Extended x-ray absorption fine structure study on the cerium(IV)-induced DNA hydrolysis: Implication to the roles of 4f orbitals in the catalysis

At various Ce(IV)/DPP (DPP: diphenyl phosphate) molar ratios, the structure of Ce clusters in aqueous solutions was studied by extended x-ray absorption fine structure measurements. Although the Ce–Ce bond was strong in the absence of DPP, the corresponding signal was significantly reduced when the Ce(IV)/DPP molar ratio was 1. The result indicates the existence of a strong interaction between Ce(IV) ions and DPP, and suggests the formation of a Ce–DPP structure in solution. With an increase in the molar ratio, the signal of Ce–Ce bonds increased again, and exceeded that observed in the absence of DPP, suggesting the formation of a Ce–Ce–DPP structure. Ce(IV) ions in the complexes were found to have ~ 0.67 4f electrons. On the other hand, no measurable charge transfer was observed in the case of Ce(III) ions. These results demonstrate the role of the 4f orbital of Ce(IV) ions for its enormous activity for DNA hydrolysis

Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification

A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (∼60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays

Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal reaction format. In the presence of target RNA, 3WJ probes (primer and template) are designed to form a 3WJ structure, from which multiple signal primers for the following PG-RCA can be generated by repeating primer extension, nicking and signal primer dissociation. Although this signal primer generation is a linear amplification process, the PG-RCA exponentially can amplify these signal primers and thus even a very small amount of RNA specimen can be detected. After optimizing the structures of 3WJ probes, the detection limit of this assay was 15.9 zmol (9.55 × 103 molecules) of synthetic RNA or 143 zmol (8.6 × 104 molecules) of in vitro transcribed human CD4 mRNA. Further, the applicability of this assay to detect CD4 mRNA in a human mRNA sample was demonstrated

Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this ‘double-duplex invasion’, a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the α-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G–C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes

Applications of PNA-Based Artificial Restriction DNA Cutters

More than ten years ago, artificial restriction DNA cutters were developed by combining two pseudo-complementary peptide nucleic acid (pcPNA) strands with either Ce(IV)/EDTA or S1 nuclease. They have remarkably high site-specificity and can cut only one predetermined site in the human genome. In this article, recent progress of these man-made tools have been reviewed. By cutting the human genome site-selectively, desired fragments can be clipped from either the termini of chromosomes (telomeres) or from the middle of genome. These fragments should provide important information on the biological functions of complicated genome system. DNA/RNA hybrid duplexes, which are formed in living cells, are also site-selectively hydrolyzed by these cutters. In order to further facilitate the applications of the artificial DNA cutters, various chemical modifications have been attempted. One of the most important successes is preparation of PNA derivatives which can form double-duplex invasion complex even under high salt conditions. This is important for in vivo applications, since the inside of living cells is abundant of metal ions. Furthermore, site-selective DNA cutters which require only one PNA strand, in place of a pair of pcPNA strands, are developed. This progress has opened a way to new fields of PNA-based biochemistry and biotechnology

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates

Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination

<p>By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.</p