Nucleic acid with guanidinium modification exhibits efficient cellular uptake.
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DNA↗
Biomedical subjects
Tatsuo Ohmichi
Publications and source records attributed to Tatsuo Ohmichi.
DNA-based biosensor for monitoring pH in vitro and in living cells.
DNA is a promising material for the construction of a biosensor or bioindicator because its structure is sensitive to the binding of cofactors. In the current studies, we found that a combination of two DNA oligonucleotides, 5'-TCTTTCTCTTCT-3' and 5'-AGAAAGAGAAGA-3', exhibit a novel structural transition from a Watson-Crick antiparallel duplex to a parallel Hoogsteen duplex as the pH changes from pH 7.0 to 5.0. By labeling this DNA for fluorescence resonance energy transfer, we were able to develop a sensitive pH indicator that can detect changes between pH 7.0 and 5.0. Moreover, using DNA-based hairpin parallel-stranded duplex in conjunction with fluorescence microscopy, we were able to observe the pH changes in living cells during apoptosis as an easily detected change in color. These results indicate that the DNA-based pH indicator should be useful for detecting pH changes between pH 7.0 and 5.0 in living cells.
Base Pairing↗
A nano-circular single-stranded DNA as a novel tool for SNPs detection.
Analysis of single nucleotides polymorphisms (SNPs) is very important for the elucidation of gene-based physiological differences. For high-throughput applications, detection systems need to be highly selective, highly sensitive, and simple. In this study, we investigated whether synchronous transcription and translation from nano-circular single-stranded DNA (ssDNA) might be useful for the detection of SNPs. A nano-circular probe ssDNA was designed to contain codons for a (His)6 peptide, and the sense mRNA transcribed from this ssDNA contains a Shine-Dalgarno sequence, a start AUG codon, 6 His codons (CAU), and a stop UAG codon. The entire circular ssDNA is 55 nt and contains T20 as a linker sequence and a binding site for the target SNP-containing DNAs. Our results that show SNPs can be detected by the cell-free synthesis of the (His)6 peptide from this ssDNA. Because this method allows sequence distinction, signal amplification, and easy detection in one system, it should improve the efficacy of high-throughput gene analysis.
Base Pair Mismatch↗
The effect of molecular crowding with nucleotide length and cosolute structure on DNA duplex stability.
The thermodynamics of DNA duplex structures in the presence of high concentrations of cosolutes in solution were investigated to discern nucleic acid structures and functions in living cells. In the presence of ethylene glycol (EG) and poly(ethylene glycol) (PEG) (MW = 200-8000), the stability of the oligomer DNA duplexes with differing nucleotide length varied, depending on the nucleotide length as well as the size of PEG. It was also revealed that the decrease of water activity is the primary factor for destabilization of the short (8-mer) duplex by addition of high molecular weight PEGs as well as low molecular weight PEGs and other low molecular weight cosolutes. In addition, the number of water molecules taken up per base pair formation was the same for all the PEGs and for 1,2-dimethoxyethane, which was greater than in the cases of glycerol, EG, 1,3-propanediol, and 2-methoxyethanol, suggesting that the solvation of nucleotides may differ, depending on the cosolute structure. These findings are useful not only for understanding nucleic acid structures and functions in cells but also for the design of oligonucleotides applicable for cells, such as antisense nucleic acids, RNAi, and DNA chips.
DNA↗
Stabilization of a DNA duplex under molecular crowding conditions of PEG.
A living cell generally contains macromolecules occupying 20-40% of the total volume. To mimic the crowded cellular condition, we prepared solutions including poly(ethylene glycol) (PEG) as a cosolute and investigated the influence of the cosolute on the DNA duplex stability. In the presence of PEG 200 or PEG 8000, the Tm (melting temperature) of a self-complementary duplex of 5'-dATGCGCAT-3' decreased by 11.8 degrees C in the presence of 20 wt% PEG200 and by 1.5 degrees C in the presence of 20 wt% PEG 8000. The dln K(obs) vs. dln a(w) plots for PEG 200 and PEG 8000 were linear with a negative slope, suggesting the association of water molecules upon the duplex formation. Interestingly, when the NaCl concentration decreased from 1 M to 400 mM, the Tm increased in the presence of PEGs. Our results imply that the nucleic acid stabilities in a living cell may be different from those in in vitro conditions.
Base Pairing↗
A novel approach of cell-free peptide synthesis and SNPs detection using nano-circular single-stranded DNA.
Development of peptide/protein synthesis is very important for investigating the functions of peptides and proteins. The advantages of cell-free synthesis, which has been extensively developed, include the ability to synthesize toxic proteins, aggregated proteins and proteins containing unnatural amino acids. However, this promising approach does not have a high reaction yield. In this study, we examine whether the rolling synchronization system, which produces long RNAs with a repeated sequence encoded by very small circular ssDNAs without the promoter sequence for RNA polymerase, can be used for cell-free peptide synthesis to improve the reaction yield. As a result, we have found that rolling synchronization is useful for large-scale cell-free peptide/protein synthesis.
Base Sequence↗
Long RNA dangling end has large energetic contribution to duplex stability.
Long terminal unpaired nucleotides known as dangling ends play interesting roles in biological systems. Previous studies, however, only dealt with the energy contributions of single dangling bases. The energy contributions of long dangling ends on the stability of duplexes have not been systematically studied. We now report a quantitative increase in stability of RNA-RNA and DNA-DNA duplexes containing a long dangling end. We found a larger enhancement of the stability by the long RNA dangling end of the RNA-RNA duplex than has been observed for the DNA duplexes. It is also found that structural stabilizations by long dangling ends seem to originate from the single-stranded stacking interactions of nucleotides. These results indicate that RNA stability can be achieved by increasing the length of the dangling end. The thermodynamic parameters of the long dangling ends are useful for designing ribozymes and antisense oligonucleotides, and for the prediction of the RNA secondary structure like the pseudoknot.
DNA↗
Immobilized small deoxyribozyme to distinguish RNA secondary structures.
The RNA folding variation due to one or more mutations leads to different RNA splicing, RNA processing, and translational controls as a result of differences in the primary and higher-ordered structures that interact with other cellular molecules. Thus, distinguishing RNA folding is one of the guides to detect the gene functions related to disease and drug responses. We found, previously, a small Ca(2+)-dependent deoxyribozyme with its site-specific RNA cleavage [Sugimoto, N., Okumoto, Y., and Ohmichi, T. (1999) J. Chem. Soc., Perkin Trans. 2, 1382-1388]. In this study, we report the potential of this deoxyribozyme as a useful tool to distinguish RNA foldings. It is found that the immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The systematic design for the target RNA hairpin loops shows that the immobilized deoxyribozyme is able to cleave them with a > or =17 nucleotide loop size at only one site under single-turnover conditions. Furthermore, an RNA cleavage reaction is detected using the immobilized deoxyribozyme on a surface plasmon resonance (SPR) sensor chip. These results show that the immobilized deoxyribozymes on a column and on an SPR sensor chip become a novel and useful tool to distinguish the RNA foldings.
Base Sequence↗
Effect of secondary structure of short double-stranded RNA on RNAi efficiency.
The dangling nucleotides are very important for the RNA interference (RNAi). We investigate the effect of the dangling end on both the thermodynamic property of the RNA helix and efficiency of gene regulation in RNAi pathway. The result indicates that the four dangling adenines, which are more stable than two or three dangling residues, is not suitable for the RNAi efficiency.
Gene Expression Regulation↗
Efficient bacterial transcription of DNA nanocircle vectors with optimized single-stranded promoters.
We describe experiments aimed at establishing whether circular single-stranded DNAs can form promoters for bacterial transcription from small folded motifs. In vitro selection experiments were carried out on circular 103-nt DNA libraries encoding 40-nt randomized sequences as well as self-processing hammerhead ribozymes. Rounds of rolling circle transcription, reverse transcription-PCR, and recyclization were carried out to optimize transcription efficiency. Sequences were identified that are 80-fold more actively transcribed than the initial library by E. coli RNA polymerase (RNAP). The selected motifs were found to be more active than canonical E. coli promoters in the same context. Experiments also demonstrated that a single-stranded pseudopromoter identified by this selection can be transplanted to other circular DNA contexts and retain transcriptional activity. Results suggest that the promoter is localized in a short ( approximately 40 nt) hairpin, which is smaller than canonical E. coli promoters. To test whether this pseudopromoter was active in bacterial cells, a synthetic DNA nanocircle vector encoding a ribozyme targeted to a site in the marA drug resistance gene was constructed to contain an optimized single-stranded promoter. It is shown that this DNA circle can act as a "Trojan horse" in E. coli, being actively transcribed by the cellular RNAP and producing ribozymes that cleave a sequence in the marA drug resistance gene. The use of optimized single-stranded promoters in combination with synthetic nanocircle DNA vectors represents a potentially useful way to engender the synthesis of biologically active RNAs in living cells.
Bacterial Physiological Phenomena↗