Nucleic Acid Signal Research Articles

Signal-amplified detection of biomarker transcription factors with improved accuracy based on T4 DNA ligase protection and rolling circle amplification

Transcription factors (TFs) are emerging tumor biomarkers that regulate gene expression. The detection of TFs depends on the transduction of TF-DNA binding to the nucleic acid signal, which mostly relies on exonuclease III (Exo III) protection assays. However, the processive digestion of Exo III usually causes undesired dissociation of the TF-DNA complex, resulting in low assay accuracy. We proposed a strategy, named T4 DNA ligase protection (T4LiP), to transduce the binding signal and then coupled it with rolling circle amplification (RCA) to fluorescently detect TFs. TFs bound to the nick region of dsDNA can prevent the association of T4 DNA ligase with the nick, thus inhibiting the ligation. Because of the mild activity of T4 DNA ligase, negligible dissociation of the TFs from dsDNA was observed. Using this to our advantage, we transduced the TF-DNA binding signal to generate circular DNA and then loaded it with RCA to sensitively detect p50 protein, yielding a detection limit of 0.5 pM. By sequence engineering, the detection of multiple TFs including MITF, p53, AP-1, and c-Myc with low picomolar sensitivities was accomplished. This is the first report of ligase-assisted transduction of TF-DNA binding, which shows high stability and versatility and holds the potential to improve the performance of TF assays.

Computational Design of RNA Toehold-Mediated Translation Activators.

Translation activators are an important class of riboregulators that respond to nucleic acid signals by activating gene expression. Toehold switches and single-nucleotide-specific programmable riboregulators (SNIPRs) are two types of translation activators that can detect nearly any nucleic acid sequence using interactions initiated by single-stranded domains known as toeholds. Toehold switches operate with high dynamic range, orthogonality, and programmability, making them capable of detecting a variety of pathogens in paper-based cell-free diagnostic assays. SNIPRs are designed to enable the accurate detection of single-nucleotide mutations, making them valuable tools for mutation and drug-resistance assays. Here we describe the computational design process for generating toehold switches and SNIPRs active against different pathogens and mutations of interest. Such riboregulators can be deployed in paper-based diagnostic assays to enable rapid and low-cost disease detection.

Nucleic acid–protein interfaces studied by MAS solid-state NMR spectroscopy

Solid-state NMR (ssNMR) has become a well-established technique to study large and insoluble protein assemblies. However, its application to nucleic acid–protein complexes has remained scarce, mainly due to the challenges presented by overlapping nucleic acid signals. In the past decade, several efforts have led to the first structure determination of an RNA molecule by ssNMR. With the establishment of these tools, it has become possible to address the problem of structure determination of nucleic acid–protein complexes by ssNMR. Here we review first and more recent ssNMR methodologies that study nucleic acid–protein interfaces by means of chemical shift and peak intensity perturbations, direct distance measurements and paramagnetic effects. At the end, we review the first structure of an RNA–protein complex that has been determined from ssNMR-derived intermolecular restraints.

Programmable High-Speed and Hyper-Efficiency DNA Signal Magnifier.

Herein, a programmable dual‐catalyst hairpin assembly (DCHA) for realizing the synchronous recycle of two catalysts is developed, displaying high reaction rate and outstanding conversion efficiency beyond traditional nucleic acid signal amplifications (NASA). Once catalyst I interacts with the catalyst II, the DCHA can be triggered to realize the simultaneous recycle of catalysts I and II to keep the highly concentrated intermediate product duplex I‐II instead of the steadily decreased one in typical NASA, which can accomplish in about only 16 min and achieves the outstanding conversion efficiency up to 4.54 × 108, easily conquering the main predicaments of NASA: time‐consuming and low‐efficiency. As a proof of the concept, the proposed DCHA as a high‐speed and hyper‐efficiency DNA signal magnifier is successfully applied in the rapid and ultrasensitive detection of miRNA‐21 in cancer cell lysates, which exploits the new generation of universal strategy for the applications in biosensing assay, clinic diagnose, and DNA nanobiotechnology.

Integrated Point-of-Care Molecular Diagnostic Devices for Infectious Diseases.

The global outbreaks of deadly infectious diseases caused by pathogenic microorganisms have threatened public health worldwide and significantly motivated scientists to satisfy an urgent need for a rapid and accurate detection of pathogens. Traditionally, the culture-based technique is considered as the gold standard for pathogen detection, yet it has a long turnaround time due to the overnight culturing and pathogen isolation. Alternatively, nucleic acid amplification tests provide a relatively shorter turnaround time to identify whether pathogens exist in individuals with high sensitivity and high specificity. In most cases, nucleic acid amplification tests undergo three steps: sample preparation, nucleic acid amplification, and signal transduction. Despite the explosive advancement in nucleic acid amplification and signal transduction technologies, the complex and labor-intensive sample preparation steps remain a bottleneck to create a transformative integrated point-of-care (POC) molecular diagnostic device. Researchers have attempted to simplify and integrate the sample preparations for nucleic acid-based molecular diagnostic devices with innovative progress in integration strategies, engineered materials, reagent storages, and fluid actuation. Therefore, understanding the know-how and obtaining truthful knowledge of existing integrated POC molecular diagnostic devices comprising sample preparations, nucleic acid amplification, and signal transduction can generate innovative solutions to achieve personalized precision medicine and improve global health.In this Account, we discuss the challenges of automated sample preparation solutions integrated with nucleic acid amplification and signal transduction for rapid and precise home diagnostics. Blood, nasal swab, saliva, urine, and stool are emphasized as the most commonly used clinical samples for integrated POC molecular diagnostics of infectious diseases. Even though these five types of samples possess relatively correlated biomarkers due to the human body's circulatory system, each shows unique properties and exclusive advantages for molecular diagnostics in specific situations, which are included in this Account. We examine different integrated POC devices for sample preparation, which includes pathogen isolation and enrichment from the crude sample and nucleic acid purification from isolated pathogens. We present the promising on-chip integration approaches for nucleic acid amplification. We also investigate the on-chip integration methods for reagent storage, which is crucial to simplify the manual operation for end-users. Finally, we present several integrated POC molecular diagnostic devices for infectious diseases. The integrated sample preparation and nucleic acid amplification approach reviewed here can potentially impact the next generation of POC molecular home diagnostic chips, which will significantly impact public health, emergency medicine, and global biosecurity.

Dual 3D DNA Nanomachine-Mediated Catalytic Hairpin Assembly for Ultrasensitive Detection of MicroRNA.

Herein, we designed a dual 3D DNA nanomachine (DDNM)-mediated catalytic hairpin assembly (DDNM-CHA) to construct an electrochemical biosensor for ultrasensitive detection of miRNA, which possesses quite a faster reaction rate and much higher amplification efficiency than those of traditional catalytic hairpin assembly (CHA). Impressively, since the DDNM skillfully increases the local concentration of reactants and decreases the steric hindrance of substrates simultaneously, the DDNM-CHA could be endowed with higher collision efficiency and more effective reaction compared with traditional CHA, resulting in a hyper conversion efficiency up to 2.78 × 107 only in 25 min. This way, the developed DDNM-CHA could easily conquer the main predicaments: long reaction time and low efficiency. As a proof of the concept, we adopt the gold nanoparticles (AuNPs) and the magnetic nanoparticle (Fe3O4) as the kernel of DNM-A and DNM-B, respectively, and harness the magnetic electrode to directly adsorb the products H1-H2/Fe3O4 for constructing an immobilization-free biosensor for high-speed and ultrasensitive detection of miRNA with a detection limit of 0.14 fM. As a result, the DDNM-CHA we developed carves out a new insight to design a functional DNA nanomachine and evolve the analysis method for practical amplification in the sensing area and promotes the deeper exploration of the nucleic acid signal amplification strategy and DNA nanobiotechnology.

Versatile Luminol/Dissolved Oxygen/Fe@Fe2O3 Nanowire Ternary Electrochemiluminescence System Combined with Highly Efficient Strand Displacement Amplification for Ultrasensitive microRNA Detection.

Herein, a versatile ECL biosensor was fabricated for ultrasensitive detection of microRNA-21 (miRNA-21) from cancer cells based on a novel H2O2-free electrochemiluminescence (ECL) system (luminol/dissolved oxygen/Fe@Fe2O3 nanowires). Compared with the previously reported coreaction accelerator that needed a negative potential to produce reactive oxygen species (ROS), these newly discovered Fe@Fe2O3 nanowires could generate ROS in the detection solution immediately without the application of voltage, which narrowed down the detection potential range to avoid side reactions, favoring their practical application in biological systems. Especially, the Fe@Fe2O3 nanowires could produce H• for activating dissolved oxygen into ROS to improve the ECL intensity dramatically, which initiates a novel pathway to promote the generation of ROS for the ECL system. In addition, an original strand displacement amplification coupled with strand displacement reaction (SDA-SDR) was developed to improve the conversion efficiency of the target for sensitive detection of miRNA-21. By virtue of the SDR, a quadruple quenching effect was achieved through each output DNA strand of SDA; hence, the nucleic acid signal amplification efficiency was effectively enhanced. As expected, on account of the superb activation performance of Fe@Fe2O3 nanowires and the outstanding amplification efficiency of the SDR-SDA strategy, the fabricated ECL biosensor realized ultrasensitive detection of miRNA-21 with a detection limit down to 52.5 aM. The established ECL sensing platform ushered a new route for H2O2-free detection and a promising biomarker assay method for clinical diagnosis.

A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL−1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.

The point-of-care-testing of nucleic acids by chip, cartridge and paper sensors

Point-of-care nucleic acid testing (POCNAT) has played an important role in the outbreak of infectious diseases (e.g., COVID-19) over recent years. POCNAT aims to realize the rapid, simple and automatic detection of nucleic acid. Thanks to the development of manufacturing technology, electronic information technology, artificial intelligence technology, and biological information technology in recent years, the development of the POCNAT device has led to significant advancement. Instead of the normal nucleic acid detection methods used in the laboratory, some novel experimental carriers have been applied, such as chips, cartridges and papers. The application of these experimental carriers has realized the automation and integration of nucleic acid detection. The entire process of nucleic acid detection is normally divided into three steps (nucleic acid extraction, target amplification and signal detection). All of the reagents required by the process can be pre-stored on these experimental carriers, without unnecessary manual operation. Furthermore, all of the processes are carried out in this experimental carrier, with the assistance of a specific control device. Although they are complicated to manufacture and precise in design, their application provides a significant step forwards in nucleic acid detection and realizes the integration of nucleic acid detection. This technology has great potential in the field of point-of-care molecular diagnostics in the future. This paper focuses on the relevant content of these experimental carriers.

Proteomics Investigation of the Time Course Responses of RAW264.7 Macrophages to Infections With the Wild-Type and Twin-Arginine Translocation Mutant Strains of Brucella melitensis.

Brucella, a notorious intracellular pathogen, causes chronic infections in many mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane; protein substrates translocated by Brucella include ABC transporters, oxidoreductases, and cell envelope biosynthesis proteins. Previously, we showed that a Tat mutant of Brucella melitensis M28 exhibits reduced survival within murine macrophages. In this study, we compared the host responses elicited by wild-type M28 and its Tat-mutant strains ex vivo. We utilized label-free quantitative proteomics to assess proteomic changes in RAW264.7 macrophages after infection with M28 and its Tat mutants. A total of 6085 macrophage proteins were identified with high confidence, and 79, 50, and 99 proteins were differentially produced upon infection with the Tat mutant at 4, 24, and 48 hpi, respectively, relative to the wild-type infection. Gene ontology and KEGG enrichment analysis indicated that immune response-related proteins were enriched among the upregulated proteins. Compared to the wild-type M28 infection, the most upregulated proteins upon Tat-mutant infection included the cytosolic nucleic acid signaling pathway-related proteins IFIH1, DHX58, IFI202, IFI204, and ISG15 and the NF-κB signaling pathway-related proteins PTGS2, CD40, and TRAF1, suggesting that the host increases the production of these proteins in response to Tat mutant infection. Upregulation of some proteins was further verified by a parallel reaction monitoring (PRM) assay. ELISA and qRT-PCR assays indicated that Tat mutant infection significantly induced proinflammatory cytokine (TNF-α and IL-6) and nitric oxide (NO) production. Finally, we showed that the Tat mutant displays higher sensitivity to nitrosative stress than the wild type and that treatment with the NO synthase inhibitor L-NMMA significantly increases the intracellular survival of the Tat mutant, indicating that NO production contributes to restricting Tat mutant survival within macrophages. Collectively, this work improves our understanding of host immune responses to Tat mutants and provides insights into the mechanisms underlying the attenuated virulence of Tat mutants.

Ultrasensitive detection of trace Hg2+ by SERS aptasensor based on dual recycling amplification in water environment

Due to the nonbiodegradability and accumulation of mercury ion, even in extremely small amount, it will cause varying degrees of harm to environment and human health. Although researchers have developed many strategies to detect and monitor trace Hg2+, only a few provide sensitivities of less than 1.0 pM. Surface Enhanced Raman Spectroscopy (SERS) is a common method to detect mercury ion due to its high sensitivity, rapid detection and easy operation. In this work, we report a new SERS aptasensor based on dual recycling amplification for the detection of trace mercury ion, which combines SERS with nucleic acid signal amplification through functional aptamer and elaborately designed hairpin DNA. Under the optimal experimental conditions, this SERS aptasensor exhibits excellent selectivity and high sensitivity. A linear range (0.2–125 fM) and a low detection limit (0.11 fM) are obtained. By using specific aptamers, the strategy will provide a new idea for the trace detection of toxic contaminants in water environment.

Molecular insight into somaclonal variation phenomena from transcriptome profiling of cucumber (Cucumis sativus L.) lines

Somaclonal variation during in vitro culture is often an undesirable phenomenon but may also be a source of genetic variation useful for breeders. The molecular mechanisms underlying this phenomenon remain uncertain. In this study, we analyzed the fruit transcriptome of three independent cucumber lines using RNA-sequencing technology and performed an extensive in silico study to determine how somaclonal variation altered gene expression. Comparison of the transcriptome profiles with the wild-type cucumber ‘Borszczagowski B10’ revealed 418, 364, and 273 genes that were differentially regulated. We performed bioinformatic functional analysis, gene ontology classification, molecular network analysis, and explored differentially expressed genes associated with processes such as protein and nucleic acid binding, enzyme activity, signaling, transport, sugar and lipid metabolism. We determined that the differential gene expression may be caused by polymorphism in the genic region and may also be a result of interaction among molecular networks, which triggers specific pathways.

Extracellular Vesicles Derived From Apoptotic Cells: An Essential Link Between Death and Regeneration.

Apoptosis is a universal and continuous event during tissue development, restoration, repair, and regeneration. Mounting evidence has demonstrated that apoptosis is essential for the activation of tissue regeneration. However, the underlying mechanism remains elusive. A striking development in recent years comes from research on extracellular vesicles (EVs) derived from apoptotic cells. During apoptosis, cells secrete vesicles of various sizes containing various components. Apoptotic cell-derived EVs (ApoEVs) have been found to transit to neighboring cells or cells in distant tissues through the circulation. These vesicles could act as containers to transmit the nucleic acid, protein, and lipid signals to target cells. ApoEVs have been shown to promote regeneration in the cardiovascular system, skin, bone, muscle, kidney, etc. Moreover, several specific signaling pathways mediating the anabolic effects of ApoEVs have been classified. In this review, we comprehensively discussed the latest findings on the function of ApoEVs in tissue regeneration and disease prevention. These findings may reveal unexpected clues regarding the regulatory network between cell death and tissue regeneration and suggest novel targets for regenerative medicine. The findings discussed here also raise the question whether and to what extent ApoEVs contribute to embryonic development. This question is all the more urgent because the exact functions of apoptotic events during numerous developmental processes are still largely unclear.

Highly Specific Recognition of Guanosine Using Engineered Base‐Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na+ -binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na+ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm. Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.

Probe-lengthening amplification-assisted microchip electrophoresis for ultrasensitive bacteria screening

Probe-lengthening amplification (PLA) is a target-specific nucleic acid amplification method that realizes good selectivity by effectively avoiding nonspecific amplification. Here, we propose a probe-lengthening amplification-assisted microchip electrophoresis (MCE) strategy for sensitive analysis of 16S rRNA genes of five bacteria. In this assay, four specific short probes were designed for a target bacterium to recognize its bacterial 16S rRNA gene, integrated into longer DNA ligation duplexes using Ampligase, and subsequently separated and detected by MCE. Along with the rapid generation of ligation duplexes, this approach provides exponential amplification of nucleic acid signals that are useful for sensitive bacterial quantification. Through tactfully combining PLA and MCE, the detection sensitivity of bacterial genes was significantly improved, and a limit of detection (LOD) of 30 fM was realized for the artificial target DNA. This approach was also applied to detect actual bacterial genomic samples with excellent results, demonstrating the potential application of this methodology in infection diagnosis.

CRISPR-Cas12a based aptasensor for sensitive and selective ATP detection

CRISPR-Cas based detection platform opens up a new avenue for biosensing owing to its programmability, sequence specificity and high base resolution upon nucleic acid targets. In current study, we utilised the target DNA-induced non-specific single-stranded DNA cutting activity (trans-cleavage) of CRISPR-Cas12a to fabricate an aptamer mediated fluorescent biosensor for sensitive and selective detection of adenosine triphosphate (ATP). In this sensor, we designed an ATP-binding aptamer as the target ssDNA for Cas12a, thereby in the presence of ATP, the ATP-binding aptamer would be occupied by ATP, and less target ssDNA can be processed by Cas12a, which gave rise to an ATP concentration dependent change in fluorescence resulted from trans-cleavage of doubly labeled ssDNA reporter. In this way, we successfully “translated” ATP signals to nucleic acid signals that can be amplified by Cas12a process. The dynamic detection range was from 1 μM to 200 μM and the limit of detection was 400 nM. The entire sample-to-answer time for this biosensor was around 40 min. Overall, this novel biosensor balanced sensitivity, specificity, detection time and ease of use. Our proposed biosensor provided a principle-of-proof for detecting small molecule by using CRISPR-Cas12a system, which would unlock its potential and further its futuristic applications in the field of diagnostics.

Accurate MRSA identification through dual-functional aptamer and CRISPR-Cas12a assisted rolling circle amplification

Infectious diseases have become one of the most threatening global challenge with high morbidity and mortality, bringing great difficulties to clinical diagnosis and treatment. New strategy for high-specific and sensitive bacteria detection are urgently needed in facing the crisis of worldwide antibiotic resistance. Herein, a novel method through the integration of dual aptamer technology and CRISPR-Cas12a assisted rolling circle amplification (RCA) was present to obtain both accurate identification and high-sensitive detection of Methicillin-Resistant Staphylococcus Aureus (MRSA). The specificity inherited from the dual functionalized aptamers initiated bioconjugation to specifically recognize the protein targets on the surface of bacteria. Besides the target activity, the functionalized aptamer could also convert the protein recognition to nucleic acids signals. Through the integration of attached RCA and CRISPR-Cas12a assisted trans-cleavage, dual amplification of the nucleic acid signal was obtained. Based on this, we have extended the application of CRISPR-Cas12a from the nucleic acid detection to bacteria detection. As a result, the proposed method was demonstrated to be with significantly improved sensitivity towards MRSA detection. We believe that the novel integrated strategy would diversify the existing pool of bacterial detection and inspire the development of promising drug candidates in the future.

SLAF-seq Uncovers the Genetic Diversity and Adaptation of Chinese Elm (Ulmus parvifolia) in Eastern China

The Chinese elm is an important tree ecologically; however, little is known about its genetic diversity and adaptation mechanisms. In this study, a total of 107 individuals collected from seven natural populations in eastern China were investigated by specific locus amplified fragment sequencing (SLAF-seq). Based on the single nucleotide polymorphisms (SNPs) detected by SLAF-seq, genetic diversity and markers associated with climate variables were identified. All seven populations showed medium genetic diversity, with PIC values ranging from 0.2632 to 0.2761. AMOVA and Fst indicated that a low genetic differentiation existed among populations. Environmental association analyses with three climate variables (annual rainfall, annual average temperature, and altitude) resulted in, altogether, 43 and 30 putative adaptive loci by Bayenv2 and LFMM, respectively. Five adaptive genes were annotated, which were related to the functions of glycosylation, peroxisome synthesis, nucleic acid metabolism, energy metabolism, and signaling. This study was the first on the genetic diversity and local adaptation in Chinese elms, and the results will be helpful in future work on molecular breeding.

Proteomic Analysis of Differentially Expressed Proteins in the Placenta of Anticardiolipin Antibody‐ (ACA‐) Positive Pregnant Mice after Anzi Heji Treatment

Anzi Heji (AZHJ) has been used to treat anticardiolipin antibody- (ACA-) positive pregnant women at risk of spontaneous abortion for many years. The aim of this study was to investigate the protective mechanism of AZHJ in a mouse model of ACA-positive pregnancy at risk of spontaneous abortion using label-free quantitative proteomics. Mice were divided into three groups: normal pregnant mice (control group), ACA-positive pregnant mice administered normal saline (model group), and ACA-positive pregnant mice administered AZHJ (AZHJ group). The model was established by injecting β2-glycoprotein I (GPI) into mice for 18 days. The DEPs and their functions were analyzed by label-free quantitative proteomic and bioinformatic analyses. The levels of IL-6, IL-10, ACA, and TNF-α in the serum and placentas of the mice were measured by enzyme-linked immunosorbent assays (ELISAs). Proteomic data were validated by western blot analysis. The abnormal serum and placental levels of IL-6, ACA, and TNF-α in the model group were reversed by AZHJ. There were 39 upregulated and 10 downregulated DEPs in the AZHJ group relative to the model group. Bioinformatic analysis revealed that the DEPs were mainly involved in nucleic acid binding, signal conduction, and posttranslational modification. The placental levels of T-cell immunoglobulin mucin 3 (Tim-3) and Toll-like receptor 4 (TLR4) expression and AKT phosphorylation in the three groups were consistent with the proteomic findings. Tim-3/AKT signaling is involved in maternal-fetal immune tolerance, while TLR4 is associated with inflammatory responses. Collectively, these results indicate that AZHJ may exert its protective effect in ACA-positive pregnant mice by regulating the maternal-fetal immune tolerance and inflammatory response.

Role of skeletal muscle homeostasis of functional food material

Functional food material, polyamines are considered to be essential for growth factors in virtually all cells. The polyamines putrescine, spermidine and spermine are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. The proposed functions of polyamines are the regulation of ion channels, nucleic acid packaging, signal transduction, cell proliferation, and differentiation, as well as gene expression. In skeletal muscle, regulation of polyamine levels is associated with muscle hypertrophy and atrophy, yet detailed studies are remained to be undergoing. Here, we studied how polyamines may affect the proliferation and/or differentiation of murine myoblast progenitor C2C12 cell line. Upon polyamine treatment of C2C12 cells during induction of myogenic differentiation, the number of myotubes significantly increased. Morphologically, polyamine-treated myotubes exhibited elongated cell body and contained larger amount of nuclei in the cell. On the other hand, the polyamine did not have influence on myoblasts proliferation. Furthermore, compensatory muscle hypertrophy of C57BL6 mice that underwent sciatic nerve transection of the left hindlimb was enhanced by administration of polyamines. Therefore, our study demonstrates that polyamines may play an important role in regulating myogenic differentiation rather than myoblasts proliferation.