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N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5′-DRACH-3′ in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation., Graphical Abstract Graphical AbstractThe RNA methyltransferase METTL3/METTL14 heterodimer was shown to preferentially bind to RNA G-quadruplex (rG4) structures and methylate adenosines close to rG4, providing insights into the role of rG4 in epitranscriptomic regulation.

G-quadruplexes have important biologic functions that are regulated by G-quadruplex-binding proteins. In particular, G-quadruplex structures are folded or unfolded by their binding proteins and affect transcription and other biologic functions. Here, we investigated the effect of the RNA recognition motif (RRM) and arginine-glycine-glycine repeat (RGG) domain of nucleolin on G-quadruplex formation. Our findings indicate that Phe in the RGG domain of nucleolin is responsible for G-quadruplex binding and folding. Moreover, the RRM of nucleolin potentially binds to a guanine-rich single strand and folds the G-quadruplex with a 5'-terminal and 3'-terminal single strand containing guanine. Our findings contribute to our understanding of how the RRM and RGG domains contribute to G-quadruplex folding and unfolding.

Translocated in liposarcoma (TLS)/fused in sarcoma (FUS) is a multitasking DNA/RNA binding protein implicated in cancer and neurodegenerative diseases. Upon DNA damage, TLS is recruited to the upstream region of the cyclin D1 gene (CCND1) through binding to the promotor associated non-coding RNA (pncRNA) that is transcribed from and tethered at the upstream region. Binding to pncRNA is hypothesized to cause the conformational change of TLS that enables its inhibitive interaction with histone acetyltransferases and resultant repression of CCND1 expression, although no experimental proof has been obtained. Here, the closed-to-open conformational change of TLS on binding pncRNA was implied by fluorescence resonance energy transfer. A small fragment (31 nucleotides) of the full-length pncRNA (602 nucleotides) was shown to be sufficient for the conformational change of TLS. Dissection of pncRNA identified the G-rich RNA sequence that is critical for the conformational change. The length of RNA was also revealed to be critical for the conformational change. Furthermore, it was demonstrated that the conformational change of TLS is caused by another target DNA and RNA, telomeric DNA and telomeric repeat-containing RNA. The conformational change of TLS on binding target RNA/DNA is suggested to be essential for biological functions.

Stereoselective and efficient synthesis of Gly-Gly-type (E)-methylalkene and (Z)-chloroalkene dipeptide isosteres is realized by organocuprate-mediated single electron transfer reduction. The synthetic isosteres can be used in Fmoc-based solid phase peptide synthesis, resulting in the preparation of the 14-mer RGG peptidomimetics containing an (E)-methylalkene or a (Z)-chloroalkene unit.

The G-quadruplex structure in the genome is an important drug target because it regulates gene expression and the genome structure. Several small molecules that bind the G-quadruplex have been developed, but few artificial G-quadruplex binding proteins have been reported. We previously reported a novel G-quadruplex DNA binding protein (RGGF) engineered using the Arg-Gly-Gly repeat (RGG) domain of TLS (translocated in liposarcoma). Here we show that RGGF recognizes DNA loops in the G-quadruplex and preferentially binds G-quadruplex DNA with long loops. Furthermore, RGGF binds to G-quadruplex DNA of the c-myc promoter in vitro and represses c-myc transcription in vivo. On the basis of these findings, G-quadruplex binding protein engineered from the RGG domain will be useful for investigating G-quadruplex transcriptional function in the genome.

Coryneazolicin is a plantazolicin family peptide, belonging to linear azole-containing peptides (LAPs). Although coryneazolicin was previously synthesized by in vitro experiments, its biological activity has not been evaluated. In this report, the heterologous production of coryneazolicin was accomplished to obtain enough coryneazolicin for biological activity tests. The structure of coryneazolicin was confirmed by ESI-MS and NMR analyses. The biological activity tests indicated that coryneazolicin possessed potent antibacterial activity and cytotoxicity. Although antibacterial activity of plantazolicin was previously reported, cytotoxicity was newly found in coryneazolicin among plantazolicin type peptides. In addition, we revealed that coryneazolicin induced apoptosis on HCT116 and HOS cancer cell lines.

Peroxidase-proximity protein labeling was performed using a hemin-parallel G-quadruplex (G4) complex. A tyrosine labeling reaction using an N-methyl luminol derivative was accelerated in close proximity to the hemin with enhanced peroxidase activity by binding to parallel G4. The TERRA-hemin complex activated the labeling of many RNA-binding proteins, including heterogeneous nuclear ribonucleoproteins, in a HeLa cell lysate.

Stereoselective and efficient synthesis of Gly-Gly-type (

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles as well as being related to cancer and other genetic diseases. It is therefore important to investigate Z-DNA structure and related biological events in living cells. However, the development of molecular probes for the observation of Z-DNA structures inside living cells has not yet been realized. Here, we have succeeded in developing site-specific trifluoromethyl oligonucleotide DNA by incorporation of 8-trifluoromethyl-2′-deoxyguanosine (FG). 2D NMR strongly suggested that FG adopted a syn conformation. Trifluoromethyl oligonucleotides dramatically stabilized Z-DNA, even under physiological salt concentrations. Furthermore, the trifluoromethyl DNA can be used to directly observe Z-form DNA structure and interaction of DNA with proteins in vitro, as well as in living human cells by19F NMR spectroscopy for the first time. These results provide valuable information to allow understanding of the structure and function of Z-DNA.

Using genome mining, a new cytotoxic peptide named curacozole was isolated from Streptomyces curacoi. Through ESI-MS and NMR analyses, curacozole was determined to be a macrocyclic peptide containing two isoleucine, two thiazole and three oxazole moieties. Curacozole exhibited potent cytotoxic activity against HCT116 and HOS cancer cells. The proposed biosynthetic gene cluster of curacozole was identified and compared with that of the related compound YM-216391.

The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system.

A recent finding demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA (referred to as TERRA) in mammalian cells. The existence of TERRA RNA may reveal a new level of regulation and protection of chromosome ends that could promote valuable insight into fundamental biological processes such as cancer and aging. Revealing the structure and function of telomere RNA will be essential for understanding telomere biology and telomere-related diseases. In fact, others and we have shown that human telomere RNA forms G-quadruplex structures by NMR spectroscopy and X-ray crystallography. In the present study, we employed the 19F NMR spectroscopy to investigate the interaction of RNA G-quadruplex with ligand molecule and protein. We showed that 19F NMR analysis of telomere RNA is useful for identifying the binding of RNA G-quadruplex with ligand molecule. We further demonstrated that 19F NMR can be used to monitor the interaction of RNA G-quadruplex and the telomeric protein TRF2.

We attached D-Ala residues to cross-linked helical peptides based on the pro-apoptotic protein Bad at their C-termini. The D-Ala attachment had little influence on the secondary structures and binding abilities against Bcl-XL. The D-Ala attached helical peptides were much more stable in cells than original ones and efficiently induced apoptosis of the cells.

The functions of local conformations of non-B form DNA and RNA, such as the G-quadruplex, are thought to be regulated by their specific binding proteins. They regulate the formation of G-quadruplexes in cells and affect the biological functions of G-quadruplexes. Recent studies reported that G-quadruplexes regulate epigenetics through these G-quadruplex binding proteins. We discuss regulation of histone modifications through G-quadruplex RNA and its binding proteins which modulate the G-quadruplex conformations. G-quadruplex RNA is involved in telomere maintenance and transcription via histone modification. Furthermore, G-quadruplex binding proteins regulate formation and biological functions of G-quadruplexes through regulating their folding or unfolding. In this review, we will focus on the G-quadruplex binding proteins containing RRM and RGG domains.

The RGG domain, defined as closely spaced Arg-Gly-Gly repeats, is a DNA and RNA-binding domain in various nucleic acid-binding proteins. Translocated in liposarcoma (TLS), which is also called FUS, is a protein with three RGG domains, RGG1, RGG2 and RGG3. TLS/FUS binding to G-quadruplex telomere DNA and telomeric repeat-containing RNA depends especially on RGG3, comprising Arg-Gly-Gly repeats with proline- and arginine-rich regions. So far, however, only non-specific DNA and RNA binding of TLS/FUS purified with buffers containing urea and KCl have been reported. Here, we demonstrate that protein purification using a buffer with high concentrations of urea and KCl decreases the G-quadruplex binding abilities of TLS/FUS and RGG3, and disrupts the β-spiral structure of RGG3. Moreover, the Arg-Gly-Gly repeat region in RGG3 by itself cannot form a stable β-spiral structure that binds to the G-quadruplex, because the proline- and arginine-rich regions induce the β-spiral structure and the G-quadruplex-binding ability of RGG3. Our findings suggest that the G-quadruplex-specific binding abilities of TLS/FUS require RGG3 with a β-spiral structure stabilized by adjacent proline- and arginine-regions.

Human telomere DNA (Htelo) and telomeric repeat-containing RNA (TERRA) are integral telomere components containing the short DNA repeats d(TTAGGG) and RNA repeats r(UUAGGG), respectively. Htelo and TERRA form G-quadruplexes, but the biological significance of their G-quadruplex formation in telomeres is unknown. Compounds that selectively bind G-quadruplex DNA and RNA are useful for understanding the functions of each G-quadruplex. Here we report that engineered Arg-Gly-Gly repeat (RGG) domains of translocated in liposarcoma containing only Phe (RGGF) and Tyr (RGGY) specifically bind and stabilize the G-quadruplexes of Htelo and TERRA, respectively. Moreover, RGGF inhibits trimethylation of both histone H4 at lysine 20 and histone H3 at lysine 9 at telomeres, while RGGY inhibits only H3 trimethylation in living cells. These findings indicate that G-quadruplexes of Htelo and TERRA have distinct functions in telomere histone methylation.

We attached d-Ala residues to cross-linked helical peptides based on the pro-apoptotic protein Bad at their C-termini. The d-Ala attachment had little influence on the secondary structures and binding abilities against Bcl-X

Guanine is the most easily oxidized among the four DNA bases, and some guanine-rich sequences can form quadruplex structures. In a previous study using 6-mer DNA d(TGGGGT), which is the shortest oligomer capable of forming quadruplex structures, we demonstrated that guanine oxidation products of quadruplex DNA differ from those of single-stranded DNA. Therefore, the hotooxidation products of double-stranded DNA (dsDNA) may also differ from that of quadruplex or single-stranded DNA, with the difference likely explaining the influence of DNA structures on guanine oxidation pathways. In this study, the guanine oxidation products of the dsDNA d(TGGGGT)/d(ACCCCA) were analyzed using HPLC and electrospray ionization-mass spectrometry (ESI-MS). As a result, the oxidation products in this dsDNA were identified as 2,5-diamino-4H-imidazol-4-one (Iz), 8-oxo-7,8-dihydroguanine (8oxoG), dehydroguanidinohydantoin (Ghox), and guanidinohydantoin (Gh). The major oxidation products in dsDNA were consistent with a combination of each major oxidation product observed in single-stranded and quadruplex DNA. We previously reported that the kinds of the oxidation products in single-stranded or quadruplex DNA depend on the ease of deprotonation of the guanine radical cation (G•+) at the N1 proton. Similarly, this mechanism was also involved in dsDNA. Deprotonation in dsDNA is easier than in quadruplex DNA and more difficult in single-stranded DNA, which can explain the formation of the four oxidation products in dsDNA.

Mammalian telomeres containing TTAGG repeats are bound by a multiprotein complex with a telomeric repeat-containing RNA (TERRA) containing UUAGGG repeats, which is a long noncoding RNA transcribed from the telomeres. Telomere DNA and TERRA form a G-quadruplex in vitro. The functions of the G-quadruplex structures in the telomere, however, are not clear, because little is known about G-quadruplex specific binding proteins and G-quadruplex RNA-binding molecules without binding to G-quadruplex telomere DNA. We have reported that the Arg-Glu-Gly motif in Translocated in Liposarcoma (TLS) forms G-quadruplex telomere DNA and TERRA simultaneously in vitro. Furthermore, TLS promotes the methylation of hinstone H4 and H3 at lysine and regulates telomere length. These finding suggest that the G-quadruplex functions as a scaffold for telomere-binding protein, TLS. Moreover, we have shown that substitution of Tyr for Phe in the RGG motif of TLS converts its binding specificity solely toward G-quadruplex TERRA. This molecule binds to loops within the G-quadruplexes of TERRA by recognizing the 2′-OH of the riboses. It will be useful for investigating biological roles of the G-quadruplex in long noncoding RNA.

An amphiphilic low-molecular-weight hydrogelator 1 was synthesized. A tris-glycine-SDS solution gel of 1 was applied for electrophoresis to separate proteins. Centrifugation of a mixture of protein and a hydrogel of 1 enabled the recovery of protein. Various combinations of proteins were applied for supramolecular gel electrophoresis (SUGE), and remarkably poor mobility for small proteins (

Amphiphilic tris-urea molecules self-assemble into a supramolecular hydrogel in tris(hydroxymethyl)aminomethane–glycine buffer. The supramolecular hydrogel is used as a matrix for the electrophoresis of acidic native proteins, in which proteins are separated based on their isoelectric points rather than their molecular weights. The proteins remain in their native forms during migration, and their activities are retained after electrophoresis. Glucoside substituents on the amphiphilic tris-urea molecule allow for the affinity electrophoresis of a carbohydrate-binding protein to be performed. The proteins can be efficiently recovered from the supramolecular hydrogel using a simple procedure. This is a major advantage of using this noncovalent, self-assembled material.

Managing protein–protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side-chain-crosslinked helical peptides based on the binding domain of a pro-apoptotic protein (Bad) that induces programed cell death. The peptide showed high helical content and bound to its target, Bcl-XL, more strongly than its non-crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl-XL localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70 % of HeLa cells died by the apoptosis pathway (without evidence of necrosis).

Telomeric repeat-containing RNA (TERRA), which contains tandem arrays of short RNA repeats, r(UUAGGG), is an integral component of the telomere and contributes to telomeric heterochromatin formation and telomere-length regulation. TERRA forms a G-quadruplex, but the biologic significance of its G-quadruplex formation is unknown. Compounds that selectively bind to G-quadruplex RNA are useful for understanding G-quadruplex TERRA. Here we report that an engineered RGG domain translocated in liposarcoma (TLS) specifically binds to G-quadruplex TERRA. The Arg-Gly-Gly repeat (RGG) TLS binds to G-quadruplex human telomere DNA and TERRA simultaneously, but we show that substitution of Tyr for Phe in the RGG domain of TLS (TLSRGG3Y) converts its binding specificity solely toward G-quadruplex TERRA. TLSRGG3Y binds to dG tetrads with abasic RNA loops, but fails to bind to rG tetrads without loops or dG tetrads with abasic DNA loops. These findings suggest that TLSRGG3Y binds to loops within the G-quadruplexes of TERRA by recognizing the 2'-OH of the riboses. To our knowledge, TLSRGG3Y is the first known molecule that specifically recognizes the 2'-OH of the riboses of loops in the G-quadruplex. TLSRGG3Y will be useful for investigating the role of the G-quadruplex form of TERRA without affecting G-quadruplex telomere DNA functions.

Summary Mammalian telomeres comprise noncoding TTAGGG repeats in double-stranded regions with a single-stranded TTAGGG repeat 3′ overhang and are bound by a multiprotein complex with a telomeric repeat-containing RNA (TERRA) containing a UUAGGG repeat as a G-quadruplex noncoding RNA. TLS/FUS is a human telomere-binding protein that was first identified as an oncogenic fusion protein in human myxoid and round-cell liposarcoma. Here, we show that the Arg-Gly-Gly domain in the C-terminal region of TLS forms a ternary complex with human telomere G-quadruplex DNA and TERRA in vitro. Furthermore, TLS binds to G-quadruplex telomere DNA in double-stranded regions and to G-quadruplex TERRA, which regulates histone modifications of telomeres and telomere length in vivo. Our findings suggest that the G-quadruplex functions as a scaffold for the telomere-binding protein, TLS, to regulate telomere length by histone modifications.

Transcription from DNA sequence-specifically alkylated by a hairpin polyamide (ImPyPy-gamma-ImPyLDu86, 1) was investigated. High-resolution polyacrylamide gel electrophoresis demonstrated that conjugate 1 alkylated a 993-bp DNA fragment, in accordance with the Py-Im recognition rule, predominantly at the one match site on the GFP-encoding strand and at four sites (I'-IV') on the noncoding strand. Alkylation of DNA inhibited the formation of full-length mRNA and caused the transcription of truncated mRNA. Polyacrylamide gel electrophoresis demonstrated that the length of the truncated mRNA was consistent with the alkylated site on the coding strand. Complete inhibition of full-length mRNA synthesis was observed in the presence of 50 nM 1. In clear contrast, the hydrolyzed derivative of 1, designated 2, produced no truncated mRNA, nor did it significantly retard transcription:80% transcription of full-length mRNA was observed at 50 nM. These results clearly indicate that inhibition of transcription can be achieved with alkylating Py-Im polyamide even in the coding regions of genes.

The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs), resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma) with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT) activity in human cells. Transcription regulator EWS (Ewing's sarcoma), which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG) repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro. Here, we show that the RGG of EWS binds preferentially to G-quadruplexes with longer loops, which is not related to the topology of the G-quadruplex structure. Moreover, the G-quadruplex DNA binding of the RGG in EWS depends on the phosphate backbone of the loops in the G-quadruplex DNA. We also investigated the G-quadruplex DNA binding activity of the N- and C-terminally truncated RGG to assess the role of the regions in the RGG in G-quadruplex DNA binding. Our findings indicate that the RGG and the other arginine-rich motif of residues 617-656 of the RGG in EWS are important for the specific binding to G-quadruplex DNA. These findings will contribute to the development of molecules that selectively target different G-quadruplex DNA.

The Ewing's sarcoma (EWS) oncogene contains an N-terminal transcription activation domain and a C-terminal RNA-binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich single-stranded DNA and RNA fold in the G-quadruplex structure. Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration-dependent manner by the formation of a stabilized G-quadruplex DNA-RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg-Gly-Gly sites and RGG containing Arg methylated by protein arginine N-methyltransferase 3 decrease the binding ability of EWS to G-quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G-quadruplex DNA and RNA via the Arg residues in it.

Gene silencing was examined by sequence-specific alkylation of DNA by N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides. Polyamides ImImPyPygammaImImPyLDu86 (A) and ImImPyPygammaImPyPyLDu86 (B) selectively alkylated the coding regions of the renilla and firefly luciferases, respectively, according to the base pair recognition rule of Py-Im polyamides. Two different plasmids, encoding renilla luciferase and firefly luciferase, were used as vectors to examine the effect of alkylation on gene silencing. Transfection of the alkylated luciferase vectors-by polyamide A or B-into HeLa, 293, and NIH3T3 cells demonstrated that these sequence-specific DNA alkylations lead to selective silencing of gene expression. Next, the vectors were cotransfected into HeLa cells and the cells were treated with polyamide A or B. Selective reduction of luciferase activities was caused by both polyamides. On the basis of this sequence-specific alkylation and gene silencing activity, these alkylating Py-Im polyamides thus have potential as antitumor drugs to target specific gene expression in human cells.

DNA local conformations are thought to play an important biological role in processes such as gene expression by altering DNA-protein interactions. Although left-handed Z-form DNA is one of the best-characterized and significant local structures of DNA, having been extensively investigated for more than two decades, the biological relevance of Z-form DNA remains unclear. This is presumably due to the lack of a versatile detection method in a living cell. Previously, we demonstrated that the incorporation of a methyl group at the guanine C8 position (m(8)G) dramatically stabilizes the Z-form of short oligonucleotides in a variety of sequences. To develop a photochemical method to detect Z-form DNA, we examined the photoreaction of 5-iodouracil-containing Z-form d(CGCG(I)UGCG)(ODN 1)/d(Cm(8)GCAm(8)GCG)(ODN 2) in 2 M NaCl and found stereospecific C2'alpha-hydroxylation occurred at G(4) to provide d(CGCrGUGCG), 5. Recently, Rich and co-workers [Schwartz et al. Science 1999, 284, 1841. Schwartz et al. Nat. Struct. Biol. 2001, 8, 761] found that an ubiquitous RNA editing enzyme, adenosine deaminase 1 (ADAR1), and tumor-associated protein DML-1 specifically bind to Z-form DNA. In the present study, we investigate the photoreactivity of octanucleotide ODN 1-2 in Z-form induced by Zalpha, which is the NH(2)-terminal domain of ADAR1 responsible for tight binding of ADAR1. Detailed product analysis revealed that the C2'alpha-hydroxylated products 5 and 6 produced significantly higher yields in Z-form ODN 1-2 induced by Zalpha compared with that in 2 M NaCl. Upon treatment with ribonuclease T1, 5 and 6 were quantitatively hydrolyzed at the 3'-phosphodiester bond of the rG residue to provide d(UGCG) as a common hydrolyzed fragment on the 3' side. Quantitative analysis demonstrated that the amount of photochemically formed 5 and 6 from ODN 1-2 directly correlated with the proportion of Z-form induced by Zalpha or NaCl. These results suggest that this photochemical and enzymatic procedure can be used as a specific probe for the existence of local Z-form structure in cellular DNA.

Photoirradiation of 5-iodouracil-containing DNA, d(GTAAT(I)UAC)(2) with Sso7d protein, possessing significant kink in DNA in the crystal structure induces an unprecedented intrastrand H abstraction at the methyl group of T(5), together with selective photooxidations at Met29 of Sso7d. The reactivity of the deoxyuridin-5-yl radical can be explained by the crystal structure of the d(GTAATTAC)(2)-Sso7d complex, suggesting that the interaction of DNA-Sso7d in solution is substantially similar to its crystal structure.

Binding structures of metal complexes of deglyco-peplomycin (dPEP) on DNA were investigated by comparing dPEP complexes with those of bleomycin (BLM) using DNA fiber EPR spectroscopy. A low spin species of Fe(III)dPEP observed in the DNA pellet changed irreversibly to several high spin species after the fabrication of the DNA fibers. The g values of the high spin species were different from those of Fe(III)BLM. The high spin species could not be nitrosylated reductively to ON–Fe(II)dPEP, suggesting that some nitrogen atoms coordinated to the Fe(III) were displaced on the DNA fibers. On the other hand, O2–Co(II)dPEP remained intact on the fibers similarly to O2–Co(II)BLM but with an increased randomness in the orientation on the DNA. In contrast to Cu(II)BLM, a considerable amount of Cu(II)dPEP bound almost randomly on B-form DNA fibers. These results indicated that the sugar moiety in peplomycin or bleomycin is playing an important role in enhancing the stability of the metal-binding domain and in the stereospecificity of the binding on DNA. Electronic supplementary material is available if you access this article at http://www.dx.doi.org/10.1007/s00775-002-0398-3. On that page (frame on the left side), a link takes you directly to the supplementary material.

X-ray structure of DNA-Sso7d complex indicated that binding of this protein causes sharp DNA bending. In order to examine whether this protein also causes DNA bending in solution, photoreactions of 1U-substituted DNA in the presence and the absence of Sso7d protein were investigated. It was found that the unusual intrastrand hydrogen abstraction at methyl of adjacent thymine occurred efficiently at the observed bending site of crystal structure.

TLS (Translocated in liposarcoma) has been characterized as a rearranged gene in chromosomal translocations specific of human myxoid liposarcoma. The various cellular functions of TLS participating either in transcription or splicing processes are thought the involvement of an interaction of TLS with DNA and/or RNA. To investigate insight into DNA-TLS interaction, we performed Electrophoretic mobility shift assay of TLS with G-quadruplex DNA. It revealed that TLS especially bound to single stranded human telomeric DNA in the presence of potassium ion while it was not able to bind double stranded human telomeric DNA and single stranded human telomeric DNA in the presence of sodium ion.

The TET-family proteins (TAF15, EWS and TLS) are the RNA binding proteins involved in multiple levels of cellular functions. The RNA binding domain of those proteins is known as the important region for cellular functions. But little is known about the RNA binding specificity of TET-family proteins. In order to investigate the RNA binding properties of the TET-family proteins, we performed electrophoretic mobility shift assay using recombinant Flag-tagged TLS and guanine-rich and RNAs. It was found that TLS binds to human telomeric RNA in the presence of KCl, but not in the presence of LiCl.

The formation of quadruplex structure changed the site reactivity and the kinds of guanine photooxidation products of d(TGGGGT). In quadruplex DNA, 8-oxo-7,8-dihydroguanine (8oxoG) and dehydroguanidinohydantoin (Ghox) were mainly formed, although 2,5-diamino-4H-imidazol-4-one (Iz) was mainly formed in single-stranded DNA. In addition, 3′-guanine was specifically oxidized in quadruplex DNA compared with single-stranded DNA, which depended on the localization of the HOMO.

In order to investigate the inhibition of gene expression by a new type of hairpin polyamide-CPI conjugate 1, its ability to inhibit transcription in cell free system was investigated. Sequence-selective alkylation of double-stranded DNA by 1 was investigated by denaturing gel electrophoresis using 1000 bp DNA fragment which codes for green fluorescence protein (GFP) under the control of T7 promoter. Analysis of DNA sequence indicated that 1 alkylated predominantly at the site of 5'-AGTCA-3' in coding region of GFP. The transcript by T7 RNA polymerase using the alkylated DNA as a template was analyzed by PAGE. The results clearly indicate that 1 inhibits transcription by alkylation of coding region at a nanomolar concentration.

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3′-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5′-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K+ and d(TAGGGT)4T +2K+ localized at the 5′-guanine, and that the HOMO shifted from the 5′-guanine to the 3′-guanine by the addition of a 5′-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K+ and d(TAGGGT)4T +2K+ matched the experimental results and suggest that d(TGGGGT)4 contains a 5′-capping cation in solution.