Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes.

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3' UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5' and 3' termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3'-side CL improves nuclease resistance, whereas the 5'-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3' part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Bienzyme reactions on cross-linked DNA scaffolds for electrochemical analysis.

Enzymes play an essential role in various detection technologies. We show here that interstrand cross-linked oligodeoxynucleotides (CL-ODNs) can provide stable scaffolds for efficiently coupling two types of enzymatic reactions on an electrode. Glucose can be electrochemically detected using glucose oxidase (GOx) and horseradish peroxidase (HRP). When both GOx and HRP were immobilized on an electrode surface by attachment at the termini of CL-ODNs, the current value was markedly increased compared with that obtained on a standard ODN scaffold. The relative orientation of the enzymes on the electrode strongly affected the current intensities. The CL-ODN also allowed GOx-HRP to form a complex on the tiny surface of a microelectrode, resulting in the imaging of local glucose distribution. These results suggest that CL-ODNs have potential utility in other sensing technologies.

Development of a 3'-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex.

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5'-termini of oligonucleotides; however, the ssH linker is not appropriate for 3'-terminal modification because it undergoes intramolecular trans-acylation under heat-aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat-alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3'-revH linker was efficiently linked with the 5'-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2'-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3'-terminal modification of oligonucleotides but also expands the utility range in combination with the 5'-ssH linker.

Development of dansyl-modified oligonucleotide probes responding to structural changes in a duplex.

We have synthesized a nonnucleoside amidite block of dansyl fluorophore to prepare dansyl-modified oligonucleotides (ONTs). The fluorescence intensities of dansyl-ONT specifically increased by the presence of adjacent guanosine residues but, significantly reduced in a dansyl-flipping duplex. These changes were caused by solvatochromism effect due to the number of guanine which is hydrophobic functional group and the external environment of dansyl group. The fluorescence intensities could be plotted as a function of the ONTs concentrations and the increase in the fluorescence was observed to equimolar concentrations of target DNA. This duplex exhibited higher melting temperature relative to the corresponding duplexes containing other base pairs. Similar changes in fluorescence could be detected upon hybridization with complementary RNAs. Thus, the dansyl-modified ONTs provide sequence specific fluorescent probe of DNA and RNA.

Development of a scanning electrochemical microscopy-based micropipette and its application to analysis of topographic change of single-cell.

Scanning electrochemical microscopy (SECM) is useful for analyzing various cellular responses. We have combined a micropipette (MP) with SECM to perform quantitative solution delivery to single cells. In this system, since the concentrations of electrochemical mediators are changed by the volume of solution delivered from the MP, we constructed a feedback control system to regulate MP delivery by SECM-detected signals. Cellular responses induced by MP delivery could be monitored by the SECM, and cell apoptosis was successfully detected by adding a kinase inhibitor of two orders of magnitude less than what is required in the conventional method. The SECM-based MP can activate a target cell, requiring a minimal amount of agent, and can continually examine target cell responses. This system improves the accuracy of delivery from the MP and is useful for single-cell analysis.

Electrochemical properties of interstrand cross-linked DNA duplexes labeled with Nile blue.

DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.

Interstrand cross-link of DNA by covalently linking a pair of abasic sites.

A pair of apurinic/apyrimidinic sites formed in DNA has been covalently connected with bis(aminooxy) derivatives. The efficacy of the interstrand cross-link is associated with the structural tethering of two aminooxy groups. The interstrand cross-link constructed stable DNA scaffolds for enzyme alignment.

Comparison of enzymatic recycling electrodes for measuring aminophenol: development of a highly sensitive natriuretic peptide assay system.

Several redox enzymes were examined for enzymatic/electrochemical-recycling systems in order to measure p-aminophenol (PAP) with high sensitivity. Glucose oxidase (GOD) and diaphorase (DI) worked well as catalysts for recycling electrode systems: these enzymes effectively reduced p-iminoquinone (PIQ), the electrochemically-oxidized form of PAP, and caused an enhancement in the electrochemical signals (anodic currents in the voltammogram and amperogram) by approximately 100 fold. The lower detection limits for PAP were estimated to be 50 nM with the GOD system and 2 nM with the DI system. We combined the enzymatic-recycling electrode using DI with an enzyme immunoassay system to measure atrial natriuretic peptide (ANP), an important marker peptide hormone involved in heart diseases. ANPs from serum samples at ppt-levels were determined appropriately using the present assay system.

Construction of time-lapse scanning electrochemical microscopy with temperature control and its application to evaluate the preservation effects of antifreeze proteins on living cells.

Antifreeze proteins (AFPs) can protect cells from hypothermic damage; however, their mechanism of action remains unclear. Scanning electrochemical microscopy (SECM) can evaluate the size and activities of cells, although long-term continuous monitoring has been unsuccessful. We constructed a novel, fully automated, time-lapse SECM system and investigated the cell preservation effect of AFPs by analyzing single cellular topography at low temperatures. From the SECM measurements, mammalian cells (HepG2), treated in Euro-Collins (EC) solution at 4 degrees C, began to swell at 8 h and then immediately ruptured. In AFP-containing EC solution, the cellular size did not change until 16 h and then gradually increased and finally ruptured. In addition, the cellular height at rupture point significantly increased in the presence of AFPs. These results suggest that AFPs stabilize the cellular membrane and protect cells from hypothermic damage. This SECM system allowed us to observe the single cellular response to hypothermia by long-term automatic scanning and will be applicable for analysis to other cellular activities and topographies.