[Identification and expression analysis of members of R2R3-MYB transcription factors family in Artemisia argyi].

The MYB(v-myb avian myeloblastosis viral oncogene homolog) family of transcription factors is the largest class of genes among higher plant transcription factors, which can be divided into four subfamilies, with the R2R3-MYB being the most common subfamily type. R2R3-MYB transcription factors are widely involved in the regulation of organ development and secondary metabolite biosynthesis in plants. To investigate the role of R2R3-MYB family transcription factors in the synthesis of flavonoids and glandular trichome development in Artemisia argyi, this study screened and identified 92 R2R3-MYB transcription factors based on the whole genome data of A. argyi, and predicted their potential functions based on bioinformatics. The results showed that the amino acid lengths of the 92 transcription factors ranged from 168 to 547 aa, with relative molecular weights ranging from 19. 6 to 60. 5 kDa, all of which were hydrophilic proteins. Subcellular localization analysis showed that 89 AaMYB proteins were located in the nucleus, while three proteins were simultaneously located in the nucleus and cytoplasm. According to the classification of Arabidopsis R2R3-MYB family, the 92 A. argyi R2R3-MYB proteins were divided into 26 subfamilies, with similar gene structures within the same subfamily.Cis-acting element prediction results showed that light-responsive elements, methyl jasmonate elements, and abscisic acid elements were widely distributed in the promoter regions of R2R3-MYB genes. Transcriptome expression analysis results showed that the expression of AaMYB60, AaMYB63, and AaMYB86 in leaves was higher than that in stems and roots, indicating that these three transcription factors mainly function in leaves. Additionally, five candidate R2R3-MYB transcription factors involved in A. argyi flavonoid biosynthesis or glandular trichome development were selected through phylogenetic analysis. This study provides important genetic resources for the breeding of superior varieties and germplasm innovation of A. argyi in the future.

Comparative Analysis of Casparian Strip Membrane Domain Protein Family in Oryza sativa (L.) and Arabidopsis thaliana (L.).

The Casparian strip membrane domain proteins (CASPs) are pivotal for the formation of the Casparian strip (CS) in endodermal cells and play a crucial role in a plant's response to environmental stresses. However, existing research on the CASP gene family in rice and Arabidopsis lacks a comprehensive bioinformatics analysis and necessitates further exploration. In this study, we identified 41 OsCASP and 39 AtCASP genes, which were grouped into six distinct subgroups. Collinearity analysis underscored the pivotal roles of WGD and TD events in driving the evolution of CASPs, with WGDs being the dominant force. On the one hand, the analysis of cis-elements indicated that most OsCASP and AtCASP genes contain MYB binding motifs. On the other hand, RNA-seq revealed that the majority of OsCASP and AtCASP genes are highly expressed in roots, particularly in endodermal cells, where OsCASP_like11/9 and AtCASP_like1/31 demonstrated the most pronounced expression. These results suggest that OsCASP_like11/9 and AtCASP_like1/31 might be candidate genes involved in the formation of the endodermis CS. RT-qPCR results demonstrated that OsCASP_like2/3/13/17/21/30 may be candidate genes for the ion defect process. Collectively, this study offers a theoretical foundation for unraveling the biological functions of CASP genes in rice and Arabidopsis.

The complete chloroplast genome of Swertia davidii (gentianaceae) and its phylogenetic analysis.

To elucidate the genetic information and evolutionary relationships of Swertia, we initiated the sequencing of the complete chloroplast genome of Swertia davidii Franch. 1888, complemented by comparative analyses with closely related species. The chloroplast genome of S. davidii was 153,516 bp in length and exhibited a typical quadripartite structure. It contained two regions with Inverted Repeat lengths of 25,767 bp, located between one Large Single-Copy region (83,617 bp) and one Short Single-Copy region (18,365 bp). The chloroplast genome of S. davidii encoded 132 genes, including 87 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes. The overall GC content was 38.15%. Maximum likelihood phylogenetic analysis of Swertia based on 26 available plastomes showed a close relationship between S. davidii and S. kouitchensi. This study will contribute to the genetic preservation of the species and the phylogenetic study of Swertia.

Assembly and comparative analysis of the complete mitochondrial and chloroplast genome of Cyperus stoloniferus (Cyperaceae), a coastal plant possessing saline-alkali tolerance.

BACKGROUND: Cyperus stoloniferus is an important species in coastal ecosystems and possesses economic and ecological value. To elucidate the structural characteristics, variation, and evolution of the organelle genome of C. stoloniferus, we sequenced, assembled, and compared its mitochondrial and chloroplast genomes. RESULTS: We assembled the mitochondrial and chloroplast genomes of C. stoloniferus. The total length of the mitochondrial genome (mtDNA) was 927,413 bp, with a GC content of 40.59%. It consists of two circular DNAs, including 37 protein-coding genes (PCGs), 22 tRNAs, and five rRNAs. The length of the chloroplast genome (cpDNA) was 186,204 bp, containing 93 PCGs, 40 tRNAs, and 8 rRNAs. The mtDNA and cpDNA contained 81 and 129 tandem repeats, respectively, and 346 and 1,170 dispersed repeats, respectively, both of which have 270 simple sequence repeats. The third high-frequency codon (RSCU > 1) in the organellar genome tended to end at A or U, whereas the low-frequency codon (RSCU < 1) tended to end at G or C. The RNA editing sites of the PCGs were relatively few, with only 9 and 23 sites in the mtDNA and cpDNA, respectively. A total of 28 mitochondrial plastid DNAs (MTPTs) in the mtDNA were derived from cpDNA, including three complete trnT-GGU, trnH-GUG, and trnS-GCU. Phylogeny and collinearity indicated that the relationship between C. stoloniferus and C. rotundus are closest. The mitochondrial rns gene exhibited the greatest nucleotide variability, whereas the chloroplast gene with the greatest nucleotide variability was infA. Most PCGs in the organellar genome are negatively selected and highly evolutionarily conserved. Only six mitochondrial genes and two chloroplast genes exhibited Ka/Ks > 1; in particular, atp9, atp6, and rps7 may have undergone potential positive selection. CONCLUSION: We assembled and validated the mtDNA of C. stoloniferus, which contains a 15,034 bp reverse complementary sequence. The organelle genome sequence of C. stoloniferus provides valuable genomic resources for species identification, evolution, and comparative genomic research in Cyperaceae.

DNA Meets Protein─Development, Characterization, and Application of Aptamers against Peanut Allergen.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Assembly and comparative analysis of the complete mitochondrial genome of Vaccinium carlesii Dunn.

Vaccinium L. is an important fruit tree with nutritional, medicinal, and ornamental values. However, the mitochondrial (mt) genome of Vaccinium L. remains largely unexplored. Vaccinium carlesii Dunn is an endemic wild resource in China, which is crucial for blueberry breeding. The V. carlesii mt genomes were sequenced using Illumina and Nanopore, which total length was 636,904 bp with 37 protein coding genes, 20 tRNA genes, and three rRNA genes. We found four pairs of long repeat fragments homologous recombination mediated the generation of substructures in the V. carlesii mt genome. We predicted 383 RNA editing sites, all converting cytosine (C) to uracil (U). According to the phylogenetic analysis, V. carlesii and V. macrocarpon of the Ericaceae exhibited the closest genetic relationship. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of Vaccinium germplasm resources.

Comparative chloroplast genomics and phylogenetic analysis of Oreomecon nudicaulis (Papaveraceae).

Oreomecon nudicaulis, commonly known as mountain poppy, is a significant perennial herb. In 2022, the species O. nudicaulis, which was previously classified under the genus Papaver, was reclassified within the genus Oreomecon. Nevertheless, the phylogenetic status and chloroplast genome within the genus Oreomecon have not yet been reported. This study elucidates the chloroplast genome sequence and structural features of O. nudicaulis and explores its evolutionary relationships within Papaveraceae. Using Illumina sequencing technology, the chloroplast genome of O. nudicaulis was sequenced, assembled, and annotated. The results indicate that the chloroplast genome of O. nudicaulis exhibits a typical circular quadripartite structure. The chloroplast genome is 153,903 bp in length, with a GC content of 38.87%, containing 84 protein-coding genes, 8 rRNA genes, 38 tRNA genes, and 2 pseudogenes. The genome encodes 25,815 codons, with leucine (Leu) being the most abundant codon, and the most frequently used codon is AUU. Additionally, 129 microsatellite markers were identified, with mononucleotide repeats being the most abundant (53.49%). Our phylogenetic analysis revealed that O. nudicaulis has a relatively close relationship with the genus Meconopsis within the Papaveraceae family. The phylogenetic analysis supported the taxonomic status of O. nudicaulis, as it did not form a clade with other Papaver species, consistent with the revised taxonomy of Papaveraceae. This is the first report of a phylogenomic study of the complete chloroplast genome in the genus Oreomecon, which is a significant genus worldwide. This analysis of the O. nudicaulis chloroplast genome provides a theoretical basis for research on genetic diversity, molecular marker development, and species identification, enriching genetic information and supporting the evolutionary relationships among Papaveraceae.

Evolutionary game analysis of factors influencing green innovation in Enterprises under environmental governance constraints.

The key to controlling environmental pollution is to promote green innovation in relevant enterprises and achieve a healthy development of the environmental governance system. This paper constructs a tripartite evolutionary game model of environmental protection enterprises, polluting enterprises, and governments, and conducts in-depth research on the influencing factors that promote green innovation in two types of enterprises. MATLAB software is used to analyze the impact of different degrees of influencing variables on system evolution. It has found that (1) increasing the intensity of environmental governance and the level of innovation subsidies by the government can effectively promote green innovation in both types of enterprises. (2) The varying degrees of innovation compensation from polluting enterprises to environmental protection enterprises have a significant impact on system evolution. (3) The initial intention and population size of two types of enterprise entities will have a significant impact on system evolution. In the initial state, subjects with more green innovation are less willing to change their strategies during the evolution process, while the willingness of the other party to green innovation will be suppressed.

Screen and Optimization of an Aptamer for Alexandrium tamarense-A Common Toxin-Producing Harmful Alga.

Among all the paralytic shellfish toxins (PSTs)-producing algae, Alexandrium tamarense is one of the most widespread harmful species posing a serious threat to marine resources and human health. Therefore, it is extremely important to establish a rapid and accurate monitoring method for A. tamarense that can provide early warnings of harmful algal blooms (HABs) caused by this alga and limit the contamination due to PSTs. In this study, an ssDNA library was first obtained by whole cell systematic evolution of ligands by exponential enrichment after 18 consecutive rounds of iterative screening. After sequencing in combination with subsequent multiple alignment of sequences and secondary structure simulation, the library could be classified into 2 families, namely, Family1 and Family2, according to sequence similarity. Flow cytometry was used to test the affinity and cross-reactivity of Ata19, Ata6, Ata25 and Ata29 belonging to Family2. Ata19 was selected to be modified by truncation, through which a new resultant aptamer named as Ata19-1-1 was obtained. Ata19-1-1 with a KD of 75.16 ± 11.10 nM displayed a much higher affinity than Ata19. The specificity test showed that Ata19-1-1 has the same discrimination ability as Ata19 and can at least distinguish the target microalga from other microalgae. The observation under a fluorescence microscopy showed that the A. tamarense cells labeled with Ata19-1-1 are exhibiting bright green fluorescence and could be easily identified, factually confirming the binding of the aptamer with target cells. In summary, the aptamer Ata19-1-1 produced in this study may serve as an ideal molecular recognition element for A. tamarense, which has the potential to be developed into a novel detection method for this harmful alga in the future.

Selection of aptamers using ß-1,3-glucan recognition protein-tagged proteins and curdlan beads.

RNA aptamersare nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilize target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins but also to the protein tags and matrix beads. In this study, we performed SELEX using ß-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilize the acute myeloid leukaemia 1 (AML1) Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Assembly and analysis of the first complete mitochondrial genome of Punica granatum and the gene transfer from chloroplast genome.

Pomegranate (Punica granatum L.) is one of the oldest fruits with edible, medicinal and ornamental values. However, there is no report on the mitochondrial genome of pomegranate. In this study, the mitochondrial genome of P. granatum was sequenced, assembled and analyzed in detail, while the chloroplast genome was assembled using the same set of data. The results showed that the P. granatum mitogenome had a multi branched structure, using BGI + Nanopore mixed assembly strategy. The total genome length was 404,807 bp, with the GC content of 46.09%, and there were 37 protein coding genes, 20 tRNA genes and three rRNA genes. In the whole genome, 146 SSRs were identified. Besides, 400 pairs of dispersed repeats were detected, including 179 palindromic, 220 forward and one reverse. In the P. granatum mitochondrial genome, 14 homologous fragments of chloroplast genome were found, accounting for 0.54% of the total length. Phylogenetic analysis showed that among the published mitochondrial genomes of related genera, P. granatum had the closest genetic relationship with Lagerstroemia indica of Lythraceae. The 580 and 432 RNA editing sites were predicted on 37 protein coding genes of mitochondrial genome using BEDTools software and online website PREPACT respectively, but all were from C to U, of which ccmB and nad4 gene were most frequently edited, with 47 sites. This study provides a theoretical basis for understanding the evolution of higher plants, species classification and identification, and will also be useful for further utilization of pomegranate germplasm resources.

The first chloroplast sequence of Rosa davurica Pall. var. Davurica.

Rosa davurica Pall. var. davurica is a member of the plant family Rosaceae. Although R. davurica has high application value, its chloroplast genome sequence has not been reported. This study aims to reveal the genetic characteristics of the chloroplast genome of Rosa roxburghii. The length of its total chloroplast DNA is 156,971 bp, with 37.22% G/C content. Its chloroplast genome has two inverted repeat (IRa and IRb) regions totaling 26,051 bp which are separated by a large single copy (LSC) region of 86,032 bp and a small single copy (SSC) region of 18,837 bp. The genome contains 131 independent genes (86 protein-coding, 37 tRNA, and 8 rRNA), and there are 18 repeated genes within the IR region. Among these genes, 17 genes contained one or two introns. The phylogenetic analysis showed that R. davurica was relatively close to other Rosa species, such as the Rosa hybrid.

A novel aptamer-G-quadruplex/hemin self-assembling color system: rapid visual diagnosis of invasive fungal infections.

BACKGROUND: The clinical symptoms of invasive fungal infections (IFI) are nonspecific, and early clinical diagnosis is challenging, resulting in high mortality rates. This study reports the development of a novel aptamer-G-quadruplex/hemin self-assembling color system (AGSCS) based on (1 → 3)-ß-D-glucans' detection for rapid, specific and visual diagnosis of IFI. METHODS: We screened high affinity and specificity ssDNA aptamers binding to (1 → 3)-ß-D-glucans, the main components of cell wall from Candida albicans via Systematic Evolution of Ligands by EXponential enrichment. Next, a comparison of diagnostic efficiency of AGSCS and the (1 → 3)-ß-D-glucans assay ("G test") with regard to predicting IFI in 198 clinical serum samples was done. RESULTS: Water-soluble (1 → 3)-ß-D-glucans were successfully isolated from C. albicans ATCC 10,231 strain, and these low degree of polymerization glucans (< 1.7 kD) were targeted for aptamer screening with the complementary sequences of G-quadruplex. Six high affinity single stranded DNA aptamers (A1, A2, A3, A4, A5 and A6) were found. The linear detection range for (1 → 3)-ß-D-glucans stretched from 1.6 pg/mL to 400 pg/mL on a microplate reader, and the detection limit was 3.125 pg/mL using naked eye observation. Using a microplate reader, the sensitivity and specificity of AGSCS for the diagnosis of IFI were 92.68% and 89.65%, respectively, which was higher than that of the G test. CONCLUSION: This newly developed visual diagnostic method for detecting IFI showed promising results and is expected to be developed as a point-of-care testing kit to enable quick and cost effective diagnosis of IFI in the future.

Selection of a Novel DNA Aptamer Specific for 5-Hydroxymethylfurfural Using Capture-SELEX.

A capture systematic evolution of ligands by exponential enrichment (Capture-SELEX) was described to discover novel aptamers specific for 5-hydroxymethylfurfural (5-HMF), and a biosensor based on molecular beacon was constructed to detect 5-HMF. The ssDNA library was immobilized to streptavidin (SA) resin to select the specific aptamer. The selection progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing (HTS). Candidate and mutant aptamers were selected and identified by Isothermal Titration Calorimetry (ITC). The FAM-aptamer and BHQ1-cDNA were designed as the quenching biosensor to detect 5-HMF in milk matrix. After the 18th round selection, the Ct value decreased from 9.09 to 8.79, indicating that the library was enriched. The HTS results indicated that the total sequence numbers for 9th, 13th, 16th, and 18th were 417054, 407987, 307666, and 259867, but the number of sequences for the top 300 sequences was gradually increased from 9th to 18th, and the ClustalX2 analysis showed that there were four families with high homology rate. ITC results indicated that the Kd values of H1 and its mutants H1-8, H1-12, H1-14, and H1-21 were 2.5 µM, 1.8 µM, 1.2 µM, 6.5 µM, and 4.7 µM. The linear range of the quenching biosensor was from 0 µM to 75 µM, and it had a similar linear range in the 0.1% milk matrix. This is the first report to select a novel aptamer specific for 5-HMF and develop quenching biosensor for the rapid detection of 5-HMF in milk matrix.

Genomic SELEX Reveals Pervasive Role of the Flagella Master Regulator FlhDC in Carbon Metabolism.

Flagella are vital bacterial organs that allow microorganisms to move to favorable environments. However, their construction and operation consume a large amount of energy. The master regulator FlhDC mediates all flagellum-forming genes in E. coli through a transcriptional regulatory cascade, the details of which remain elusive. In this study, we attempted to uncover a direct set of target genes in vitro using gSELEX-chip screening to re-examine the role of FlhDC in the entire E. coli genome regulatory network. We identified novel target genes involved in the sugar utilization phosphotransferase system, sugar catabolic pathway of glycolysis, and other carbon source metabolic pathways in addition to the known flagella formation target genes. Examining FlhDC transcriptional regulation in vitro and in vivo and its effects on sugar consumption and cell growth suggested that FlhDC activates these new targets. Based on these results, we proposed that the flagella master transcriptional regulator FlhDC acts in the activation of a set of flagella-forming genes, sugar utilization, and carbon source catabolic pathways to provide coordinated regulation between flagella formation, operation and energy production.

Aptamer Selection Based on Microscale Electrophoretic Filtration Using a Hydrogel-Plugged Capillary Device.

This study reports a novel aptamer selection method based on microscale electrophoretic filtration. Aptamers are versatile materials that recognize specific targets and are attractive for their applications in biosensors, diagnosis, and therapy. However, their practical applications remain scarce due to issues with conventional selection methods, such as complicated operations, low-efficiency separation, and expensive apparatus. To overcome these drawbacks, a selection method based on microscale electrophoretic filtration using a capillary partially filled with hydrogel was developed. The electrophoretic filtration of model target proteins (immunoglobulin E (IgE)) using hydrogel, the electrokinetic injection of DNAs to interact with the trapped proteins, the elimination of DNAs with weak interactions, and the selective acquisition of aptamer candidates with strong interactions were successfully demonstrated, revealing the validity of the proposed concept. Two aptamer candidates for IgE were obtained after three selection cycles, and their affinity for the target was confirmed to be less than 1 nM based on their dissociation constant (KD) values. Therefore, the proposed method allows for the selection of aptamers with simple operations, highly effective separation based on electrophoresis and filtration, and a relatively cheap apparatus with disposable devices.

Aptamer-based enzyme-linked oligonucleotide assay for specific detection of clinical bacterial strains isolated from cerebrospinal fluid samples.

Meningitis, acute infection of the meninges, is the 10th leading cause of mortality among infectious diseases. Although many different causes for meningitis (viruses and bacteria) have been diagnosed, the most common ones are Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. The effort to find a new method for detection of bacterial meningitis is an urgent need for clinical treatment. DNA aptamers generated by cell-systematic evolution of ligands by exponential enrichment (SELEX) against bacterial cells provide a novel cell labeling and biosensing technique. Here, we isolated single-stranded DNA aptamers during the SELEX method with a high affinity for different bacterial genera. This approach was demonstrated on H. influenzae type B, N. meningitidis serogroups A, B, C, and Y, and Streptococcus pneumoniae serotypes 18, 14, 19A, 6A, and 6B which served as targets in 20 rounds of cell-SELEX. After 20 rounds of SELEX, a total of 93 aptamers were identified. Among these, aptamers C65 and C50 showed the highest affinity toward targets with a dissociation constant of 6.98 and 15.79, respectively. Selected aptamers were able to successfully detect clinical bacterial strains isolated from cerebrospinal fluid samples of meningitis patients by double-aptamer sandwich enzyme-linked oligonucleotide assay (ELONA). Our findings demonstrated that aptamers with broad affinity to bacterial taxa in different genera can be isolated for the development of diagnostic tools for multiple targets. We further showed that sandwich ELONA based on single-stranded DNA aptamer is sensitive and specific enough for detection of the superior cause of bacterial meningitis.

Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses.

In this study, 52 AAAP genes were identified in the L. chinense genome and divided into eight subgroups based on phylogenetic relationships, gene structure, and conserved motif. A total of 48 LcAAAP genes were located on the 14 chromosomes, and the remaining four genes were mapped in the contigs. Multispecies phylogenetic tree and codon usage bias analysis show that the LcAAAP gene family is closer to the AAAP of Amborella trichopoda, indicating that the LcAAAP gene family is relatively primitive in angiosperms. Gene duplication events revealed six pairs of segmental duplications and one pair of tandem duplications, in which many paralogous genes diverged in function before monocotyledonous and dicotyledonous plants differentiation and were strongly purification selected. Gene expression pattern analysis showed that the LcAAAP gene plays a certain role in the development of Liriodendron nectary and somatic embryogenesis. Low temperature, drought, and heat stresses may activate some WRKY/MYB transcription factors to positively regulate the expression of LcAAAP genes to achieve long-distance transport of amino acids in plants to resist the unfavorable external environment. In addition, the GAT and PorT subgroups could involve gamma-aminobutyric acid (GABA) transport under aluminum poisoning. These findings could lay a solid foundation for further study of the biological role of LcAAAP and improvement of the stress resistance of Liriodendron.

Improving aptamer performance with nucleic acid mimics: de novo and post-SELEX approaches.

Aptamers are structural single-stranded oligonucleotides generated in vitro to bind to a specific target molecule. Aptamers' versatility can be enhanced with nucleic acid mimics (NAMs) during or after a selection process, also known as systematic evolution of ligands by exponential enrichment (SELEX). We address advantages and limitations of the technologies used to generate NAM aptamers, especially the applicability of existing engineered polymerases to replicate NAMs and methodologies to improve aptamers after SELEX. We also discuss the limitations of existing methods for sequencing NAM sequences and bioinformatic tools to predict NAM aptamer structures. As a conclusion, we suggest that NAM aptamers might successfully compete with molecular tools based on proteins such as antibodies for future application.

Novel nucleic acid aptamer gold (Au)-nanoparticles (AuNPs-AptHLA-G5-1 and AuNPs-AptHLA-G5-2) to detect the soluble human leukocyte antigen G5 subtype (HLA-G5) in liquid samples.

BACKGROUND: The human leukocyte antigen G5 subtype (HLA-G5) is a major histocompatibility complex (MHC) molecule that is selectively expressed at the maternal-foetal tissue interface and is required for the successful implantation of the in vitro fertilized embryo. It is critical to detect HLA-G5, especially HLA-G5 expression in embryo fluid, during in vitro embryo incubation and culture. However, the specificity and sensitivity of traditional ELISA methods to detect sHLA-G5 are insufficient. This work aimed to explore novel nucleic acid aptamer gold (Au)-nanoparticles to detect soluble HLA-G5 in liquid samples. METHODS: Soluble HLA-G5 was obtained using a prokaryotic expression system, and two novel aptamers (HLA-G5-Apt1 and HLA-G5-Apt2) detecting HLA-G5 were screened by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. Small (10 nm) gold nanoparticles (AuNPs) were incubated with AptHLAs to form two novel nucleic acid aptamers: Au-nanoparticles (AuNPs-AptHLA-G5-1 and AuNPs-AptHLA-G5-2). RESULTS: The results showed that AptHLA-G5-1 and AptHLA-G5-2 have a high affinity for HLA-G5 and can detect its presence in liquid samples. Using the colorimetric sensing method, AuNPs-AptHLA-G1 had a detection limit as low as 20 ng/mL (recovery range between 98.7% to 102.0%), while AuNPs-AptHLA-G2 had a detection limit as low as 20 ng/mL (recovery range between 98.9% to 103.6%). CONCLUSIONS: Our work demonstrates that novel AuNPs are efficient detectors for HLA-G5 and are useful for diagnosis and treatment in the field of obstetrics-gynaecology.