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We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and (3)H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (90% 1 day after injection of 2.5 mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.Molecular Therapy-Nucleic Acids (2013) 2, e123; doi:10.1038/mtna.2013.50; published online 17 September 2013.

Analysis of unusual gel mobility patterns formed by certain DNA triplexes has revealed that intermolecular triplex formation can occur during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. Control experiments showed that this capture of the third strand occurs by sequence-specific hybridization rather than some nonspecific retardation. This phenomenon can be used to detect triplexes by a gel-shift assay even if their lifetime is much shorter than the time of gel electrophoresis.

Until recently, oligonucleotide-directed triplex formation has been limited to oligopurine tracts of target DNA. Triplex formation by alternate-strand recognition relaxes this limitation by allowing triplexes to form at 5'-(Pu)m(Py)n-3' and 5'-(Py)m(Pu)n-3' sequences, with the third strand pairing first with purines on one strand and then switching to pair with purines on the other strand. In this study, the interaction of several oligonucleotides with the potential to form triplexes by alternate-strand recognition at the sequence 5'-A8C8A8-3' was studied by chemical probing and affinity cleaving. The results show that triplex formation can be readily accomplished at the 5'-A8C8-3' part of the sequence; however, base triplet formation is disrupted on either side of the strand switch and the Watson-Crick helix is distorted in such a way as to expose the N7 positions of purines adjoining the strand switch. Triplex formation is weak or nonexistent at the 3'-most A8 block, despite the opportunity for recruiting a spacer sequence for the second (C8-A8) strand switch by "slippage". This finding indicates that the C8-A8 strand switch is energetically unfavorable, although pairing at other 5'-(Py)n(Pu)n-3' sequences has been observed, with or without a spacer [Beal, P. A., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478; Jayasena, S. D., & Johnston, B. H. (1992) Nucleic Acids Res. 20, 5279-5288]. Thus, alternate-strand recognition may not be feasible for certain sequences of 5'-(Py)m(Pu)n-3', at least under the conditions examined.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.

Publisher Summary This chapter discusses the structure of DNA using hydroxylamine and methoxylamine as probes. In the presence of oxygen, the treatment of DNA with low concentrations of hydroxylamine (0.01–0.1 M) results in reaction with all four bases and extensive cleavage of the polynucleotide chain. These reactions are inhibited at higher concentrations of hydroxylamine (>0.1 M) by the removal of oxygen or by the addition of catalase or peroxidase, and they appear to be mediated by hydroxyl radicals produced through the oxidation of hydroxylamine. The oxygen-dependent reactions are probably mostly responsible for the mutagenicity and other biological effects of hydroxylamine. When hydroxylamine-reactive sites in DNA are visualized by backbone cleavage by piperidine, a faint guanine ladder is frequently seen. This ladder is probably mainly due to depurination during the reaction with hot piperidine, because it is seen to some extent when unmodified DNA is treated with piperidine.

Publisher Summary This chapter presents protocols for the use of both DNA and RNA polymerases to detect adducts. DNA polymerases are used to extend an annealed primer, an approach applied to the analysis of altered DNA structures. Recently, a procedure was developed for using RNA polymerases from phages to map adducts by transcribing modified DNA. The chapter refers to the RNA polymerase approach as transcription and the DNA polymerase approach as primer extension. The principal requirement for the transcription approach is that an appropriate promoter should exist near the sequence to be analyzed. Commonly used promoters are those derived from SP6, T3, and T7 phages. The transcription termination procedure has been successfully tested using SP6, T3, and T7 RNA polymerases, each of which has a high specificity for its cognate promoter. The use of SP6 and T7 RNA polymerases, in separate reactions for each modified sample in a pGEM vector, allows analysis of each strand of the inserted DNA. Many of the generalizations made about transcription also apply to primer extension. The main differences are that the copying enzymes used are different, and have somewhat different sensitivities to the presence of adducts, and that an annealed primer is required for primer extension. Because primers can be directed at any segment of DNA, the analysis of modified DNA is no longer limited to a region contained between two phage promoters. Some genomic sequences can readily be examined with a highly radioactive primer or by multiple rounds of DNA synthesis.

Publisher Summary Z-DNA is the only known helical conformation of DNA that is left-handed. The Z-DNA helix is longer and thinner than B-DNA, and the bases lie relatively farther from the helix axis, creating a single deep, narrow groove. This chapter discusses the techniques available for inducing the formation of Z-DNA and detecting it. The left-handed helical sense means that any torsional stress directed opposite the right-handed helical sense of B-DNA will tend to favor the formation of Z-DNA. The syn-anti alternation dictates that alternating purine–pyrimidine sequences most easily form Z-DNA because pyrimidines favor the anti orientation, whereas purines can accommodate either the syn or the anti conformation. Because of the displacement of the bases from the helix axis, Z-DNA can be stabilized by adding bulky groups at sites that are sterically hindered in B-DNA but become more accessible in the Z conformation. Similarly, certain chemicals that react at those accessible sites can be used to recognize Z-DNA. The overall conformational differences between B-DNA and Z-DNA permit the two structures to be distinguished by many nucleases and DNA-binding proteins, which generally recognize right-handed DNA, and by antibodies specifically directed against Z-DNA.

The boundary between two segments of Z-DNA that differ in the phase of their syn-anti alternation about the glycosidic bond is termed a Z-Z junction. Using chemical probes and two-dimensional gel electrophoresis, we examined a Z-Z junction consisting of the sequence d[(CG)8C(CG)8] inserted into a plasmid and used energy minimization techniques to devise a three-dimensional model that is consistent with the available data. We show that both alternating CG segments undergo the B-Z transition together to form a Z-Z junction. The junction is very compact, displaying a distinctive reactivity signature at the two base pairs at the junction. In particular, the 5' cytosine of the CC dinucleotide at the junction is hyperreactive toward hydroxylamine, and the two guanines of the GG dinucleotide on the complementary strand are less reactive toward diethyl pyrocarbonate than are the surrounding Z-DNA guanines. Statistical mechanical treatment of the 2-D gel data yields a delta G for forming the Z-Z junction equal to 3.5 kcal, significantly less than the cost of a B-Z junction and approximately equal to the cost of a base out of alternation (i.e., a Z-DNA pyrimidine in the syn conformation). The computer-generated model shows little distortion of the Z helix outside of the central two base pairs, and the energy of the structure and the steric accessibility of the reactive groups are consistent with the data.

If a mixture of T4 deoxyribonucleic acid (DNA) and 4'-(aminomethyl)-4,5',8-trimethylpsoralen is irradiated with two closely spaced pulses of long-wave UV laser light, the resulting cross-linking is dependent on the time delay between the pulses. As the delay lengthens to 1 mus, a rise in the number of cross-links is observed which follows first-order kinetics. This delay, the time required for most monoadducts to be able to absorb a second photon and thereupon form a cross-link, is interpreted in terms of a conformational change in the DNA at the psoralen intercalation site which may occur upon monoadduct formation.

While many intercalated psoralens require a 1.3-mus relaxation time between absorption of the first and second photons for cross-link formation to occur, some psoralens can form cross-links within the lifetime of a 10-ns laser pulse. This effect is largely or completely oxygen independent. Structural, kinetic, and energetic considerations suggest that the 1.3-mus delay may be due to a conformational change in the deoxyribonucleic acid (DNA) at the intercalation site which could be required for proper alignment of the double bonds which react in the second photoreaction. The cross-links which can form with single pulses of light may result from intercalation complexes which are already in a conformation such that, within 20 ns after absorption of an initial photon, a monoadduct is formed which can absorb a second photon and thence result in a cross-link. These intercalation sites may be distinguished by the type and sequence of base pairs at the site or, alternatively, at the moment of the pulse, random motions of the DNA may have brought those sites into a conformation which allows cross-linking without the 1.3-mus delay. Unlike "ordinary" cross-links, these rapidly forming cross-links appear to be monophotonic; i.e., they increase linearly with laser pulse energy. This suggests that the second photostep for these adducts effectively saturates at much lower laser intensities than is the case for ordinary cross-links.

We have analyzed, at nucleotide resolution, the progress of the B-to-Z transition as a function of superhelical density in a 2.2-kilobase plasmid containing the sequence d(C-A)31.d(T-G)31. The transition was monitored by means of reactivity to two chemical probes: diethyl pyrocarbonate, which is sensitive to the presence of Z-DNA, and hydroxylamine, which detects B-Z junctions. At a threshold negative superhelical density between about 0.048 and 0.056, hyper-reactivity to diethyl pyrocarbonate appears throughout the CA/TG repeat and remains as the superhelical density is further increased. However, there is no reactivity characteristic of B-Z junctions until the superhelical density reaches 0.084, when single cytosines at each end of the repeat become hyper-reactive to hydroxylamine. A two-dimensional gel analysis of this system by others (Haniford, D. B., and Pulleyblank, D. E. (1983) Nature 302, 632-634) indicates that only about half of the 62 base pairs of the CA/TG repeat undergo the initial transition at omega = 0.056. Our results indicate that this region of Z-DNA is free to exist anywhere along the CA/TG repeat and is probably constantly in motion. Well defined B-Z junctions are seen only when there is sufficient supercoiling to convert the entire CA/TG sequence to Z-DNA. The implications for possible B-Z transitions in chromosomal domains of different sizes are discussed.

Synthese de t-butyldiphenylsiloxy-2″ ethyl-6' [hydroxymethyl-1' ethyl]-6 perhydro trimethyl-3,5,5' spirobi-2:2'-pyrane via la reaction du dihydro-3,4 methoxymethoxymethyl-1' ethyl-2 methyl-3 pyranne avec le bromo-1 t-butyldimethylsiloxy-4 t-butyldiphenylsiloxy-6 methyl-3 hexane. Application a la synthese de la calcimycine

Chemical probes sensitive to alterations in DNA conformation, especially Z-DNA, have been identified. These permit cleavage of DNA at sites of unusual structure, the results of which can be displayed on a sequencing gel. Using supercoiled plasmids containing Inserts of d(C-G) 16 and d(C-A) 31 ·d(T-G) 31 , it was found that hydroxylamine and osmium tetraoxide react preferentially with cytosines and thymines, respectively, near B-DNA-Z-DNA junctions; diethylpyrocarbonate reacts more strongly with purines within Z-DNA regions; and dimethylsulfate and diethylsulfate react more strongly with guanines in Z-DNA that are out of phase with the usual pattern of purine-pyrimidine alternation. Our results show that B-Z boundaries are mobile and that with increasing torsional strain, the Z-DNA regions can expand to include nonalternating nucleotide sequences.

Samples of DNA which contain adducts of a new psoralen derivative, but no cross-links, have been prepared by irradiating mixtures of DNA and the derivative with single, 15-nanosecond pulses of laser light. Succeeding pulses introduce cross-links. The ability to rapidly and selectively create monoadducts and corss-links may allow the use of psoralens as probes of dynamic processes in living cells.

An analysis of the B-to-Z transition as a function of supercoiling for a natural Z-DNA-forming sequence found in plasmid pBR322 is presented at nucleotide resolution. The analysis is based on reactivity to four chemical probes which exhibit hyperreactivity in the presence of Z-DNA: hydroxylamine, osmium tetroxide, diethyl pyrocarbonate and dimethyl sulfate. We find that the initial transition occurs largely within a 14 base pair region which is mostly alternating purines and pyrimidines. With increasing negative supercoiling. Z-DNA extends into flanking regions having less and less alternating character, first one direction and then in the other. Evidence of B-Z junctions is seen at four sites bracketing these three adjacent regions. One of these Z-forming regions contains the non-alternating sequence CTCCT, suggesting that such sequences can form Z-DNA without great difficulty if they are adjacent to alternating sequences. A plasmid containing three copies of a 61 base pair fragment bearing the entire Z-forming region shows equal reactivity of all three copies at any given superhelical density, implying that they compete equally and independently for the torsional strain energy which promotes the B-Z transition, and are unaffected by adjacent sequences more than 20-30 base pairs away.

Homopurine-homopyrimidine sequences that flank certain actively transcribed genes are hypersensitive to single strand-specific nucleases such as S1. This has raised the possibility that an unusual structure exists in these regions that might be involved in recognition or regulation. Several of these sequences, including d(C-T)n.d(A-G)n, are known to undergo a transition in plasmids to an underwound state that is hypersensitive to single strand-specific nucleases; this transition occurs under conditions of moderately acid pH and negative supercoiling. Chemical probes were used to examine the reactivity of a restriction fragment from a human U1 gene containing the sequence d(C-T)18.d(A-G)18 as a function of supercoiling and pH, and thus analyze the structure in this region. Hyperreactivity was seen in the center and at one end of the (C-T)n tract, and continuously from the center to the same end of the (A-G)n tract, in the presence of supercoiling and pH less than or equal to 6.0. These results provide strong support for a triple-helical model recently proposed for these sequences and are inconsistent with other proposed structures.

Rates of trifluoroacetylation of a structurally diverse range of aliphatic alcohols in neat trifluoroacetic acid at 35°C have been measured using 1H n.m.r. spectroscopy. The reaction mechanism changes from reverse AAC2 for primary and secondary alcohols (except Ph2CHOH) to reverse AAL1 for t-butanol.