flow fluorescence data to measure how different cations modify folding kinetics of the 60 nucleotide E. coli rRNA GTPase Center. The folding pathway of the GAC RNA in the presence of Mg2+, Ca2+, and Sr2+, appears identical. Rate constants for the five folding transitions span timescales from sub-microsecond to tens of seconds, and are ion-dependent for transition times longer than >10 ms. Divalent ions appear to modulate intermediate populations in conformational ensembles along the folding pathway and at equilibrium. The sensitivity of RNA tertiary structure to cation identity impacts all but the fastest events in RNA folding.

Simple G-quadruplex-based 2-aminopurine fluorescence probe for highly sensitive and amplified detection of microRNA-21

Based on 2-aminopurine (2-AP) probe in conjunction with a G-quadruplex structure and signal amplification technique, a simple and highly sensitive fluorescence sensor for detecting microRNA (miRNA) is developed for high signal-to-background ratio and wide linear range. The proposed sensor contains two hairpins DNA: H1 and H2. H1 is labeled by 2-AP incorporated into a G-rich sequence. Upon the addition of a target miRNA, H1 is unfolded and forms DNA/RNA complexes that contain a G-quadruplex, thereby significantly enhancing 2-AP fluorescence due to the protection provided by the G-quadruplex. Subsequently, H2 can displace the miRNA from the DNA/RNA complexes and induce signal amplification, resulting in further enhanced fluorescence intensity. Hence, the sensor is highly sensitive and its low limit of detection (L.O.D.) can reach as low as 1.48pM. Furthermore, the proposed sensor is used to detect overexpressed miRNA-21 from human breast cancer cell lysate. The result demonstrates the potential of the proposed sensor to monitor different miRNA biomarkers for the early diagnosis of various cancers.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Two Completely Different Mechanisms for Highly Specific Na+ Recognition by DNAzymes

Our view of the interaction between Na+ and nucleic acids was changed by a few recently discovered Na+ -specific RNA-cleaving DNAzymes. In addition to nonspecific electrostatic interactions, highly specific recognition is also possible. Herein, two such DNAzymes, named EtNa and Ce13d, are compared to elucidate their mechanisms of Na+ binding. Mutation studies indicate that they have different sequence requirements. Phosphorothioate (PS) substitution at the scissile phosphate drops the activity of EtNa 140-fold, and it cannot be rescued by thiophilic Cd2+ or Mn2+ , whereas the activity of PS-modified Ce13d can be rescued. Na+ -dependent activity assays indicate that two Na+ ions bind cooperatively in EtNa, and each Na+ likely interacts with a nonbridging oxygen atom in the scissile phosphate, whereas Ce13d binds only one Na+ ion in a well-defined Na+ aptamer, and this Na+ ion does not directly interact with the scissile phosphate. Both DNAzymes display a normal pH-rate profile, with a single deprotonation reaction required for catalysis. For EtNa, Na+ fails to protect the conserved nucleotides from dimethyl sulfate attack, and no specific Na+ binding is detected by 2-aminopurine fluorescence, both of which are different from those observed for Ce13d. This work suggests that EtNa binds Na+ mainly through its scissile phosphate without significant involvement of the nucleotides in the enzyme strand, whereas Ce13d has a well-defined aptamer for Na+ binding. Therefore, DNA has at least two distinct ways to achieve highly selective Na+ binding.

Folding of the silver aptamer in a DNAzyme probed by 2-aminopurine fluorescence

The RNA-cleaving Ag10c DNAzyme was recently isolated via in vitro selection and it can bind two Ag+ ions for activity. The Ag10c contains a well-defined Ag+ binding aptamer as indicated by DMS footprinting. Since aptamer binding is often accompanied with conformational changes, we herein used 2-aminopurine (2AP) to probe its folding in the presence of Ag+. The Ag10c was respectively labeled with 2AP at three different positions, both in the substrate strand and in the enzyme strand, one at a time. Ag+-induced folding was observed at the substrate cleavage junction and the A9 position of the enzyme strand, consistent with aptamer binding. The measured Kd at the A9 position was 18 μM Ag+ with a Hill coefficient of 2.17, similar to those obtained from the previous cleavage activity based assays. However, labeling a 2AP at the A2 position inhibited the activity and folding. Compared to other metal ions, Ag+ has a unique sigmoidal folding profile indicative of multiple silver binding cooperatively. This suggests that Ag+ can induce a local folding in the enzyme loop and this folding is important for activity. This study provides important biophysical insights into this new DNAzyme, suggesting the possibility of designing folding-based biosensors for Ag+.

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl.

The cocaine aptamer has been seen as a good candidate for development as a probe for cocaine in many contexts. Here, we demonstrate that the aptamer binds cocaine, norcocaine, and cocaethylene with similar affinities and aminoglycosides with similar or higher affinities in a mutually exclusive manner with cocaine. Analysis of its affinities for a series of cocaine derivatives shows that the aptamer specificity is the consequence of its interaction with all faces of the cocaine molecule. Circular dichroism spectroscopy and 2-aminopurine (2AP) fluorescence studies show no evidence of large structural rearrangement of the cocaine aptamer upon ligand binding, which is contrary to the general view of this aptamer. The aptamer's affinity for cocaine and neomycin-B decreases with the inclusion of physiological NaCl. The substitution of 2AP for A in position 6 (2AP6) of the aptamer sequence eliminated the effect of NaCl on its affinities for cocaine and analogues, but not for neomycin-B, showing a selective effect of 2AP substitution on cocaine binding. The affinity for cocaine also decreased with increasing concentrations of serum or urine, with the 2AP6 substitution blunting the effect of urine. Its low affinities for cocaine and metabolites and its ability to bind irrelevant compounds limit the opportunities for application of this aptamer in its current form as a selective and reliable sensor for cocaine. However, these studies also show that a small structural adjustment to the aptamer (2AP exchanged for adenine) can increase its specificity for cocaine in physiological NaCl relative to an off-target ligand.

ATP Is an Allosteric Inhibitor of Coxsackievirus B3 Polymerase.

The RNA-dependent RNA polymerases from positive-strand RNA viruses, such as picornaviruses and flaviviruses, close their active sites for catalysis via a unique NTP-induced conformational change in the palm domain. Combined with a fully prepositioned templating nucleotide, this mechanism is error-prone and results in a distribution of random mutations in the viral progeny often described as a quasi-species. Here we examine the extent to which noncognate NTPs competitively inhibit single-cycle elongation by coxsackievirus B3 3D(pol), a polymerase that generates three to four mutations per 10 kb of RNA synthesized during viral infection. Using an RNA with a templating guanosine combined with 2-aminopurine fluorescence as a reporter for elongation, we find that the cognate CTP has a Km of 24 μM and the three noncognate nucleotides competitively inhibit the reaction with Kic values of 500 μM for GTP, 1300 μM for ATP, and 3000 μM for UTP. Unexpectedly, ATP also acted as an uncompetitive inhibitor with a Kiu of 1800 μM, resulting in allosteric modulation of 3D(pol) that slowed the polymerase elongation rate ≈4-fold. ATP uncompetitive inhibition required the β- and γ-phosphates, and its extent was significantly diminished in two previously characterized low-fidelity polymerases. This led to further mutational analysis and the identification of a putative allosteric binding site below the NTP entry channel at the interface of conserved motifs A and D, although cocrystallization failed to reveal any density for bound ATP in this pocket. The potential role of an ATP allosteric effect during the virus life cycle is discussed.

Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA.

SummaryWe present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction.

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging.

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG–DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.

2-Aminopurine Fluorescence as a Probe of Local RNA Structure and Dynamics and Global Folding.

The biology of an RNA is encoded in its structure and dynamics, whether that be binding to a protein, binding to another RNA, enzymatic catalysis, or becoming a substrate. In solution, most RNA molecules are sampling conformations, and their structures are best described as conformational ensembles. For larger RNAs, experiments that can describe the conformations of their domains can be particularly daunting, especially when the RNA is novel and not well characterized. Here, we explain how we have used site-specific 2-aminopurine as a fluorescent probe of the secondary and tertiary structures of a 60 nucleotide RNA, and what new findings we have about its Mg(2+)-dependent conformational changes. We focus on this RNA from prokaryotic ribosome as a proof of concept as well as a research project. Its tertiary structure is known from a cocrystal, and its secondary structure is modeled from phylogenetic conservation, but there are virtually no data describing the motions of its nucleotides in solution, or its folding kinetics. It is a perfect system to illustrate the unique information that comes from a comprehensive fluorescence study of this intricate RNA.

Quencher-free fluorescent method for homogeneously sensitive detection of microRNAs in human lung tissues.

Sensitive and accurate analysis of microRNA (miRNA) expression is imperative for understanding the biological functions of miRNAs and the early diagnosis of human cancers. Here, we develop a quencher-free fluorescent method for homogeneously sensitive detection of let-7a miRNA using the target-triggered recycling signal amplification in combination with a 2-aminopurine probe. The 2-aminopurine probe is characterized by the substitution of 2-aminopurine for adenine in the DNA strand and the quenching of 2-aminopurine fluorescence through its stacking interaction with the adjacent bases. The binding of target miRNA with the 2-aminopurine probe initiates the extension reaction in the presence of polymerase to produce the DNA duplexes. These DNA duplexes can be further cleaved by lambda exonuclease through the recycling digestion to release abundant free 2-aminopurines, leading to an enhanced fluorescence signal. The proposed method exhibits high sensitivity with a detection limit of 0.3 fmol, and it can even discriminate the single-base difference among the miRNA family members. More importantly, this method can accurately distinguish the expression of let-7a miRNA in human lung tissues between ten non small cell lung cancer (NSCLC) patients and ten healthy persons, holding a great potential for further application in early clinical diagnosis.