Improved Anti-Prion Nucleic Acid Aptamers by Incorporation of Chemical Modifications.

Nucleic acid aptamers are innovative and promising candidates to block the hallmark event in the prion diseases, that is the conversion of prion protein (PrP) into an abnormal form; however, they need chemical modifications for effective therapeutic activity. This communication reports on the development and biophysical characterization of a small library of chemically modified G-quadruplex-forming aptamers targeting the cellular PrP and the evaluation of their anti-prion activity. The results show the possibility of enhancing anti-prion aptamer properties through straightforward modifications.

Development and structural determination of an anti-PrP aptamer that blocks pathological conformational conversion of prion protein.

Prion diseases comprise a fatal neuropathy caused by the conversion of prion protein from a cellular (PrP) to a pathological (PrP) isoform. Previously, we obtained an RNA aptamer, r(GGAGGAGGAGGA) (R12), that folds into a unique G-quadruplex. The R12 homodimer binds to a PrP molecule, inhibiting PrP-to-PrP conversion.

RNA sequence and length contribute to RNA-induced conformational change of TLS/FUS.

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained.

The anti-prion RNA aptamer R12 disrupts the Alzheimer's disease-related complex between prion and amyloid β.

The neurodegenerative disorder Alzheimer's disease (AD) is associated with the accumulation of misfolded proteins. Some recent studies suggested that amyloid beta (Aβ) forms soluble oligomers, protofibrils, and fibrils; the Aβ oligomers being more toxic than the fibrils. Surprisingly, these Aβ oligomers reportedly bind to prion protein (PrP), which acts as a receptor on the cell membrane, possibly resulting in AD.

Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5-10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection.

Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA.

The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA.

DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease.

Development of an RNA aptamer that acquires binding capacity against HIV-1 Tat protein via G-quadruplex formation in response to potassium ions.

For the development of K-responsive RNA aptamers, we proposed a new general strategy that makes use of a G-quadruplex formation in response to K. This is the first report of developing an RNA aptamer that demonstrates ON/OFF switching of its target-binding activity by sensing the addition/removal of K.

Arid5a regulates naive CD4+ T cell fate through selective stabilization of Stat3 mRNA.

Balance in signal transducer and activator of transcription (STAT) activation is a key factor in regulating the fate of naive CD4(+)T cells. Here, we demonstrate that AT-rich interactive domain-containing protein 5a (Arid5a) in T cells directs naive CD4(+)T cells to differentiate into inflammatory CD4(+)T cells, especially Th17 cells, through selective stabilization of Stat3(but not Stat1 and Stat5) mRNA in an IL-6-dependent manner. Loss of Arid5a in T cells led to reduction of STAT3 level under Th17-polarizing conditions, whereas STAT1 and STAT5 in Arid5a-deficient T cells were highly activated compared with those of WT T cells under the same conditions.

The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1.

Background: Translocated in LipoSarcoma (TLS, also known as FUsed in Sarcoma) is an RNA/DNA binding protein whose mutation cause amyotrophic lateral sclerosis. In previous study, we demonstrated that TLS binds to long noncoding RNA, promoter-associated ncRNA-D (pncRNA-D), transcribed from the 5' upstream region of cyclin D1 (CCND1), and inhibits the expression of CCND1.

Results: In order to elucidate the binding specificity between TLS and pncRNA-D, we divided pncRNA-D into seven fragments and examined the binding with full-length TLS, TLS-RGG2-zinc finger-RGG3, and TLS-RGG3 by RNA pull down assay.

K(+)-Responsive off-to-on switching of hammerhead ribozyme through dual G-quadruplex formation requiring no heating and cooling treatment.

Functional RNAs that switch their activities in response to K(+) may sense the intracellular (100 mM) and extracellular (5 mM) K(+) concentrations and regulate their functions accordingly. Previously, we developed a quadruplex hammerhead ribozyme (QHR) whose conformational change, from a duplex to a G-quadruplex, triggered by K(+) results in expression of the activity. However, this QHR required heating and cooling treatment (annealing) to induce the K(+)-responsive conformational change and activity.

Bifacial Base-Pairing Behaviors of 5-Hydroxyuracil DNA Bases through Hydrogen Bonding and Metal Coordination.

A novel bifacial ligand-bearing nucleobase, 5-hydroxyuracil (U(OH) ), which forms both a hydrogen-bonded base pair (U(OH) -A) and a metal-mediated base pair (U(OH) -M-U(OH) ) has been developed. The U(OH) -M-U(OH) base pairs were quantitatively formed in the presence of lanthanide ions such as Gd(III) when U(OH) -U(OH) pairs were consecutively incorporated into DNA duplexes. This result established metal-assisted duplex stabilization as well as DNA-templated assembly of lanthanide ions.

Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts.

DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine.

Boosting of activity enhancement of K(+)-responsive quadruplex hammerhead ribozyme.

Two second-generation quadruplex hammerhead ribozymes, whose activity enhances in response to K(+)via quadruplex formation of embedded r(GGA)3GG, were developed. Different strategies were applied to suppress basal activity when K(+) is absent. As a result, the activity enhancement upon the addition of K(+) has reached as high as 21-fold.

Binding of an RNA aptamer and a partial peptide of a prion protein: crucial importance of water entropy in molecular recognition.

It is a central issue to elucidate the new type of molecular recognition accompanied by a global structural change of a molecule upon binding to its targets. Here we investigate the driving force for the binding of R12 (a ribonucleic acid aptamer) and P16 (a partial peptide of a prion protein) during which P16 exhibits the global structural change. We calculate changes in thermodynamic quantities upon the R12-P16 binding using a statistical-mechanical approach combined with molecular models for water which is currently best suited to studies on hydration of biomolecules.

Structure of perosamine-containing polysaccharide, a component of the sheath of Thiothrix fructosivorans.

A sheath-forming and sulfur-oxidizing bacterium, Thiothrix fructosivorans, was heterotrophically cultured. The sheath, which is an extracellular microtube, was prepared by selectively removing the cells using lysozyme, sodium dodecyl sulfate, and sodium hydroxide. Solid-state (13)C-nuclear magnetic resonance (NMR) spectrum revealed that the sheath is assembled from a glycan possessing acetyl and methyl groups.

Anti-prion activity of an RNA aptamer and its structural basis.

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases.

Conformational analysis of an extracellular polysaccharide produced by Sphaerotilus natans.

Sphaerotilus natans is a filamentous sheath-forming bacterium, commonly found in bulking activated sludge. The bulky nature of this bacterium is caused by an extracellular polysaccharide (EPS). EPS is a linear acidic polysaccharide with the following chemical structure: [ → 4)-α-D-Glcp-(1 → 2)-β-D-GlcpA-(1 → 2)-α-L-Rhap-(1 → 3)-β-L-Rhap-(1 → ](n).

'Intelligent' ribozyme whose activity is altered in response to K+ as a result of quadruplex formation.

The structure of r(GGAGGAGGAGGA) (R12) changes from a single-stranded form to a compact quadruplex one in response to K(+). In a hammerhead ribozyme, two portions of the catalytic core are linked with the stem and are located in close proximity in order to exert activity. In this study, the stem was replaced by R12 (or R11, which lacks the terminal A residue) with or without linker residues.

Presence of alternating glucosaminoglucan in the sheath of Thiothrix nivea.

A sheath-forming sulfa oxidizer, Thiothrix nivea, was mixotrophically cultured in a medium supplemented with acetic acid and sodium disulfide. Its sheath, a microtube-like extracellular supermolecule, was prepared by selectively removing the cells with lysozyme, sodium dodecyl sulfate, and sodium hydroxide. The sheath was not visibly affected by hydrazine treatment, suggesting that it is not a proteinous supermolecule.

Structural aspects for the recognition of ATP by ribonucleopeptide receptors.

A modular structure of ribonucleopeptide (RNP) affords a framework to construct macromolecular receptors and fluorescent sensors. We have isolated ATP-binding RNP with the minimum of nucleotides for ATP binding, in which the RNA consensus sequence is different from those reported for RNA aptamers against the ATP analogues. The three-dimensional structure of the substrate-binding complex of RNP was studied to understand the ATP-binding mechanism of RNP.

Study of a novel glycoconjugate, thiopeptidoglycan, and a novel polysaccharide lyase, thiopeptidoglycan lyase.

A typical filamentous bacterium, Sphaerotilus natans, secretes a thiolic glycoconjugate which is assembled into a microtube, so called sheath. The glycoconjugate is known to consist of a pentasaccharide-dipeptide repeating unit, but its chemical structure has not been completely elucidated. In order to determine its chemical structure, the sheath was broken down by performic acid oxidation.

Solubilization and structural determination of a glycoconjugate which is assembled into the sheath of Leptothrix cholodnii.

The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already determined, its modifications with amino acids and other molecules are not fully resolved. In this study, we aimed to determine the chemical structure of the glycoconjugate as a whole.