Yasuo Komatsu

Construction of hairpin ribozymes with a three-way junction.

Hairpin ribozymes with high cleavage activities were designed. An extra sequence was introduced at the 3-end of the hairpin ribozyme to increase the binding to the substrate RNA, as compared to the wild-type hairpin ribozyme. A three-way junction (TWJ) was formed between the newly designed ribozyme and the substrate RNA. The complex with a solid TWJ showed less RNA cleavage activity than the wild-type hairpin ribozyme. However, the ribozyme with a TWJ with five unpaired bases or propandiol phosphate linkers had higher cleavage activity than the parent ribozyme without the TWJ. When a cis-cleavage system, in which the 5-end of the substrate RNA was conjugated to the 3-end of the ribozyme, was employed, the complex with the TWJ containing unpaired bases was also cleaved faster than the complex with the solid TWJ. This suggested that these differences in the cleavage activities were derived from the confirmation, and this was proven by nondenaturing gel electrophoresis. The TWJ hairpin ribozyme containing unpaired bases is able to bind strongly with substrate RNAs and to cleave them efficiently. Since the three-way ribozyme presented here is more active than the wild-type ribozyme, this type of ribozyme can serve as a more efficient tool to control RNA activities in vitro and in vivo.

Design and Anti-HIV-1 Activity of Hammerhead and Hairpin Ribozymes Containing a Stable Loop#

Three ribozymes, a hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5C(UUCG)G3 loop were designed to cleave the U5 region in the long terminal repeat (LTR) of HIV-1 RNA. The t1/2 values of chemically synthesized substrates mediated by three ribozymes were measured. The transformed CEM cells possessing these three ribozyme-encoding genes were challenged with a HIV-1IIIB strain, and two of these three ribozymes, HR112 and RZ119, were shown to possess strong anti-HIV-1 activity.

31P NMR study of the interactions between oligodeoxynucleotides containing (6-4) photoproduct and Fab fragments of monoclonal antibodies specific for (6-4) photoproduct

A 31P nuclear magnetic resonance (NMR) study of the interactions between oligonucleotides containing the (6‐4) photoproduct and the Fab fragments of monoclonal antibodies (64M3 and 64M5) recognizing the (6‐4) photoproduct is reported. The 31P chemical shift data indicate that backbone conformation of (6‐4) adduct is affected by the presence of flanking oligodeoxynucleotides, and (6‐4) adducts with different backbone conformations are accommodated in the antigen binding sites of these antibodies. It was also revealed that epitopes for these antibodies consist of not only the (6‐4) adduct but the flanking di‐ or tri‐deoxynucleotides on both the 5′ and 3′ sides as well.

Sequence-specific cleavage of RNA by a hybrid ribonuclease H

Site‐specific cleavage of the 22‐, 132‐ and 534‐base RNAs by the DNA/protein hybrid R Nase H were examined. The 22‐base RNA was chemically synthesized, and 132‐ and 534‐base RNAs were prepared by run‐off transcription. The hybrid enzyme cleaves these RNAs, which contain a single target sequence, primarily at the unique phosphodiester bond within the target sequence. The hybrid enzyme performs multiple turnovers, and at a substrate/enzyme ratio of 10:1 the RNAs are almost completely cleaved by the hybrid enzyme at 37°C within 1 h. We propose that hybrid RNase H molecules with various oligodeoxyribonucleotides function as RNA restriction enzymes and are useful for structural and functional studies of RNA.

Effects of a High‐Affinity Antibody Fragment on DNA Polymerase Reactions Near a (6–4) Photoproduct Site

Abstract— Pyrimidine (6–4) pyrimidone photodimers are major photoproducts that have mutagenic and carcinogenic consequences. One major reason for these biological effects of (6–4) photoproducts may be base mispairing/DNA replication errors due to hydrogen bonding to bases opposite these damaged sites. We synthesized a modified 41‐mer DNA containing a (6–4) photoproduct using a preformed building block, then employed it as a template for primer extension reactions catalyzed by Klenow fragment and DNA polymerases α, β and δ (pol α, pol β and pol δ). None of these DNA polymerases were able to bypass the (6–4) photoproduct and elongation terminated at or near the 3′‐pyrimidone of the photoproduct, depending on the dNTP concentration. When a single‐chain Fv (scFv) with high affinity for the (6–4) photoproduct was included in the polymerization reaction, DNA synthesis was inhibited at base positions four, six, eight or eight nucleotides prior to the 3′‐pyrimidone by Klenow fragment, pol α, pol β or pol δ, respectively. These results suggest that the scFv can bind to the template DNA containing a (6–4) photoproduct and inhibit extension reactions by polymerases.

DNA-binding properties of the antibody specific for the Dewar photoproduct of thymidylyl-(3-5')-thymidine.

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3′-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.

Efficient chemical synthesis of a pyrimidine (6–4) pyrimidone photoproduct analog and its properties

Abstract We synthesized an analog of a pyrimidine (6–4) pyrimidone photoproduct with a formacetal linkage instead of the phosphodiester in the (6–4) photoproduct, and investigated the interaction between a monoclonal antibody specific to the natural (6–4) photoproduct and a DNA containing the photoproduct analog.

Interactions of High Affinity Anti (6-4) Photoproduct Antibody Fragments with Damaged DNA

Abstract The interactions between chemically synthesized DNA fragments containing a T(6-4)T and antigen binding fragments (Fab) or single-chain antibodies (scFv) were investigated by X-ray crystallography, NMR, and surface plasmon resonance. The high affinity scFv protein was found to bind to the template DNA near the (6-4) photoproduct site and to interfere with DNA polymerase reactions in vitro.

Cleavage of Short RNAs Containing Higher Ordered Structures by Hammerhead Ribozymes

Abstract Cleavage of two types of secondary structure-forming substrates by their cognate hammerhead ribozymes were studied by measuring their kinetic parameters. A substrate with a self-complementary structure (GGUCCUAGGA, CL-3) was slowly cleaved by a two-stranded ribozyme. An isomer having no complementary sequence (GGUCGUAGCA, CL-3N) was cleaved more than 10 times faster than the self-complementary substrate. A newly designed ribozyme which contained a stable loop and stem cleaved the self-complementary decamer 40 times faster than the two-stranded ribozyme. A 15 mer which derived from a ras mRNA was found to have an intermolecular base pairs and was used to design more efficient ribozymes. Gel mobility shift assay was employed to investigate the binding properties of substrates to ribozymes. Investigations of the thermodynamic stability of the ribozyme-substrate complex are essential in the design of ribozymes that efficiently cleave RNA. #This paper is dedicated to Professor Y. Mizuno on the occasio...

Biochemical analysis of human tRNAHis guanylyltransferase in mitochondrial tRNAHis maturation

Mitochondria contain their own protein synthesis machinery, which includes mitochondrial tRNA maturation. It has been suggested that mammalian mitochondrial tRNAHis (mtRNAHis) is matured by post-transcriptional addition of guanosine at thexa0-1 position (G-1), which serves as an identity element for mitochondrial histidyl-tRNA synthetase. However, the exact maturation process of mammalian mtRNAHis remains unclear. In cytoplasmic tRNAHis (ctRNAHis) maturation, tRNAHis guanylyltransferase (Thg1) adds a GTP onto the 5-terminal of ctRNAHis and then removes the 5-pyrophosphate to yield the mature 5-monophospholylated G-1-ctRNAHis (pG-1-ctRNAHis). Although mammalian Thg1 is localized to both the cytoplasm and mitochondria, it remains unclear whether mammalian Thg1 plays a role in mtRNAHis maturation in mitochondria. Here, we demonstrated that human Thg1 (hThg1) catalyzes the G-1 addition reaction for both human ctRNAHis and mtRNAHis through recognition of the anticodon. While hThg1 catalyzed consecutive GTP additions to mtRNAHisinxa0vitro, it did not exhibit any activity toward mature pG-1-mtRNAHis. We further found that hThg1 could add a GMP directly to the 5-terminal of mtRNAHis in a template-dependent manner, but fungal Thg1 could not. Therefore, we hypothesized that acceleration of the pyrophosphate removal activity before or after the G-1 addition reaction is a key feature of hThg1 for maintaining a normal 5-terminal of mtRNAHis in human mitochondria. This study provided a new insight into the differences between tRNAHis maturation in the cytoplasm and mitochondria of humans.