Site-directed cleavage of DNA by an oligonucleotide conjugate with o-bromobenzoic acid in the presence of copper ions

Site-directed cleavage of single- and double-stranded DNAs by an oligonucleotide conjugate with 5-[N-(3-aminopropyl)sulfamoyl]-2-bromobenzoic acid was investigated. When forming duplex complexes with a single-stranded DNA and triplex complexes with a double-stranded DNA, this conjugate cleaves DNA near the binding site in the presence of copper ions and free o-bromobenzoic acid. The efficacy and specificity of DNA cleavage by this conjugate and other oligonucleotide conjugates bearing tetracarboxyphthalocyanine Co^II and bleomycin A5 as reactive groups were compared.     

Highly efficient modification of DNA polymerase β under conditions of direct and sensitized activation of photoreactive DNAs. Modification of cell extract proteins

dUTP and dCTP derivatives containing a 4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy group were incorporated into the 3′-end of the DNA primer within complexes with the DNA-matrix as analogs of natural dTTP by virtue of catalytic activity of DNA polymerase β or endogenous DNA polymerases of the cell extract. The photoreactive DNAs synthesized in situ were used for affinity modification of DNA polymerase β and DNA-binding proteins of the cell extract. For the photoreactive DNA based on these analogs, the efficiency of formation of covalent adducts with DNA polymerase β under the highest degree of DNA complexation with the enzyme was determined. The yield of covalent DNA adducts with the enzyme was 28–47%, depending on the type of the analog. The effect of the sequence of the DNA template near the localization of the photoreactive group on the redistribution of covalent cross-links between the possible targets was demonstrated. A possibility of increasing the efficiency of DNA polymerase β modification in the presence of a substantial excess of photoreactive DNA using a sensitizer, a dUTP derivative containing a pyrene residue, was studied. When photoreactive DNA containing a 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was used, about 60% of DNA polymerase β was covalently attached to DNA. Photoreactive dNTP analogs ensuring a high level of protein modification in the cell extract were found. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1273–1283, May, 2005.     

Spherical Nucleic Acid Nanoparticle Conjugates Enhance G‐Quadruplex Formation and Increase Serum Protein Interactions

To understand the effect of three‐dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence‐specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid‐based nanostructures, and SNAs in particular, function in complex biological milieu.     

Synthesis of oligonucleotides carrying thiol groups using a simple reagent derived from threoninol

Oligonucleotides carrying thiol groups are useful intermediates for a remarkable number of applications involving nucleic acids. In this study, DNA oligonucleotides carrying tert-butylsulfanyl (t-BuS) protected thiol groups have been prepared. A building block derived from threoninol has been developed to introduce a thiol group at any predetemined position of an oligonucleotide. The resulting thiolated oligonucleotides have been used for the preparation of oligonucleotide conjugates and for the functionalization of gold nanoparticles using the reactivity of the thiol groups.     

Opening Enediyne Scissors Wider: pH‐Dependent DNA Photocleavage by meta‐Diyne Lysine Conjugates

Photochemical activation of meta‐diynes incapable of Bergman and C1–C5 cyclizations still leads to efficient double‐strand DNA cleavage. Spatial proximity of the two arylethynyl groups is not required for efficient DNA photocleavage by the enediyne‐lysine conjugates. Efficiency of the cleavage is a function of the external pH and DNA damage is strongly enhanced at pH < 7. The pH‐dependence of the DNA photocleavage activity stems from the protonation states of lysine amino groups, the internal electron donors responsible for intramolecular PET quenching and deactivation of the photoreactive excited states. DNA‐binding analysis suggests intercalative DNA binding for phenyl substituted conjugate and groove binding for TFP‐substituted conjugate. Additional insights in the possible mechanism for DNA damage from the ROS (Reactive Oxygen Species) scavenger experiments found that generation of singlet oxygen is partially involved in the DNA damage.     

S,S‐Chiral Linker Induced U Shape with a Syn‐facial Sensitizer and Photocleavable Ethene Group

There is a major need for light‐activated materials for the release of sensitizers and drugs. Considering the success of chiral columns for the separation of enantiomer drugs, we synthesized an S,S‐chiral linker system covalently attached to silica with a sensitizer ethene near the silica surface. First, the silica surface was modified to be aromatic rich, by replacing 70% of the surface groups with (3‐phenoxypropyl)silane. We then synthesized a 3‐component conjugate [chlorin sensitizer, S,S‐chiral cyclohexane and ethene building blocks] in 5 steps with a 13% yield, and covalently bound the conjugate to the (3‐phenoxypropyl)silane‐coated silica surface. We hypothesized that the chiral linker would increase exposure of the ethene site for enhanced ¹O₂‐based sensitizer release. However, the chiral linker caused the sensitizer conjugate to adopt a U shape due to favored 1,2‐diaxial substituent orientation; resulting in a reduced efficiency of surface loading. Further accentuating the U shape was π–π stacking between the (3‐phenoxypropyl)silane and sensitizer. Semiempirical calculations and singlet oxygen luminescence data provided deeper insight into the sensitizer's orientation and release. This study has lead to insight on modifications of surfaces for drug photorelease and can help lead to the development of miniaturized photodynamic devices.     

Synthesis and Cytotoxic Activity of Macromolecular Prodrug of Cisplatin Using Poly(ethylene glycol) with Galactose Residues or Antennary Galactose Units

To provide a macromolecular prodrug with recognition ability for hepatoma cells, we synthesized new conjugates of cisplatin (CDDP) and poly(ethylene glycol) (PEG) with galactose residues or antennary galactose units (Gal4A, four branched galactose residues) at the chain terminus, Gal‐PEG‐DA/CDDP or Gal4A‐PEG‐DA/CDDP conjugates. An antennary (branched) structure of Gal4A was designed based on the fact that saccharide clusters with branched structures show highly effective binding with saccharide receptors, a phenomenon known as the ‘cluster effect’. The cytotoxic activity of the conjugates was investigated against HepG2 human hepatoma cells in vitro and compared with a control conjugate without galactose, MeO‐PEG‐DA/CDDP. Gal‐PEG‐DA/CDDP and Gal4A‐PEG‐DA/CDDP conjugates showed lower IC₅₀ values (3.1×10^–4 and 2.3×10^–4 M , respectively) than the MeO‐PEG‐DA/CDDP conjugate (10.5×10^–4 M ). The cytotoxic activities of these conjugates with galactose residues or antennary galactose units were inhibited as a result of the addition of galactose and strongly inhibited by the addition of Gal4A, however the inclusion of a methoxy group (the MeO‐PEG‐DA/CDDP conjugate) did not affect the activity. These results suggest that the Gal4A unit introduced to the conjugate has effective recognition ability against HepG2 human hepatoma cells.     

Effect of concentrations of salts on the formation of protein monolayers

Effects of salts present in the subphase on the properties of monolayers,viz., molecular areas of proteins and Gibbs elasticities, were studied by the monolayer method.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 2038–2042, October, 1995.     

Discontinuous reversed-phase high performance liquid chromatography increases load capacity of analytical columns. Separation of ribosomal proteins from the archaebacteriumSulfolobus acidocaldarius

Summary Since ribosomes are a fundamental feature component of all organisms, they present a good model for studying evolutionary diversity. We investigated the ribosomal proteins of the archaebacteriumSulfolobus acidocaldarius, which contains slightly more ribosomal proteins thanEscherichia coli. While the ribosomal proteins of most organisms contain a high proportion of lysine and arginine residues, these basic amino acids are particularly prevelent in the thermaacidophile organismSulfolobus, a possible reason for poor separations of total ribosomal proteins ofS. acidocaldarius by single column HPLC. To solve this complex separation problem, we developed the discontinuous reversed-phase HPLC (Disc RPC) method. Discontinuus chromatography combines at least two different stationary phases in a sequence of increasing retention times for the elution of proteins. Unlike other multi-column techniques, all of the injected sample passes through this discontinous stationary phase, which is used in place of single columns, thus permitting separations to be carried out without the need any changes in the HPLC system. Here we present Disc RPC separations of 30S ribosomal proteins fromS. acidocaldarius with a variety of stationary phases of different legnths and diameters. Each of five Disc RPC categories presented in this paper has some special application possibilities when used in protein HPLC.     

Hybridization and Melting Behavior of Peptide Nucleic Acid (PNA) Oligonucleotide Chimeras Conjugated to Gold Nanoparticles

Peptide nucleic acids (PNA) and PNA–DNA chimeras carrying thiol groups were used for surface functionalization of Au nanoparticles. Conjugation of PNA to citrate‐stabilized Au nanoparticles destabilized the nanoparticles causing them to precipitate. Addition of a tail of glutamic acid to the PNA prevented destabilization of the nanoparticles but resulted in loss of interaction with complementary sequences. Importantly, PNA–DNA chimeras gave stable conjugates with Au nanoparticles. The hybridization and melting properties of complexes formed from chimera–nanoparticle conjugates and oligonucleotide–nanoparticle conjugates are described for the first time. Similar to oligonucleotide–nanoparticle conjugates, conjugates with PNA–DNA chimeras gave sharper and more‐defined melting profiles than those obtained with unmodified oligonucleotides. In addition, mismatch discrimination was found to be more efficient than with unmodified oligonucleotides.     

Controlling the activity of peptides and proteins with smart nucleic acid-protein hybrids

Oligonucleotide-peptide conjugates have frequently been used to control the localisation of the conjugate molecule. For example, the oligonucleotide segment has allowed spatially addressed immobilization of peptides and proteins on DNA-arrays via hybridisation while the peptide part has most frequently been used to confer transfer of oligonucleotide cargo into live cells. The regulation of functional properties such as the affinity of these bioconjugates for protein targets has rarely been addressed. This review article describes the current developments in the application of smart oligonucleotide-peptide hybrids. The mutual recognition between nucleic acid segments is used to constrain the structure or control the distance between peptide and protein segments. Application of these new type of oligonucleotide-peptide hybrids allowed not only the regulation of binding affinity of peptide ligands but also control of enzymatic and optical activity of proteins.     

Synthesis of nitrilotriacetic acid terminated tethers for the binding of His-tagged proteins to lipid bilayers and to gold

Nitrilotriacetic acid terminated tethers for trapping of His-tagged proteins have been synthesized and characterized. Compound 1, containing a lipophilic cholesterol anchor, hydrophilic oligoethoxy chain linker, and nitrilotriacetic acid terminus, can be used for attaching His-tagged proteins to phospholipid bilayers. Compound 2, containing a gold binding thioacetate anchor, hydrophilic oligoethoxy chain, and nitrilotriacetic acid terminus, can be used to tether His-tagged proteins to a gold surface.     

Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates

DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.     

A high-complexity, multiplexed solution-phase assay for profiling protease activity on microarrays

We have developed a miniaturized and multiplexed solution assay for the measurement of protease activity in complex samples. This technology can accelerate research in functional proteomics and enable biologists to carry out multiplexed protease inhibitor screens on a large scale. The assay readout is based on Illumina's universal Sentrix BeadArrays. The peptide sequences that serve as protease substrates are conjugated to oligonucleotide sequences complementary to the oligo tags on randomly assembled and decoded bead arrays. The peptide portion is C-terminally labeled with a biotin residue and contains a sequence of five histidine residues on the amino terminus. The unique oligonucleotide part of each oligonucleotide-peptide conjugate is attached to amino terminus of the peptide sequence. Upon protease cleavage, the biotin residue is cleaved from the oligonucleotide-peptide conjugate. Following the reaction, all biotin-containing species are captured and removed by incubation with streptavidin beads. The cleaved conjugates that remain in solution are captured by hybridization of their oligo sequence to Sentrix BeadArrays and detected using a labeled antibody against pentahistidine tag of the conjugate or by an antibody sandwich assay. We have generated multiple sets of oligonucleotide tagged peptide substrates of varying complexity (100 to 1000 substrates in a mixture) and show that the response of individual substrate is independent of the complexity of the mixture. Our initial results demonstrate the feasibility of assaying proteases in a multiplexed environment with high sensitivity.     

Kinetics and Mechanism of Ruthenium(III) Catalyzed Oxidation of Dimethyl Sulfoxide by N-Chlorosuccinimde in Aqueous Alkaline Medium

The ruthenium(III) catalyzed oxidation of dimethyl sulfoxide by N-chlorosuccinimide (NCS) in aqueous alkaline medium is found to occur via substrate-catalyst complex formation followed by the interaction of active species of NCS viz., HOCl and the complex in a slow step to yield the products with regeneration of the catalyst. One of the products, succinimide, retards the rate of reaction. The reaction is first order in [NCS] and [Ru(III)], lower than first order in [DMSO] and of inverse fractional order in [OH^-]. A suitable mechanism is proposed and the reaction constants of individual steps involved in the mechanism have been evaluated. This revised version was published online in June 2006 with corrections to the Cover Date.     

A cationic dye triplet as a unique "glue" that can connect fully matched termini of DNA duplexes

In this study, we propose that three consecutive cationic p‐methylstilbazoles tethered on D ‐threoninols ( Z residues) at 5′ termini act as a unique “glue” connecting DNA duplexes by their interstrand cluster formation. Interstrand clustering of p‐methylstilbazoles ( ZZZ triplets) induces narrowing and hypsochromic shift of bands at 350 nm, which can be assigned to the absorption of p‐methylstilbazole. However, single‐stranded DNA conjugates involving a ZZZ triplet at the 5′ terminus of 8‐mer native nucleotides is found not to induce such large spectral changes, which implies that the intrinsic self‐assembling property of ZZZ triplets is weak. Interestingly, when this conjugate is hybridized with a complementary 8‐mer native oligonucleotide, a remarkable spectral change is observed, indicating the dimerization of a duplex through the interstrand clustering of ZZZ triplets. Dimerization of the duplex is also evidenced by cold‐spray ionization mass spectrometry. This interstrand clustering is observed only when a ZZZ triplet is tethered to a 5′ rather than 3′ terminus. Furthermore, the stability of the interstrand cluster increases by increasing the number of nucleobases of the DNA portion, and when mismatched base pairs are incorporated or when a base next to the Z residue is deleted, the stability substantially drops. When we apply the ZZZ triplet to the formation of a nanowire using two complementary DNA conjugates, each of which has a ZZZ triplet at the 5′ termini as overhang, we demonstrate the successful formation of a nanowire by native PAGE analysis. Since native sticky ends that have three nucleotides do not serve as “glue”, ZZZ triplets with their unique glue‐like properties are prime candidates for constructing DNA‐based nanoarchitectures.     

Zn2+ Complexes of 3,5‐Bis[(1,5,9‐triazacyclododecan‐3‐yloxy)methyl]phenyl Conjugates of Oligonucleotides as Artificial RNases: The Effect of Oligonucleotide Conjugation on Uridine Selectivity of the Cleaving Agent

2‐(3,5‐Bis{[1,5,9‐tris(trifluoroacetyl)‐1,5,9‐triazacyclododecan‐3‐yloxy]methyl}phenoxy)ethanol was synthesized and converted to a O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite building block, 12 . 2′‐O‐Methyl oligoribonucleotides incorporating a 2‐[(2S,4S,5R)‐4‐hydroxy‐5‐(hydroxymethyl)tetrahydrofuran‐2‐yl)ethyl 4‐oxopentanoate or a 2‐{2‐[2‐({[(2R,4S,5R)‐4‐hydroxy‐5‐(hydroxymethyl)tetrahydrofuran‐2‐yl]acetyl}amino)ethoxy]ethoxy}ethyl 4‐oxopentanoate non‐nucleosidic unit close to the 3′‐terminus were assembled on a solid support, the 4‐oxopentanoyl protecting groups were removed by treatment with hydrazinium acetate on‐support, and 12 was coupled to the exposed OH function. The deprotected conjugates were purified by HPLC, and their ability to cleave a complementary RNA containing either uridine or some other nucleoside at the potential cleaving site was compared. Somewhat unexpectedly, conjugation to an oligonucleotide did not enhance the catalytic activity of the Zn²⁺? bis(azacrown) complex and virtually abolished its selectivity towards the uridine sites.     

Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.     

Effect of β3‐Amino Acids on the Performance of the Peptidic Catalyst H‐dPro‐Pro‐Glu‐NH2

The effect of β³‐amino acids on the conformation and catalytic performance of the peptidic catalyst H‐d Pro‐Pro‐Glu‐NH₂ was investigated. Analogues of the peptidic catalyst bearing instead of the α‐amino acids the respective β³‐amino acids were prepared and their reactivity and stereoselectivity was investigated in conjugate addition reactions of aldehydes to nitroolefins. Additional computational studies provided insights into the preferred conformations of the peptidic catalysts. The results show that conformational flexibility at the N‐terminus has a severe effect on the stereoselectivity but is tolerated at the C‐terminus.     

Triplex-Mediated, in vitro Targeting of Psoralen Photoadducts within the Genome of a Transgenic Mouse

Abstract— Light-activated psoralens can covalently modify DNA and are widely used to study nucleic acid secondary structure and mutagenesis. Sequence specificity can be added to the photoaddition reaction by attaching the psoralen to an oligonucleotide designed to recognize a double-stranded DNA binding site through formation of a triple helix. We have previously used this strategy to study targeted psoralen modification of a triplex binding site within the bacterial supF gene carried in viral genomes. In the present work we report the targeting of psoralen photoadducts in vitro to a specific site in the genome of a transgenic mouse. Both 10 base and 16 base oligonucleotide-psoralen conjugates were capable of sequence-specific modification of genomic mouse DNA, while a truncated 8 base conjugate was not. Light activation was necessary, and a dose dependence was demonstrated for target site modification and mutagenesis. The 10 base conjugate rapidly found its target, with sequence-specific binding occurring after just 10 min incubation in the presence of mouse DNA. The ability to target psoralen photoadducts within mammalian genomes may prove useful in the study of chromatin structure and DNA repair. Moreover, this work may lead to potential in vivo applications of targeted psoralen modification.