Anne M. Noronha

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-α production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-α-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-α in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).

Interdomain Allostery Promotes Assembly of the Poly(A) mRNA Complex with PABP and eIF4G

Many RNA-binding proteins contain multiple single-strand nucleic acid-binding domains and assemble into large multiprotein messenger ribonucleic acid protein (mRNP) complexes. The mechanisms underlying the self-assembly of these complexes are largely unknown. In eukaryotes, the association of the translation factors polyadenylate-binding protein-1 (PABP) and eIF4G is essential for high-level expression of polyadenylated mRNAs. Here, we report the crystal structure of the ternary complex poly(A)(11)·PABP(1-190)·eIF4G(178-203) at 2.0 Å resolution. Our NMR and crystallographic data show that eIF4G interacts with the RRM2 domain of PABP. Analysis of the interaction by small-angle X-ray scattering, isothermal titration calorimetry, and electromobility shift assays reveals that this interaction is allosterically regulated by poly(A) binding to PABP. Furthermore, we have confirmed the importance of poly(A) for the endogenous PABP and eIF4G interaction in immunoprecipitation experiments using HeLa cell extracts. Our findings reveal interdomain allostery as a mechanism for cooperative assembly of RNP complexes.

Repair of O6-G-alkyl-O6-G interstrand cross-links by human O6-alkylguanine-DNA alkyltransferase

O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine- O (6) in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.

Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells

DNA interstrand cross-links (ICLs) are cytotoxic products of common anticancer drugs and cellular metabolic processes, whose mechanism(s) of repair remains poorly understood. In this study, we show that cross-link structure affects ICL repair in nonreplicating reporter plasmids that contain a mispaired N(4)C-ethyl-N(4)C (C-C), N3T-ethyl-N3T (T-T), or N1I-ethyl-N3T (I-T) ICL. The T-T and I-T cross-links obstruct the hydrogen bond face of the base and mimic the N1G-ethyl-N3C ICL created by bis-chloroethylnitrosourea, whereas the C-C cross-link does not interfere with base pair formation. Host-cell reactivation (HCR) assays in human and hamster cells showed that repair of these ICLs primarily involves the transcription-coupled nucleotide excision repair (TC-NER) pathway. Repair of the C-C ICL was 5-fold more efficient than repair of the T-T or I-T ICLs, suggesting the latter cross-links hinder lesion bypass following initial ICL unhooking. The level of luciferase expression from plasmids containing a C-C cross-link remnant on either the transcribed or nontranscribed strand increased in NER-deficient cells, indicating NER involvement occurs at a step prior to remnant removal, whereas expression from similar T-T remnant plasmids was inhibited in NER-deficient cells, demonstrating NER is required for remnant removal. Sequence analysis of repaired plasmids showed a high proportion of C residues inserted at the site of the T-T and I-T cross-links, and HCR assays showed that Rev1 was likely responsible for these insertions. In contrast, both C and G residues were inserted at the C-C cross-link site, and Rev1 was not required for repair, suggesting replicative or other translesion polymerases can bypass the C-C remnant.

Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis

DNA interstrand cross-links (ICLs) are products of chemotherapeutic agents and cellular metabolic processes that block both replication and transcription. If left unrepaired, ICLs are extremely toxic to cells, and ICL repair mechanisms contribute to the survival of certain chemotherapeutic resistance tumors. A critical step in ICL repair involves unhooking the cross-link. In the absence of a homologous donor sequence, the resulting gap can be filled in by a repair synthesis step involving bypass of the cross-link remnant. Here, we examine the effect of cross-link structure on the ability of unhooked DNA substrates to undergo repair synthesis in mammalian whole cell extracts. Using 32P incorporation assays, we found that repair synthesis occurs efficiently past the site of damage when a DNA substrate containing a single N4C-ethyl-N4C cross-link is incubated in HeLa or Chinese hamster ovary cell extracts. This lesion, which can base pair with deoxyguanosine, is readily bypassed by both Escherichia coli DNA polymerase I and T7 DNA polymerase in a primer extension assay. In contrast, bypass was not observed in the primer extension assay or in mammalian cell extracts when DNA substrates containing a N3T-ethyl-N3T or N1I-ethyl-N3T cross-link, whose linkers obstruct the hydrogen bond face of the bases, were used. A modified phosphorothioate sequencing method was used to analyze the ICL repair patches created in the mammalian cell extracts. In the case of the N4C-ethyl-N4C substrate, the repair patch spanned the site of the cross-link, and the lesion was bypassed in an error-free manner. However, although the N3T-ethyl-N3T and N1I-ethyl-N3T substrates were unhooked in the extracts, bypass was not detected. These and our previous results suggest that although the chemical structure of an ICL may not affect initial cross-link unhooking, it can play a significant role in subsequent processing of the cross-link. Understanding how the physical and chemical differences of ICLs affect repair may provide a better understanding of the cytotoxic and mutagenic potential of specific ICLs.

PROPERTIES OF ARABINONUCLEIC ACIDS (ANA & 20′F-ANA): IMPLICATIONS FOR THE DESIGN OF ANTISENSE THERAPEUTICS THAT INVOKE RNASE H CLEAVAGE OF RNA

Inversion of configuration of the C2′ position of RNA leads to a very unique nucleic acid structure: arabinonucleic acid (ANA). ANA, and its 2′-fluoro derivative (2′ F-ANA) form hybrids with RNA that are capable of activating RNase H, resulting in cleavage of the RNA strand. In this paper, we review the properties of duplexes formed between ANA (or 2′F-ANA) and its RNA complement. These studies support the notion that RNase H is sensitive to the minor groove dimensions of the hybrid substrate.

Reductively-sheddable cationic nanocarriers for dual chemotherapy and gene therapy with enhanced release.

The development of a versatile strategy to synthesize cationic nanocarriers capable of co-delivery and enhanced release of drugs and oligonucleotides is promising for synergic dual chemotherapy and gene therapy. Herein, we report a novel cationic amphiphilic diblock copolymer having a single reduction-responsive disulfide linkage at a junction between a FDA-approved polylactide (PLA) block and a cationic methacrylate block (C-ssABP). The amphiphilic design of the C-ssABP enables the formation of cationic micellar aggregates possessing hydrophobic PLA cores, encapsulating anticancer drugs; cationic coronas, ensuring complementary complexation with negatively-charged oligonucleotides through electrostatic interactions; and disulfides at interfaces, leading to enhanced release of both encapsulated drugs and complexed oligonucleotides. The reduction-responsive intracellular trafficking results from flow cytometry, confocal laser scanning microscopy, and cell viability, as well as in vitro gene transfection assay suggest that C-ssABP offers versatility as an effective nanocarrier platform for dual chemotherapy and gene therapy.

Influence of nucleotide modifications at the C2’ position on the Hoogsteen base-paired parallel-stranded duplex of poly(A) RNA

Abstract Polyadenylate (poly(A)) has the ability to form a parallel duplex with Hoogsteen adenine:adenine base pairs at low pH or in the presence of ammonium ions. In order to evaluate the potential of this structural motif for nucleic acid-based nanodevices, we characterized the effects on duplex stability of substitutions of the ribose sugar with 2′-deoxyribose, 2′-O-methyl-ribose, 2′-deoxy-2′-fluoro-ribose, arabinose and 2′-deoxy-2′-fluoro-arabinose. Deoxyribose substitutions destabilized the poly(A) duplex both at low pH and in the presence of ammonium ions: no duplex formation could be detected with poly(A) DNA oligomers. Other sugar C2’ modifications gave a variety of effects. Arabinose and 2′-deoxy-2′-fluoro-arabinose nucleotides strongly destabilized poly(A) duplex formation. In contrast, 2′-O-methyl and 2′-deoxy-2′-fluoro-ribo modifications were stabilizing either at pH 4 or in the presence of ammonium ions. The differential effect suggests they could be used to design molecules selectively responsive to pH or ammonium ions. To understand the destabilization by deoxyribose, we determined the structures of poly(A) duplexes with a single DNA residue by nuclear magnetic resonance spectroscopy and X-ray crystallography. The structures revealed minor structural perturbations suggesting that the combination of sugar pucker propensity, hydrogen bonding, pKa shifts and changes in hydration determine duplex stability.

Branchpoint Sugar Stereochemistry Determines the Hydrolytic Susceptibility of Branched RNA Fragments by the Yeast Debranching Enzyme (YDBR)

Abstract A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and non-natural (i.e., arabinose, xylose and acyclic nucleoside) branchpoints in order to examine the effect of sugar conformation and phosphodiester configuration on yDBR hydrolytic efficiency. The results indicate that 2′-phosphodiester scission with yDBR occurs only with a cis-arrangement of phosphate groups at the branchpoint (i.e., ribose) thereby discriminating between all other configurations.

NMR structure of an ethylene interstrand cross-linked DNA which mimics the lesion formed by 1,3-bis(2-chloroethyl)-1-nitrosourea.

The bisalkylating agent 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU), used in cancer chemotherapy to hinder cellular proliferation, forms lethal interstrand cross‐links (ICLs) in DNA. BCNU generates an ethylene linkage connecting the two DNA strands at the N1 atom of 2′‐deoxyguanosine and N3 atom of 2′‐deoxycytidine, which is a synthetically challenging probe to prepare. To this end, an ICL duplex linking the N1 atom of 2′‐deoxyinosine to the N3 atom of thymidine via an ethylene linker was devised as a mimic. We have solved the structure of this ICL duplex by a combination of molecular dynamics and high‐field NMR experiments. The ethylene linker is well‐accommodated in the duplex with minimal global and local perturbations relative to the unmodified duplex. These results may account for the substantial stabilization of the ICL duplex observed by UV thermal denaturation experiments and provides structural insights of a probe that may be useful for DNA repair studies.