ABSTRACT
Mercury pollution poses a severe threat to human health. To remove Hg(2+) from contaminated water, we synthesized Hg(2+)-trapping beads that include oligo-thymidine functionalities that can form thymine-Hg(II)-thymine base pairs on the solid support. The beads can selectively trap Hg(2+) even in the presence of other metal cations. More interestingly, Hg(2+)-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg(2+)-trapping beads can capture Hg(2+) without affecting the mineral balance of water so much. The Hg(2+)-trapping beads presented here show promise for removing Hg(2+) from environmental water.
Subject(s)
Mercury/chemistry , Thymine/chemistry , Water Pollutants, Chemical/chemistry , Base Pairing , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistryABSTRACT
A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, 'isotopically labeled guanosine 5'-monophosphate' (5'-GMP), steadily gave a 5'-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5'-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with (15)N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry.
Subject(s)
Isotope Labeling/methods , RNA/chemistry , DNA Ligases , Introns , Nuclear Magnetic Resonance, Biomolecular , RNA, Catalytic/chemistryABSTRACT
HAC1 is a transcription factor related to Unfolded Protein Response (UPR) signaling in yeast. Processing of HAC1 mRNA on Endoplasmic reticulum (ER) plays a key role in UPR signaling pathway, but the recognition mechanism of HAC1 mRNA by processing enzyme Ire1p is still unclear. Here, the solution structure of HAC1 mRNA was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy, focusing on the structure of the recognition site of Ire1p in HAC1 mRNA. From the NOESY spectrum, imino proton signals of 5' processing regions of HAC1 mRNA were assigned and it was found that this region forms the stem-loop structure.
Subject(s)
Basic-Leucine Zipper Transcription Factors/genetics , Endoribonucleases/metabolism , Membrane Glycoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/chemistry , Repressor Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Base Sequence , Basic-Leucine Zipper Transcription Factors/metabolism , Consensus Sequence , Nuclear Magnetic Resonance, Biomolecular , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Repressor Proteins/metabolismABSTRACT
Recently, in hammerhead ribozymes, newly identified loop-loop interaction was found to be important for their activation. Therefore, we chemically synthesized a hammerhead ribozyme with this extra loop sequences and its mutant ribozymes, as well as their substrate RNA strands in order to clarify their cleavable sequences. After purification with an anion exchange column chromatography, we were able to obtain 44mer and 20mer RNA.
Subject(s)
RNA, Catalytic/chemistry , Base Sequence , Molecular Sequence Data , Mutation , RNA/chemistry , RNA/metabolism , RNA, Catalytic/chemical synthesis , RNA, Catalytic/metabolismABSTRACT
The chemical structure of the mercury-mediated T-T pair (T-Hg(I)I-T) was determined with (15)N NMR spectroscopy. In order to determine the chemical structure of the T-Hg(I)I-T pair, (15)N-(15)N J-coupling across a metal center (2JNN) was employed. Notably, this is the first observation of (2)J(NN) in a biological macromolecule (DNA duplex). This pairing mode was found to be a irregular metal ion-binding mode for DNA and RNA molecules, in which imino proton-metal exchange processes are included. Accordingly, (2)J(NN) is highly important for the determination of the chemical structures of metal-mediated base pairs.
Subject(s)
Mercury/chemistry , Thymine/chemistry , Base Pairing , DNA/chemistry , Nuclear Magnetic Resonance, BiomolecularABSTRACT
Unnatural "L-DNA" containing 2'-deoxy-L-ribose backbone instead of natural 2'-deoxy-D-ribose was synthesized and evaluated by (1)H NMR, CD spectra. Proton ((1)H) NMR spectra of natural DNA (D-TpT) and L-DNA (L-TpT) are fully consistent with each other. In CD spectra, however, spectrum of L-TpT was reversed, relative to that of D-TpT. These results indicate that L-TpT is an isomer with a mirror image configuration of D-TpT. In summary, we have synthesized enantiomeric L-DNA, and the L-DNA would be materials for nanodevices and tools for exploring why the D-isomer of DNA molecules was selected as a bio-molecule.