-
ACS Applied Materials & Interfaces Jul 2023Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate...
Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate critical cellular functions such as proliferation, differentiation, and survival. At the molecular level, cell adhesion receptors, such as integrins, transmit piconewton (pN)-scale forces to the extracellular matrix, and the magnitude of the force plays a critical role in cell signaling. The most sensitive approach to measuring integrin forces involves DNA hairpin-based sensors, which are used to quantify and map forces in living cells. Despite the broad use of DNA hairpin sensors to study a variety of mechanotransduction processes, these sensors are typically anchored to rigid glass slides, which are orders of magnitude stiffer than the extracellular matrix and hence modulate native biological responses. Here, we have developed nuclease-resistant DNA hairpin probes that are all covalently tethered to PEG hydrogels to image cell traction forces on physiologically relevant substrate stiffness. Using HeLa cells as a model cell line, we show that the molecular forces transmitted by integrins are highly sensitive to the bulk modulus of the substrate, and cells cultured on the 6 and 13 kPa gels produced a greater number of hairpin unfolding events compared to the 2 kPa substrates. Tension signals are spatially colocalized with pY118-paxillin, confirming focal adhesion-mediated probe opening. Additionally, we found that integrin forces are greater than 5.8 pN but less than 19 pN on 13 kPa gels. This work provides a general strategy to integrate molecular tension probes into hydrogels, which can better mimic in vivo mechanotransduction.
Topics: Humans; Mechanotransduction, Cellular; Hydrogels; HeLa Cells; Traction; DNA Probes; Cell Adhesion; DNA; Integrins; Receptors, Cell Surface
PubMed: 37409737
DOI: 10.1021/acsami.3c04826 -
Analytical Chemistry Jun 2024Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this...
Selective and sensitive imaging of intracellular mature microRNAs (miRNAs) is of great importance for biological process study and medical diagnostics. However, this goal remains challenging because of the interference of precursor miRNAs (pre-miRNAs) and the low abundance of mature miRNAs. Herein, we develop an endogenous enzyme-driven amplified DNA nanocage probe (Acage) for the selective and sensitive imaging of mature miRNAs in living cells. The Acage consists of a microRNA-responsive probe, an endogenous enzyme-driven fuel strand, and a DNA nanocage framework with an inner cavity. Benefiting from the size selectivity of DNA nanocage, smaller mature miRNAs rather than larger pre-miRNAs are allowed to enter the cavity of DNA nanocage for molecular recognition; thus, Acage can significantly reduce the signal interference of pre-miRNAs. Moreover, with the driving force of an endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) for efficient signal amplification, Acage enables sensitive intracellular miRNA imaging without an additional external intervention. With these features, Acage was successfully applied for intracellular imaging of mature miRNAs during drug treatment. We believe that this strategy provides a promising pathway for better understanding the functions of mature microRNAs in biological processes and medical diagnostics.
Topics: MicroRNAs; Humans; DNA Probes; Nanostructures; Optical Imaging; HeLa Cells
PubMed: 38818873
DOI: 10.1021/acs.analchem.4c00704 -
Nucleic Acids Research May 2024Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear...
Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.
Topics: DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Cell Nucleolus; Cell Nucleus; DNA Probes; HeLa Cells; Molecular Chaperones; Avidin; DNA; Biotin
PubMed: 38554110
DOI: 10.1093/nar/gkae202 -
Parasites & Vectors Aug 2023Six species of apicomplexan parasites of the genus Babesia, namely B. microti, B. divergens, B. duncani, B. motasi, B. crassa-like and B. venatorum, are considered to be...
BACKGROUND
Six species of apicomplexan parasites of the genus Babesia, namely B. microti, B. divergens, B. duncani, B. motasi, B. crassa-like and B. venatorum, are considered to be the primary causal agents of human babesiosis in endemic areas. These six species possess variable degrees of virulence for their primary hosts. Therefore, the accurate identification of these species is critical for the adoption of appropriate therapeutic strategies.
METHODS
We developed a real-time PCR-high-resolution melting (qPCR-HRM) approach targeting 18S ribosomal RNA gene of five Babesia spp. based on melting temperature (T) and genotype confidence percentage values. This approach was then evaluated using 429 blood samples collected from patients with a history of tick bites, 120 DNA samples mixed with plasmids and 80 laboratory-infected animal samples.
RESULTS
The sensitivity and specificity of the proposed qPCR-HRM method were 95% and 100%, respectively, and the detection limit was 1-100 copies of the plasmid with the cloned target gene. The detection level depended on the species of Babesia analyzed. The primers designed in this study ensured not only the high interspecific specificity of our proposed method but also a high versatility for different isolates from the same species worldwide. Additionally, the Tm obtained from the prepared plasmid standard is theoretically suitable for identifying isolates of all known sequences of the five Babesia species.
CONCLUSIONS
The developed detection method provides a useful tool for the epidemiological investigation of human babesiosis and pre-transfusion screening.
Topics: Animals; Humans; Babesia; Babesiosis; Cloning, Molecular; DNA Primers; Gastropoda
PubMed: 37641091
DOI: 10.1186/s13071-023-05839-5 -
Spectrochimica Acta. Part A, Molecular... Dec 2023Hg is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg-T mismatch...
Hg is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg-T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg-T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg. Under the optimal reaction conditions, there is an excellent linear relationship between Hg concentration and fluorescence intensity in the range of 7.5-200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.
Topics: Humans; Fluorescent Dyes; Mercury; Ecosystem; DNA; Exodeoxyribonucleases; Oligonucleotides; Biosensing Techniques; Limit of Detection
PubMed: 37562208
DOI: 10.1016/j.saa.2023.123223 -
Platelets Dec 2023CD36 is a multifunctional receptor expressed on the surface of many cell types. Among healthy individuals, CD36 may be absent on platelets and monocytes (type I...
CD36 is a multifunctional receptor expressed on the surface of many cell types. Among healthy individuals, CD36 may be absent on platelets and monocytes (type I deficiency) or platelets alone (type II deficiency). However, the exact molecular mechanisms underlying CD36 deficiency remain unclear. In this study, we aimed to identify individuals with CD36 deficiency and investigate the molecular basis underlying it. Blood samples were collected from platelet donors at Kunming Blood Center. Platelets and monocytes were isolated and CD36-expression levels were analyzed using flow cytometry. DNA from whole blood and mRNA isolated from monocytes and platelets of individuals with CD36 deficiency were analyzed using polymerase chain reaction (PCR) testing. The PCR products were cloned and sequenced. Among the 418 blood donors,7 (1.68%) were CD36 deficient: 1 (0.24%) with type I deficiency and 6(1.44%) with type II deficiency. Six heterozygous mutations occurred, including c.268C>T (in type I individuals), c.120 + 1 G>T, c.268C>T, c.329_330/AC, c.1156 C>T, c.1163A>C, and c.1228_1239/ATTGTGCCTATT (in type II individuals). Mutations were not detected in one type II individual . At the cDNA level, only mutant, but not wild-type, transcripts were detected in the platelets and monocytes of type I individual. In type II individuals, only mutant transcripts were found in platelets, whereas monocytes possessed wild-type and mutant transcripts. Interestingly, only alternative splicing transcripts were observed in the individual without mutation. We report the incidence rates of type I and II CD36 deficiencies among platelet donors in Kunming. Molecular genetic analyses of DNA and cDNA demonstrated that homozygous mutations on the cDNA level in platelets and monocytes or platelets alone identified type I and II deficiencies, respectively. Furthermore, alternatively spliced products also potentially contribute to the mechanism of CD36 deficiency.
Topics: Humans; DNA, Complementary; Blood Platelets; Blood Platelet Disorders; Mutation
PubMed: 36813737
DOI: 10.1080/09537104.2023.2176168 -
Analytica Chimica Acta Oct 2023We describe a novel lateral flow DNA biosensor (LFDB) based on carbon nanotube (CNT) and triple helix DNA (THD). The carboxylated CNT was first conjugated with...
We describe a novel lateral flow DNA biosensor (LFDB) based on carbon nanotube (CNT) and triple helix DNA (THD). The carboxylated CNT was first conjugated with amine-modified auxiliary single-stranded DNA probe (P) by dehydration reaction and used as signal probe. A main DNA probe (P) was introduced to react with the P and formed the THD on the CNT surface. Because of the large spatial effect, P was in an inactive state and cannot hybridize with the capture DNA probe (P) fixed on the LFDB test area. When the target DNA was present, P in the triple helix DNA hybridized with the target DNA due to the stronger base action, and the decomposition of the triple helix structure exposed P. Therefore, P on CNT surface was activated to hybridize with P. The CNT along with P was thus captured at the test area and accumulated to show a black line, which can be observed by naked eye for qualitative analysis and recorded with a portable grayscale reader for quantitative analysis. Single-stranded DNA was used as a target to prove the feasibility of the model. Under the best experimental conditions, the THD-CNT based LFDB was able to detect the lowest DNA concentration of 15 pM, which is 2.67 times better than that of the traditional duplex CNT-based LFDB. It should be noted that the LFDB based on THD functionalized CNT can differentiate between one-base-mismatched DNA and the complementary target DNA, can detected target DNA in 10% human serum, and can be employed as a versatile platform to detect various target (proteins, small molecular) by changing the sequence of P. This biosensor platform has enormous potential in the point-of-care detection of a rich diversity of analytes for clinical diagnosis and biomedical research.
Topics: Humans; Nucleic Acids; Nanotubes, Carbon; DNA, Single-Stranded; DNA; DNA Probes; Biosensing Techniques; DNA, Complementary
PubMed: 37573103
DOI: 10.1016/j.aca.2023.341604 -
ACS Pharmacology & Translational Science Nov 2023The 7-methyl guanosine cap structure is an essential 5' end modification of eukaryotic mRNA. It plays a critical role in many aspects of the life cycle of mRNA,...
The 7-methyl guanosine cap structure is an essential 5' end modification of eukaryotic mRNA. It plays a critical role in many aspects of the life cycle of mRNA, including nuclear export, stability, and translation. Equipping synthetic transcripts with a 5' cap is paramount to the development of effective mRNA vaccines and therapeutics. Here, we report a simple and flexible workflow to selectively isolate and analyze structural features of the 5' end of an mRNA by means of DNA probe-directed enrichment with site-specific single-strand endoribonucleases. Specifically, we showed that the RNA cleavage by site-specific RNases can be effectively steered by a complementary DNA probe to recognition sites downstream of the probe-hybridized region, utilizing a flexible range of DNA probe designs. We applied this approach using human RNase 4 to isolate well-defined cleavage products from the 5' end of diverse uridylated and 1-methylpseudouridylated mRNA 5' end transcript sequences. hRNase 4 increases the precision of the RNA cleavage, reducing product heterogeneity while providing comparable estimates of capped products and their intermediaries relative to the widely used RNase H. Collectively, we demonstrated that this workflow ensures well-defined and predictable 5' end cleavage products suitable for analysis and relative quantitation of synthetic mRNA 5' cap structures by UHPLC-MS/MS.
PubMed: 37974627
DOI: 10.1021/acsptsci.3c00157 -
Langmuir : the ACS Journal of Surfaces... Jan 2024Thermodynamically, perfect DNA hybridization can be formed between probes and their corresponding targets due to the favorable energy. However, this is not the case...
Thermodynamically, perfect DNA hybridization can be formed between probes and their corresponding targets due to the favorable energy. However, this is not the case dynamically. Here, we use molecular dynamics (MD) simulations based on the oxDNA model to investigate the process of DNA microarray hybridization. In general, correlated hybrid DNA structures are formed, including one probe associated with several targets as well as one target hybrid with multiple probes leading to the target-mediated hybridization. The formation of these two types of correlated structures largely depends on the surface coverage of the DNA microarray. Moreover, DNA sequence, DNA length, and spacer length have an impact on the structural formation. Our findings shed light on the dynamics of DNA hybridization, which is important for the application of DNA microarray.
Topics: DNA; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Base Sequence; DNA Probes
PubMed: 38154122
DOI: 10.1021/acs.langmuir.3c02231 -
Mikrochimica Acta Nov 2023A new electrochemical biosensor based on the sequence of chromosome Y (SRY) has been introduced to determine the gender of the fetus. At first, the DNA probe was...
A new electrochemical biosensor based on the sequence of chromosome Y (SRY) has been introduced to determine the gender of the fetus. At first, the DNA probe was designed based on the SRY gene sequence on chromosome Y. Then, a suitable functional group was added to the DNA probe, and it has been immobilized on the surface of the electrode modified with a nanocomposite containing Cu(OH) @N-C n-boxes. This substrate causes more DNA probes to connect to the electrode surface by increasing the effective surface area. The presence of the SRY sequence in the DNA sample extracted from blood was detected by the electrochemical signal of the bio-sensor. After optimizing the parameters, the fabricated genosensor showed linear responses in the two concentration ranges containing 0.5 fM to 50 pM and 50 pM to 500 nM. The limit of detection (LOD) for the proposed method was 0.16 fM. The proposed genosensor has been successfully used to determine the gender of the fetus using cell-free fetal DNA (cffDNA) in the blood plasma of several pregnant mothers. This method has advantages such as being simple, portable, accurate, and non-invasive for early determination of the gender of the fetus and early diagnosis of X-linked genetic disorders.
Topics: Pregnancy; Female; Humans; Electric Impedance; DNA; DNA Probes; Y Chromosome; Biosensing Techniques; Cell-Free Nucleic Acids
PubMed: 38006412
DOI: 10.1007/s00604-023-06061-x