Complementary Base Research Articles

A fluorescent aptasensor for Pb2+ detection based on gold nanoflowers and RecJf exonuclease-induced signal amplification

A fluorescent aptasensor was constructed to detect lead ion (Pb2+). Complementary DNA with sulfhydryl group (SH-cDNA) were loaded on gold nanoflowers materials (Au NFs) through Au–S bonds. Then, based on complementary base pairing between FAM modified Pb2+ aptamer (FAM-Apt) and Au NFs/SH-cDNA system, an Au NFs/SH-cDNA/FAM-Apt aptasensor was constructed. The fluorescence of FAM was quenched by Au NFs due to the fluorescence resonance energy transfer (FRET). Upon addition of Pb2+ and RecJf exonuclease (RecJf Exo) for 1.5 h, the Apt changed to a G-quadruplex structure due to the high affinity between Pb2+ and its aptamer, which lead to a large recovery in the fluorescence intensity. Under the optimized conditions, the presented aptasensor revealed high selectivity toward Pb2+ in the concentration range of 0.5 nM–1 μM, with a limit of detection (LOD) of 0.285 nM. Besides, the designed aptasensor was successfully used to recognize Pb2+ with excellent selectivity, reproducibility and stability in tap water and tea, providing a potential platform for Pb2+ detection in actual samples.

Antisense therapies in neurological diseases.

Advances in targeted regulation of gene expression allowed new therapeutic approaches for monogenic neurological diseases. Molecular diagnosis has paved the way to personalized medicine targeting the pathogenic roots: DNA or its RNA transcript. These antisense therapies rely on modified nucleotides sequences (single-strand DNA or RNA, both belonging to the antisense oligonucleotides family, or double-strand interfering RNA) to act specifically on pathogenic target nucleic acids, thanks to complementary base pairing. Depending on the type of molecule, chemical modifications and target, base pairing will lead alternatively to splicing modifications of primary transcript RNA or transient messenger RNA degradation or non-translation. The key to success for neurodegenerative diseases also depends on the ability to reach target cells. The most advanced antisense therapies under development in neurological disorders are presented here, at the clinical stage of development, either at phase 3 or market authorization stage, such as in spinal amyotrophy, Duchenne muscular dystrophy, transthyretin-related hereditary amyloidosis, porphyria and amyotrophic lateral sclerosis; or in earlier clinical phase 1 B, for Huntington's disease, synucleinopathies and tauopathies. We also discuss antisense therapies at the preclinical stage, such as in some tauopathies, spinocerebellar ataxias or other rare neurological disorders. Each subtype of antisense therapy, antisense oligonucleotides or interfering RNA, has proved target engagement or even clinical efficacy in patients; undisputable recent advances for severe and previously untreatable neurological disorders. Antisense therapies show great promise, but many unknowns remain. Expanding the initial successes achieved in orphan or rare diseases to other disorders will be the next challenge, as shown by the recent failure in Huntington disease or due to long-term preclinical toxicity in multiple system atrophy and cystic fibrosis. This will be critical in the perspective of new planned applications to premanifest mutation carriers, or other non-genetic degenerative disorders such as multiple system atrophy or Parkinson disease.

Novel insights on nucleopeptide binding: A spectroscopic and in silico investigation on the interaction of a thymine-bearing tetrapeptide with a homoadenine DNA

Nucleopeptides are a class of molecules with numerous applications in the field of therapy, diagnostics and biomaterials development. Despite their nucleobase-decorated nature, their binding to natural nucleic acid targets does not necessarily involve all nucleopeptide bases, as we showed in this study. Here, we present a CD study on the interaction of a dithymine-functionalized tetra-L-serine with a homoadenine DNA (dA12) reporting an interpretation of the experimental data in light of our computational studies based on molecular docking and molecular dynamics (MD), as well as computer-assisted CD interpretation and simulation of the predicted complex structure. The stoichiometry of the complex, emerged by CD titration, accounted for a 1:2 T:A ratio. Hence, we supposed that binding did not involve a full pairing of the complementary bases but a partial thymines engagement. This hypothesis was sustained by the docking and MD simulations performed on the selected ligand and the complementary target of DNA and RNA, used for comparison. The nucleopeptide bound the DNA through a single A-T recognition involving complementary base-pairing, as well as by some interactions between its backbone (and in particular L-serine OH) and the nucleic acid. Overall, this confirmed that nucleopeptides can interact with nucleic acids leaving some of their nucleobases free for establishing further interactions with other biomolecules or for crosslinking in supramolecular structures in aqueous solution. Nevertheless, even though no typical DNA secondary structure is formed after nucleopeptide-binding, this ligand is able to induce a higher degree of structuration in the random deoxyoligonucleotide target as evidenced by CD, MD and CD simulation.

MiR-21 in Human Cardiomyopathies.

miR-21 is a 22-nucleotide long microRNA that matches target mRNAs in a complementary base pairing fashion and regulates gene expression by repressing or degrading target mRNAs. miR-21 is involved in various cardiomyopathies, including heart failure, dilated cardiomyopathy, myocardial infarction, and diabetic cardiomyopathy. Expression levels of miR-21 notably change in both heart and circulation and provide cardiac protection after heart injury. In the meantime, miR-21 also tightly links to cardiac dysfunctions such as cardiac hypertrophy and fibrosis. This review focuses on the miR-21 expression pattern and its functions in diseased-heart and further discusses the feasibility of miR-21 as a biomarker and therapeutic target in cardiomyopathies.

Optimizing training programs and opportunities for professional development in the era of digital pain interventions: A unique opportunity for collaboration

Introduction: New digital health technologies provide accessible adjuncts to alleviating pain in the general population. The advent novel digital pain interventions have resulted in a rapidly evolving learning environment. Improving knowledge and understanding of these digital patient-centric approaches to treating pain is vital for our current practitioners and new cadre of trainees. The objective of this manuscript is to initiate a discussion about digital pain intervention educational needs of residents as well as attendings in PM&R, anesthesia and neurology.
 Methodology: After reviewing Accreditation Council Graduate Medical Education (ACGME) and relevant American Board of Medical Specialties policies and best available evidence, including grey literature, we interviewed a group of practicing physicians in physiatry, anesthesiology and neurology, including program directors, to provide expert opinion, guidance and formulate recommendations on educational requirements, research endeavors, and learning techniques and opportunities in utilizing digital health interventions for management of pain. IRB approval was not required.
 Conclusions: We hope that this manuscript will serve as the basis of designing a comprehensive educational program and outlining opportunities for research that prioritizes optimal care for pain patients and leverages the unique and complementary knowledge base within our fields.
 Key words: Digital pain; Physiatry; anesthesiology; Neurology; Competency; Accreditation Council Graduate Medical education
 Citation: Nasir A, Mahmood SH. Optimizing training programs and opportunities for professional development in the era of digital pain interventions: A unique opportunity for collaboration. Anaesth. pain intensive care 2021;25(1): 78–82.
 D0I: 10.35975/apic.v25i1.1444
 Received: 24 October 2020; Reviewed: 22 November 2020; Accepted: 5 December 2020

Mixed monolayer of a nucleolipid and a phospholipid has improved properties for spectroelectrochemical sensing of complementary nucleobases

A mixed monolayer of 1,2-dipalmitoyl-sn-glycero-3-(cytidine diphosphate) nucleolipid (DG-CDP) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was transferred from the air–water interface onto an gold (1 1 1) electrode surface using the Langmuir-Schaeffer (horizontal touch) technique. Compression isotherms were recorded for the monolayer spread at the air–water interface. These measurements demonstrated that the mixture composition 7:3 of DG-CDP:DPPC displayed reduced repulsive interactions between polar heads and ideal packing of acyl chains. Chronocoulometric experiments were performed to demonstrate stability of this film at the gold–solution interface. Photon polarization modulation infrared reflection absorption spectroscopy showed that the tilt angle of the acyl chains is slightly higher, and the chains are less twisted in the mixed than in the pure nucleolipid monolayer. In the mixed film, the tilt of the cytosine moiety (the sensing element of the monolayer) assumes a large angle with respect to the surface normal, even when the monolayer becomes detached from the gold surface. The mixed monolayer has improved properties for future applications for detection of guanine, its complementary base.

Detection of Mercury Ion with High Sensitivity and Selectivity Using a DNA/Graphene Oxide Hybrid Immobilized on Glass Slides.

Excessive mercury ions (Hg2+) cause great pollution to soil/water and pose a major threat to human health. The high sensitivity and high selectivity in the Hg2+ detection demonstrated herein are significant for the research areas of analytical chemistry, chemical biology, physical chemistry, drug discovery, and clinical diagnosis. In this study, a series of simple, low-cost, and highly sensitive biochips based on a graphene oxide (GO)/DNA hybrid was developed. Hg2+ is detected with high sensitivity and selectivity by GO/DNA hybrid biochips immobilized on glass slides. The performance of the biosensors can be improved by introducing more phosphorothioate sites and complementary bases. The best limit of detection of the biochips is 0.38 nM with selectivity of over 10:1. This sensor was also used for Hg2+ detection in Dendrobium. The results show this biochip is promising for Hg2+ detection.

Identification of the 5′-Terminal Packaging Signal of the H1N1 Influenza A Virus Neuraminidase Segment at Single-Nucleotide Resolution

The genome of the influenza A virus is an eight-segmented negative-strand RNA (vRNA). Progeny vRNAs replicated in the nucleus selectively assemble into a single set of eight different segments, probably in the cytoplasm, and are packaged into progeny virions at the cell membrane. In these processes, a region of approximately 100 nucleotides at both ends of each segment is thought to function as a selective assembly/packaging signal; however, the details of the mechanism, such as the required sequences, are still unknown. In this study, we focused on the 5′-terminus of the sixth neuraminidase gene segment vRNA (Seg.6) to identify the essential sequence for selective packaging. The 5′-terminal region of the A/Puerto Rico/8/34 strain Seg.6 was divided into seven regions of 15 nucleotides each from A to G, and mutations were introduced into each region by complementary base substitutions or synonymous codon substitutions. Mutant viruses were generated and compared for infectious titers, and the relative ratios of the eight segments packaged into virions were measured. We also ascertained whether mutant vRNA was eliminated by competitive packaging with wild-type vRNA. Mutations in the A–C regions reduced infectious titers and eliminated mutant vRNAs by competition with wild-type vRNA. Even under non-competitive conditions, the packaging efficiency of the A or B region mutant Seg.6 was reduced. Next, we designed an artificial vRNA with a 50-nucleotide duplication at the 5′-terminal region. Using this, a virus library was created by randomly replacing each region, which became an untranslated region (UTR), with complementary bases. After selecting proliferative viruses from the library, nine wild-type nucleotides in the A and B regions were identified as essential bases, and we found that these bases were highly conserved in Seg.6 vRNAs encoding the N1 subtype neuraminidase. From these results, we conclude that the identified bases function as the 5′-terminal packaging signal for the N1 subtype Seg.6 vRNA.

Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing.

Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas-9 nuclease makes double-stranded breaks at a site 3 base pair upstream to protospacer adjacent motif, then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value. In medicine, it is being investigated for cancers, HIV, and gene therapy such as sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. The technology is also being utilized in the regulation of specific genes through the advanced modification of Cas-9 protein. However, immunogenicity, effective delivery systems, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. Although CRISPR/Cas-9 becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications, there are still challenges to rub in the practical applications and various improvements are needed to overcome obstacles.

Quantitative image analysis for sensing HIV DNAs based on NaGdF4:Yb,Er@NaYF4 upconversion luminescent probe and magnetic beads

A quantitative image analysis and separation system has been designed for detection of human immunodeficiency virus related DNAs by combining of upconversion luminescence nanoprobe and magnetic beads. • A quantitative image analysis and separation system was designed for detection HIV DNAs. • The image analysis was based on the upconversion luminescence total internal reflection. • The quantitative image analysis shows higher sensitivity and wider detection range. A quantitative image analysis and separation system was designed for detection of human immunodeficiency virus (HIV) related DNAs in the red region by combining of upconversion luminescence nanoprobe and magnetic beads (MBs). Carboxyl-modified NaGdF 4 :Yb,Er@NaYF 4 upconversion nanoparticles (UCNPs) were connected to single-stranded DNA to form a reporter probe, while carboxyl-modified magnetic beads were connected to another single-stranded DNA to form a capture probe. In the presence of target HIV DNAs, NaGdF 4 :Yb,Er@NaYF 4 UCNPs and magnetic beads were conjugated to form UCNPs-DNA-MBs nanocomplexes through DNA-DNA heteroduplexes based on the principle of complementary base pairing. After magnetic separation, the upconversion luminescence intensity of UCNPs-DNA-MBs nanocomplexes was related to the concentration of the target HIV DNAs. The detection methods were investigated by fluorescence spectrometry and quantitative image analysis based on upconversion luminescence total internal reflection (TIR) imaging platform. Compared to the luminescence spectral analysis, the quantitative image analysis shows higher sensitivity (linear range of 0.02−30 nM and detection limit of 0.0102 nM). More importantly, this method only needs 10 μL sample and the analysis time is about 0.1 s. The method combines quantitative image analysis with magnetic separation technology, which has the characteristics of low background interference and high separation efficiency allowing for application to the field of biology.

MiRNA-Based Potential Biomarkers and New Molecular Insights in Ulcerative Colitis.

Ulcerative colitis (UC) is a chronic non-specific inflammatory bowel disease, which usually manifests as abdominal pain, diarrhea and hematochezia. The disease often recurs and is difficult to cure. At present, the pathogenesis is not clear, but it is believed that the disease is caused by a complex interaction among immunity, heredity, environment and intestinal microflora disorders. MicroRNA (miRNA) is endogenous single-stranded non-coding RNA of 17–25 nucleotides (nts). They target the 3'Untranslated Region of a target gene and inhibit or degrade the target gene according to the extent of complementary bases. As important gene expression regulators, miRNAs are involved in regulating the expression of most human genes, and play an important role in the pathogenesis of many autoimmune diseases including UC. Studies in recent years have illustrated that abnormal expression of miRNA occurs very early in disease pathogenesis. Moreover, this abnormal expression is highly related to disease activity of UC and colitis-associated cancer, and involves virtually all key UC-related mechanisms, such as immunity and intestinal microbiota dysregulation. Recently, it was discovered that miRNA is highly stable outside the cell in the form of microvesicles, exosomes or apoptotic vesicles, which raises the possibility that miRNA may serve as a novel diagnostic marker for UC. In this review, we summarize the biosynthetic pathway and the function of miRNA, and summarize the usefulness of miRNA for diagnosis, monitoring and prognosis of UC. Then, we described four types of miRNAs involved in regulating the mechanisms of UC occurrence and development: 1) miRNAs are involved in regulating immune cells; 2) affect the intestinal epithelial cells barrier; 3) regulate the homeostasis between gut microbiota and the host; and 4) participate in the formation of tumor in UC. Altogether, we aim to emphasize the close relationship between miRNA and UC as well as to propose that the field has value for developing potential biomarkers as well as therapeutic targets for UC.

MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy.

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

Electrostatics affects formation of Watson-Crick complex between DNA bases in monolayers of nucleolipids deposited at a gold electrode surface

Chronocoulometry was applied to determine charge in a monolayer of nucleolipid (1,2-dipalmitoyl-sn‑glycero-3-(cytidine diphosphate)) deposited at a gold electrode surface. The immersion method was used to measure the potential of zero charge of the interface (Epzci), which is a sum of charge on the monolayer of the nucleolipid and charge on the gold surface. Photon polarization infrared reflection absorption spectroscopy (PM-IRRAS) was used to determine formation of the Watson-Crick complex between terminal cytidine moiety of the nucleolipid and guanine (its complementary base) added to the solution. The combination of electrochemical and spectroscopic studies allowed one to demonstrate that the Watson-Crick complex is formed when the interface is positively charged. The potential applied to the electrode affects not only the complex formation but also orientation of the cytosine moiety. The complex is formed when the cytosine moiety is oriented assuming a small angle with respect to the electrode surface.

A signal conversion system using binding-induced strand displacement for disease biomarker assay.

Using the principle of binding-induced DNA strand displacement (BINSD), a DNAzyme-powered nanomachine biosensor for multiple biomarkers via magnetic beads-based signal conversion was designed. This sensor can convert multiple biomarker recognition into release of predesigned output nucleic acids tagged with streptavidin proteins (SA-DNA) for activation of DNA nanomachines. In general, we adopted complementary base pairing rules and affinity ligand specific recognition, and three types of signal conversion systems were constructed that realized universal, sensitive, accurate, and specific detection of multiple biomarkers. Taking the advantage of the strong anti-interference capability of magnetic separation, this strategy could be used for detection of various biomarkers in clinical practice.

Structural Landscape of the Transition from an ssDNA Dumbbell Plus Its Complementary Hairpin to a dsDNA Microcircle Via a Kissing Loop Intermediate.

The experimental construction of a double-stranded DNA microcircle of only 42 base pairs entailed a great deal of ingenuity and hard work. However, figuring out the three-dimensional structures of intermediates and the final product can be particularly baffling. Using a combination of model building and unrestrained molecular dynamics simulations in explicit solvent we have characterized the different DNA structures involved along the process. Our 3D models of the single-stranded DNA molecules provide atomic insight into the recognition event that must take place for the DNA bases in the cohesive tail of the hairpin to pair with their complementary bases in the single-stranded loops of the dumbbell. We propose that a kissing loop involving six base pairs makes up the core of the nascent dsDNA microcircle. We also suggest a feasible pathway for the hybridization of the remaining complementary bases and characterize the final covalently closed dsDNA microcircle as possessing two well-defined U-turns. Additional models of the pre-ligation complex of T4 DNA ligase with the DNA dumbbell and the post-ligation pre-release complex involving the same enzyme and the covalently closed DNA microcircle are shown to be compatible with enzyme recognition and gap ligation.

Method offers new approach to DNA data storage

DNA shows promise as a material for data storage because it resists degradation and can pack a lot of information into a small volume. But reading the stored data typically requires sequencing the DNA. Researchers now show that they can use DNA origami—a method that takes advantage of DNA’s complementary base pairs to create desired shapes—and superresolution microscopy to store and read data from arrays of glowing dots without the need for time-consuming sequencing ( Nat. Commun. 2021, DOI: 10.1038/s41467-021-22277-y ). William L. Hughes and George D. Dickinson of Boise State University and their colleagues designed DNA origami sequences that fold into a 2D carpet. Each carpet contains 48 sites that act as the bits—the ones and zeros—of stored data. When the origami folds, some sites have a DNA strand sticking up and others don’t, representing one and zero, respectively. After adding fluorescently labeled DNA that is complementary to the

Terminal Sequence-Specific Interparticle Attraction between DNA Duplex-Carrying Polystyrene Microparticles in Aqueous Salt Solution Assessed by Optical Tweezers.

The dispersion behavior of DNA duplex-carrying colloidal particles in aqueous high-salt solutions shows extraordinary selectivity against the duplex terminal sequence. We investigated the interparticle force between DNA duplex-carrying polystyrene (dsDNA-PS) microparticles in aqueous salt solutions and examined their behavior in relation to the duplex terminal sequences. Force-distance (F-D) curves for a pair of dsDNA-PS particles were recorded with a dual-beam optical tweezers system with the two optically trapped particles closely approaching each other. Interestingly, only 3-5% of the oligo-DNA strands on the dsDNA-PS particles formed a duplex with complementary DNAs, and the F-D curves showed a distinct specificity to the duplex terminal sequences in the interparticle force at a high-NaCl concentration; a clear attraction peak was observed in F-D curves only when the duplex terminal was a complementary base pair. The attractive strength reached 2.6 ± 0.5 pN at 500 mM NaCl and 4.3 ± 1.0 pN at 750 mM NaCl. By sharp contrast, no significant attraction occurred for the particles with mismatched duplex terminals even at 750 mM NaCl. Similar duplex terminal-specificity in the interparticle force was also confirmed for dsDNA-PS particles in divalent MgCl2 solutions. Considering that the duplex terminal sequences on the dsDNA-PS particles showed only a negligible difference in their surface charges under identical salt conditions, we concluded that the interparticle attraction observed only for the dsDNA-PS particles with complementary duplex terminals is attributable to the salt-facilitated stacking interaction between the paired terminal nucleobases (i.e., blunt-end stacking) on the dsDNA-PS surfaces. Our results thus demonstrate the occurrence of a duplex terminal-specific interparticle force between dsDNA-PS particles under high-salt conditions.

Zeta potentials of fluoride anion result in the instability of DNA hairpin

High dose intake of fluoride results in fluorosis and our previous experiments at single-molecule level using magnetic tweezers have proved that fluoride anions destabilize DNA hairpin and its equilibrium critical force decreases with increasing concentration of fluoride anions. One possible mechanism is that a fluoride anion competes with a DNA base and forms a hydrogen bond with its complementary base. The Isothermal Titration Calorimetry experiments suggest that the fluoride anion does not form a hydrogen bond with a DNA base. With increasing concentration of fluoride anions, decreasing Zeta potentials for different oligonucleotides enhance the electrostatic repulsion between complementary oligonucleotides, which accounts for the decreasing association constant and destabilization of DNA hairpin. Molecule dynamics simulation was further performed to determine the ion distribution around the oligonucleotide, radial distribution functions (RDFs) indicate that the fluoride anion distributes far from the oligonucleotide in comparison with the chloride anion, which is the possible mechanism that decreases Zeta potentials with the increasing concentration of fluoride anions.

Directional observations of guanine and cytosine pairing structures on HOPG

Abstract The molecular self-assembly of deoxyribonucleic acid (DNA) plays an important role in understanding how nucleic acids form and can be exploited to construct nanostructures. In this study, scanning tunneling microscopy (STM) was used to observe nanostructures that form from co-adsorption of the guanine (G) and cytosine (C) complementary DNA bases on a highly oriented pyrolytic graphite surface. The high-resolution STM images show that the well-ordered co-adsorption structures are attributed to the rows formed from Watson–Crick G–C pairs, which are distinct from the structures observed for individual G–C components. The observed co-adsorption structure was modeled by density functional theory calculations, which provide information on the intermolecular interactions underlying its formation.

Analysis of small and large subunit rDNA introns from several ectomycorrhizal fungi species.

The small (18S) and large (28S) nuclear ribosomal DNA (rDNA) introns have been researched and sequenced in a variety of ectomycorrhizal fungal taxa in this study, it is found that both 18S and 28S rDNA would contain introns and display some degree variation in size, nucleotide sequences and insertion positions within the same fungi species (Meliniomyces). Under investigations among the tested isolates, 18S rDNA has four sites for intron insertions, 28S rDNA has two sites for intron insertions. Both 18S and 28S rDNA introns among the tested isolates belong to group I introns with a set of secondary structure elements designated P1-P10 helics and loops. We found a 12 nt nucleotide sequences TACCACAGGGAT at site 2 in the 3'-end of 28S rDNA, site 2 introns just insert the upstream or the downstream of the12 nt nucleotide sequences. Afters sequence analysis of all 18S and 28S rDNA introns from tested isolates, three high conserved regions around 30 nt nucleotides (conserved 1, conserved 2, conserved 3) and identical nucleotides can be found. Conserved 1, conserved 2 and conserved 3 regions have high GC content, GC percentage is almost more than 60%. From our results, it seems that the more convenient host sites, intron sequences and secondary structures, or isolates for 18S and 28S rDNA intron insertion and deletion, the more popular they are. No matter 18S rDNA introns or 18S rDNA introns among tested isolates, complementary base pairing at the splicing sites in P1-IGS-P10 tertiary helix around 5'-end introns and exons were weak.