Distinct Secondary Structure Research Articles

Disordered Conformation with Low Pii Helix in Phosphoproteins Orchestrates Biomimetic Apatite Formation.

The interplay between noncollagenous proteins and biomineralization is widely accepted, yet the contribution of their secondary structure in mineral formation remains to be clarified. This study demonstrates a role for phosvitin, an intrinsically disordered phosphoprotein, in chick embryo skeletal development, and using circular dichroism and matrix least-squares Henderson-Hasselbalch global fitting, unravels three distinct pH-dependent secondary structures in phosvitin. By sequestering phosvitin on a biomimetic 3D insoluble cationic framework at defined pHs, access is gained to phosvitin in various conformational states. Induction of biomimetic mineralization at near physiological conditions reveals that a disordered secondary structure with a low content of PII helix is remarkably efficient at promoting calcium adsorption, and results in the formation of biomimetic hydroxyapatite through an amorphous calcium phosphate precursor. By extending this finding to phosphorylated full-length human recombinant dentin matrix protein-1 (17-513 AA), this bioinspired approach provides compelling evidence for the role of a disordered secondary structure in phosphoproteins in bone-like apatite formation.

Target RNA Secondary Structure Is a Major Determinant of miR159 Efficacy.

In plants, microRNA (miRNA)-target complementarity has long been considered the predominant factor determining the silencing outcome of the miRNA-target interaction, although the efficacy of such interactions have rarely been appraised in plants. Here, we perform in planta silencing efficacy assays on seven Arabidopsis MYB genes, all of which contain conserved miR159-binding sites of analogous complementarity. These genes were found to be differentially silenced by miR159; MYB81, MYB97, MYB101, MYB104, and DUO1 were all poorly silenced, whereas MYB33 and MYB65 were strongly silenced. Curiously, this is consistent with previous genetic analysis defining MYB33 and MYB65 as the major functional targets of miR159. Neither the free energy of miR159-target complementarity, nor miRNA binding site accessibility, as determined by flanking region AU content, could fully explain the discrepancy of miR159 silencing efficacy. Instead, we found that MYB33 and MYB65 were both predicted to contain a distinctive RNA secondary structure abutting the miR159 binding site. The structure is composed of two stem-loops (SLs) that are predicted to form in MYB33/65 homologs of species as evolutionary distant as gymnosperms. Functional analysis found that the RNA structure in MYB33 correlated with strong silencing efficacy; introducing mutations to disrupt either SL attenuated miR159 efficacy, while introducing complementary mutations to restore the SLs, but not the sequence, restored strong miR159-mediated silencing. Therefore, it appears that this RNA secondary structure demarcates MYB33/65 as sensitive targets of miR159, which underpins the narrow functional specificity of Arabidopsis miR159.

A novel frameshift mutation in CX46 associated with hereditary dominant cataracts in a Chinese family.

To investigate the genetic mutations that are associated the hereditary autosomal dominant cataract in a Chinese family. A Chinese family consisting of 20 cataract patients (including 9 male and 11 female) and 2 unaffected individuals from 5 generations were diagnosed to be a typical autosomal dominant cataract pedigree. Genomic DNA samples were extracted from the peripheral blood cells of the participants in this pedigree. Exon sequence was used for genetic mutation screening. In silico analysis was used to study the structure characteristics of connexin 46 (CX46) mutant. Immunoblotting was conduceted for testing the expression of CX46. To determine the involved genetic mutations, 11 well-known cataract-associated genes (cryaa, cryab, crybb1, crybb2, crygc, crygd, Gja3, Gja8, Hsf4, Mip and Pitx3) were chosen for genetic mutation test by using exon sequencing. A novel cytosine insertion at position 1195 of CX46 cDNA (c.1194_1195ins C) was found in the samples of 5 tested cataract patients but not in the unaffected 2 individuals nor in normal controls, which resulted in 30 amino acids more extension in CX46C-terminus (cx46fs400) compared with the wild-type CX46. In silico protein structure analysis indicated that the mutant showed distinctive hydrophobicity and protein secondary structure compared with the wild-type CX46. The immunoblot results revealed that CX46 protein, which expressed in the aging cataract lens tissues, was absence in the proband lens. In contrast, CX50, alpha A-crystallin and alphaB-crystallin expressed equally in both proband and aging cataract tissues. Those results revealed that the cx46fs400 mutation could impair CX46 protein expression. The insertion of cytosine at position 1195 of CX46 cDNA is a novel mutation site that is associated with the autosomal dominant cataracts in this Chinese family. The C-terminal frameshift mutation is involved in regulating CX46 protein expression.

Using sequence signatures and kink-turn motifs in knowledge-based statistical potentials for RNA structure prediction

Kink turns are widely occurring motifs in RNA, located in internal loops and associated with many biological functions including translation, regulation and splicing. The associated sequence pattern, a 3-nt bulge and G-A, A-G base-pairs, generates an angle of ∼50° along the helical axis due to A-minor interactions. The conserved sequence and distinct secondary structures of kink-turns (k-turn) suggest computational folding rules to predict k-turn-like topologies from sequence. Here, we annotate observed k-turn motifs within a non-redundant RNA dataset based on sequence signatures and geometrical features, analyze bending and torsion angles, and determine distinct knowledge-based potentials with and without k-turn motifs. We apply these scoring potentials to our RAGTOP (RNA-As-Graph-Topologies) graph sampling protocol to construct and sample coarse-grained graph representations of RNAs from a given secondary structure. We present graph-sampling results for 35 RNAs, including 12 k-turn and 23 non k-turn internal loops, and compare the results to solved structures and to RAGTOP results without special k-turn potentials. Significant improvements are observed with the updated scoring potentials compared to the k-turn-free potentials. Because k-turns represent a classic example of sequence/structure motif, our study suggests that other such motifs with sequence signatures and unique geometrical features can similarly be utilized for RNA structure prediction and design.

Site‐directed mutagenesis studies of hydrophobic residues in the lid region of T1 lipase

T1 lipase is a potential biocatalyst for industrial application since it is highly thermostable and displays optimal activity at 60–75°C. Structural analysis of T1 lipase shows that its lid region undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation. To study structure/function of this atypical lid, we performed site‐directed mutagenesis on the hydrophobic residues in the lid region. These residues were mutated to hydrophilic ones and biochemical properties of mutants were investigated. Results showed that F181 might be an important residue for enzyme‐substrate binding. Mutants A186S and A190S had 35–50% increase in catalytic efficiencies compared to wild‐type T1, without compromising their functions at high temperatures. In general, mutagenesis did not cause large changes to chain‐length preference in T1 lipase. Mutants A186S and V187N were inactive towards long‐chain pNP esters (p‐Nitrophenyl stearate) and V187N showed lower activity towards long‐chain triacylglycerols than wt T1, which makes it a potential catalyst in dairy industry. Thermostability of mutants were affected at different extent due to the influence of hydrophobic contact between the lid and the protein core. These findings not only shed light on the lipase structure/function relationship but also lay the framework for further engineering to gain more potent, stable, and selective lipases.Practical applications: A thermostable T1 lipase owns an atypical lid and hydrophobic residues in the lid influence its catalytic properties. Here, we screened mutants A186S and A190S with 35–50% increase in catalytic efficiency, which have industrial application in the modification of fats and oils. Moreover, mutant V187N showed lower activity towards long‐chain TAGs than wt T1. This mutant can be a potential biocatalyst in dairy industry which promote generation of short‐chain fatty acids from milk fats to increase flavors in dairy products.The lid region of thermostable T1 lipase undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation (PDB ID: 2DSN, 2W22 [16,17]). Mutagenesis on hydrophobic residues in the lid affect catalytic properties of T1 lipase.

Synthesis, Thermal Stability, Biophysical Properties, and Molecular Modeling of Oligonucleotides of RNA Containing 2'-O-2-Thiophenylmethyl Groups.

Dodecamers of RNA [CUACGGAAUCAU] were functionalized with C2'-O-2-thiophenylmethyl groups to obtain oligonucleotides 10-14 and 17. The modified nucleotides were incorporated into RNA strands via solid-phase synthesis. The biophysical properties of these ONs were used to quantify the effects of this modification on RNA:RNA and RNA:DNA duplexes. A combination of UV-vis and circular dichroism were used to determine thermal stabilities of all strands, which hybridized into A-form geometries. Destabilization of the double stranded RNA was measured as a function of number of consecutive modifications, reflected in decreased thermal denaturation values (ΔTm, ca. 2.5-11.5 °C). Van't Hoff plots on a duplex containing one modification (10:15) displayed a ca. ΔΔG° of +4 kcal/mol with respect to its canonical analogue. Interestingly, hybridization of two modified strands (13:17, containing a total of eight modifications) resulted in increased stability and a distinct secondary structure, reflected in its CD spectrum. Molecular modeling based on DFT calculations shed light on the nature of this stability, with induced changes in the torsional angle δ (C5'-C4'-C3'-O3) and phosphate-phosphate distances that are in agreement with a compacted structure. The described synthetic methodology and structural information will be useful in the design of thermodynamically stable structures containing chemically reactive modifications.

Full-genome based molecular characterization of encephalitis-associated bovine astroviruses

Novel types of astrovirus have been identified recently in association with neurological disease in cattle. Among those viruses is bovine astrovirus CH13 (BoAstV CH13) that has been identified in Switzerland in a cow with encephalitis. Molecular testing by a combination of reverse transcription (RT-) PCR and in situ hybridization (ISH) indicated that astrovirus infection accounts for around one quarter of viral encephalitis cases of unknown etiology in cattle. Yet, it remained to be explored whether these animals were infected by BoAstV CH13 or other astrovirus species. In the present study we sequenced the entire astrovirus genome in brain tissues of eight RT-PCR and/or ISH positive cattle. Phylogenetic comparison of the genomic RNA and the encoded non-structural and structural proteins revealed that all these astrovirus strains were very similar to BoAstV CH13 as well as to a bovine encephalitis strain reported from the USA (BoAstV NeuroS1), and clearly distinct from other previously reported astroviruses. Conserved 5′ and 3′ untranslated regions (UTRs) were predicted to display distinct secondary RNA structures, which likely play a role in viral RNA replication and/or protein translation. Based on these data we propose that BoAstV CH13/NeuroS1 represents a new genotype species within the genus Mammastrovirus. The high degree of similarity between the strains and their relative distance to other genotype species suggest that during evolution some astroviruses acquired factors that specifically contribute to neuroinvasion.

Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17

Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na+/H+ antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. These results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H+ exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family.

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound.

RNA function is determined by its structural organization. The RNA structure consists of the combination of distinct secondary structure motifs connected by junctions that play an essential role in RNA folding. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probing is an established methodology to analyze the secondary structure of long RNA molecules in solution, which provides accurate data about unpaired nucleotides. However, the residues located at the junctions of RNA structures usually remain undetected. Here we report an RNA probing method based on the use of a novel open-paddlewheel diruthenium (OPW-Ru) compound [Ru2Cl2(µ-DPhF)3(DMSO)] (DPhF = N,N′-diphenylformamidinate). This compound has four potential coordination sites in a singular disposition to establish covalent bonds with substrates. As a proof of concept, we have analyzed the reactivity of OPW-Ru toward RNA using two viral internal ribosome entry site (IRES) elements whose function depends on the structural organization of the molecule. Our study suggests that the compound OPW-Ru preferentially attacks at positions located one or two nucleotides away from junctions or bulges of the RNA structure. The OPW-Ru fingerprinting data differ from that obtained by other chemical reagents and provides new information about RNA structure features.

Comparison of hydration behavior and conformational preferences of the Trp-cage mini-protein in different rigid-body water models.

The secondary structure conformational properties and hydration shell metrics of the Trp-cage mini-protein are examined in the folded and unfolded ensembles in mTIP3P, TIP4P, and TIP4P-Ew water models with the CHARMM22 force-field using molecular dynamics simulations at 250 K. Upon changing the water model, the conformational order metrics of the peptide show significant differences in the unfolded rather than in the folded ensemble. The unfolding temperatures for Trp-cage are observed to be around 460, 470, and 430 K in mTIP3P, TIP4P, and TIP4P-Ew, respectively. Upon comparing the results with a previous study on a 16-residue β-hairpin fragment of the 2GB1 protein, the same set of conformational order metrics are found to be insufficient in describing the free energy landscape of peptides having a distinct native secondary structure. However, the hydration shell properties of the peptide have been found to be independent of the sequence of the peptide and it changes in conformation upon unfolding. Our calculations reveal that for a particular water model, the secondary structure preferences in the unfolded ensembles of the two peptides are qualitatively different. The unfolded structures of Trp-cage prefer extended and compact structures in TIP4P-Ew and mTIP3P water, respectively, whereas the β-hairpin peptide prefers extended unfolded structures in mTIP3P. The conformational preferences of the unfolded peptide in a given water model have been found to depend on the peptide sequence, where the binding energies of the water molecules around the polar residues in the unfolded conformations show sensitivity to the multipole moments of the water models. The significance of an accurate description of peptide-solvent interactions in the parametrization of biomolecular force-fields, to obtain an accurate description of conformational preferences, in particular in the unfolded ensembles of proteins, is highlighted.

A case study to estimate the applicability of secondary structures of SSU‐rRNA gene in taxonomy and phylogenetic analyses of ciliates

Phylogenetic studies of ciliates are mainly based on the primary structure information of the nuclear genes. Some regions of the small subunit ribosomal RNA (SSU‐rRNA) gene have distinctive secondary structures, which have demonstrated value as phylogenetic/taxonomic characters. In the current work, we predict the secondary structures of four variable regions (V2, V4, V7 and V9) in the SSU‐rRNA gene of 45 urostylids. Structure comparisons indicate that the V4 region is the most effective in revealing interspecific relationships, while the V9 region appears suitable at the family level or higher. The V2 region also offers some taxonomic information, but is too conserved to reflect phylogenetic relationships at the family or lower level, at least for urostylids. The V7 region is the least informative. We constructed several phylogenetic trees, based on the primary sequence alignment and based on an improved alignment according to the secondary structures. The results suggest that including secondary structure information in phylogenetic analyses provides additional insights into phylogenetic relationships. Using urostylid ciliates as an example, we show that secondary structure information results in a better understanding of their relationships, for example generic relationships within the family Pseudokeronopsidae.

The structure of the folded domain from the signature multifunctional protein ICP27 from herpes simplex virus-1 reveals an intertwined dimer

Herpesviruses cause life-long infections by evading the host immune system and establishing latent infections. All mammalian herpesviruses express an essential multifunctional protein that is typified by ICP27 encoded by Herpes Simplex Virus 1. The only region that is conserved among the diverse members of the ICP27 family is a predicted globular domain that has been termed the ICP27 homology domain. Here we present the first crystal structure of the ICP27 homology domain, solved to 1.9 Å resolution. The protein is a homo-dimer, adopting a novel intertwined fold with one CHCC zinc-binding site per monomer. The dimerization, which was independently confirmed by SEC-MALS and AUC, is stabilized by an extensive network of intermolecular contacts, and a domain-swap involving the two N-terminal helices and C-terminal tails. Each monomer contains a lid motif that can clamp the C-terminal tail of its dimeric binding partner against its globular core, without forming any distinct secondary structure elements. The binding interface was probed with point mutations, none of which had a noticeable effect on dimer formation; however deletion of the C-terminal tail region prevented dimer formation in vivo. The structure provides a template for future biochemical studies and modelling of ICP27 homologs from other herpesviruses.

92 Shaping CRISPR repeat RNA for Cleavage by Cas6 endoribonucleases

Cellular RNAs are processed by endoribonucleases that often recognize distinct sequence or secondary structures within the RNA. A subclass of recently discovered CRISPR repeat RNA in bacteria conta...

A MYLK variant regulates asthmatic inflammation via alterations in mRNA secondary structure.

Myosin light-chain kinase (MYLK) is a gene known to be significantly associated with severe asthma in African Americans. Here we further examine the molecular function of a single-nucleotide polymorphism (SNP), located in the non-muscle myosin light-chain kinase isoform (nmMLCK), in asthma susceptibility and pathobiology. We identified nmMLCK variant (reference SNP: rs9840993, NM_053025: 721C>T, c.439C>T) with a distinct mRNA secondary structure from the other variants. The nmMLCK variant (721C) secondary structure exhibits increased stability with an elongated half-life in the human endothelial cell, and greater efficiency in protein translation initiation owing to an increased accessibility to translation start site. Finally, nmMLCK expression of 721C- and 721T-containing MYLK transgenes were compared in nmMLCK−/− mice and confirmed deleterious effects of nmMLCK expression on asthmatic indices and implicated the augmented influence of MYLK 721C>T (c.439C>T) SNP on asthma severity. The confirmation of the novel mechanism of the regulation of asthmatic inflammation by a MYLK advances knowledge of the genetic basis for asthma disparities, and further suggests the potential of nmMLCK as a therapeutic target. Our study suggests that in addition to altering protein structure and function, non-synonymous SNPs may also lead to phenotypic disparity by altering protein expression.

γδ T cells recognize the insulin B:9–23 peptide antigen when it is dimerized through thiol oxidation

The insulin peptide B:9–23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αβ T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within β cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αβ T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9–23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αβ T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-Ag7. Unlike the unstructured monomeric B:9–23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9–23 antigen.

Hepatitis delta antigen requires a flexible quasi-double-stranded RNA structure to bind and condense hepatitis delta virus RNA in a ribonucleoprotein complex.

The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2'OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.

G‐Quadruplex Structures and Their Interaction Diversity with Ligands

G-Quadruplexes are distinct nucleic acid secondary structures that are formed from G-rich DNA and RNA sequences; they are built around successive G-tetrads of Hoogsteen hydrogen-bonded guanine bases. The existence of G-quadruplex structures has been confirmed in the human telomere and genome, and their biological functions have been demonstrated. Thus, the prospect of using G-quadruplex structures as a novel target for drug design is important. This review focuses on structural polymorphism of G-quadruplexes on the basis of the classification of the strand number of the G-quadruplex formed and its interaction diversity with small molecules (G4-ligands) that display high affinity and, more importantly, selectivity. Also, we outlook some future challenges for G-quadruplex structure and ligand studies. Such information, together with the biological functions of for G-quadruplex, will be of crucial importance for the research and development of drugs that target G-quadruplexes formed from particular genes and human telomeres.

Synthesis and Properties of Polyethylene‐Bound Antioxidants

Four novel well‐defined polyethylene‐bound antioxidants (PE‐bound antioxidants) with distinct secondary structures are synthesized via transesterification reactions between methyl 3‐(3,5‐di‐tert‐butyl‐4‐hydroxyphenyl)propionate and hydroxyl‐terminated polyethylenes. The results of thermal gravimetric analysis under an air atmosphere demonstrate that the thermal stabilities of PE‐bound antioxidants are higher than that of commercial antioxidant Irganox1076 and are strongly related to their chemical structures. Particularly, the most thermally stable PE‐bound antioxidant is the one bearing both primary and secondary antioxidant moieties. The antioxidant activities of the PE‐bound antioxidants are evaluated in polypropylene by their melt flow index. image

Dynamic evolution and immunoreactivity of aptamers binding to polyclonal antibodies against MPT64 antigen of Mycobacterium tuberculosis

Antibody responses can be useful markers of tuberculosis infection. However, the established immunoassay diagnostic method is limited by antigenic variability. Replacing the recombinant proteins with aptamers may overcome these antigenic challenges. In this study, we systematically monitored the selection process of aptamers against anti-MPT64 antibodies of Mycobacterium tuberculosis to obtain more aptamers for developing a multisite system to increase the sensitivity of TB serological diagnosis. Twelve high-affinity aptamers with distinctive secondary structures were obtained by analyzing the dynamic evolution of aptamers against anti-MPT64 antibodies in the process of system evolution of ligands by exponential enrichment (SELEX). Pocket and stem-loops were found to be the basis of these aptamers binding to antibodies. Point mutations of highly conserved nucleotides in the pocket and stem-loop structures resulted in decreased affinity of aptamers to targets. To test the potential of these aptamers for future use in a serological diagnostic tool, three high-affinity aptamers with different epitope specificities were applied as capture aptamer in an enzyme-linked immunosorbent assay (ELISA) with sera of TB patients. The results showed that three aptamers all effectively bound anti-MPT64 antibodies from TB patients and had high specificity and sensitivity. These aptamers with high immunoreactivity in human sera may represent an efficient and promising analogue of MPT64 and have potential to substitute MPT64 as a nucleic acid antigen in the serological diagnosis of TB. Moreover, these aptamers with different epitope specificities may facilitate the development of a sandwich assay platform or a multisite system to effectively capture more targets in sera.

Strategies for combination of aptamer and targeted drug delivery.

Cell-specific delivery of active agents for treatment of human disease is a long cherished object for scientific researchers. Nanoscience generated nanosized carriers, such as liposome, micelle and nanoparticle, provides potential to realize such purpose based on the nanometer size effect (< 1000 nm), however, the sole nanocarrier with no specific ligands are not sufficient to deliver drugs to target sites. Aptamers are single-stranded oligonucleotides which can specifically recognize and bind to target cells by distinct secondary and tertiary structures even without knowledge of characteristic protein profiles on cell surface. Thus, aptamer, as a target moiety, provides a potential approach to realize pathological site-specific delivery of active agents. This review highlighted the strategies for combination of aptamer and targeted drug delivery, further summarized their preparation methods, strengths and weaknesses to facilitate the development of targeted drug delivery system.