Total stepwise solid-phase peptide-oligonucleotide conjugate synthesis on macroporous polystyrene

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2'-deoxynucleoside or 2'-O-methylribonucleoside phosphodiesters, or gapmers containing 2'-deoxyphosphorothioate sequences flanked by 2'-O-methyl wings.     

Synthesis of phosphorothioate oligonucleotide-peptide conjugates by solid phase fragment condensation

Phosphorothioate oligonucleotide-peptide conjugates were synthesized by solid phase fragment condensation (SPFC). Arginine rich peptides could be successfully conjugated in 2.8-13.4% isolated yields. All the products were fully characterized by reversed phase HPLC and MALDI-TOF-MS to give satisfactory results.     

Preparation of 2'-hydrazino oligonucleotides and their reaction with aldehydes and 1,3-diketones

Oligodeoxyribonucleotides that contain a hydrazino nucleoside, 2'-O-(2-hydrazinoethyl)uridine were prepared and shown to react with aldehydes or 1,3-diketones with the formation of hydrazones or pyrazoles, respectively. The method may be applicable for the preparation of oligonucleotide-peptide conjugates.     

Preparation of oligonucleotide-peptide conjugates.

A procedure for preparing oligonucleotide-peptide conjugates is presented. It is based on appending a maleimide group to the oligonucleotide for selective coupling to the thiol side chain of a cysteine residue in the peptide. A convenient chromatographic purification procedure, based on Fmoc-on/Fmoc-off, is described.     

Thermodynamic Melting Studies on Oligonucleotide-Peptide Conjugates

Abstract

A small library of oligonucleotide-peptide conjugates has been prepared and studied to explore the influence of the various peptide side chain (cationic, anionic or hydrophobic) on the hybridation properties of the DNA.     

Preparation Of 2′-Hydrazino Oligonucleotides And Their Reaction With Aldehydes And 1,3-Diketones

Oligodeoxyribonucleotides that contain a hydrazino nucleoside, 2′-O-(2-hydrazinoethyl)uridine were prepared and shown to react with aldehydes or 1,3-diketones with the formation of hydrazones or pyrazoles, respectively. The method may be applicable for the preparation of oligonucleotide-peptide conjugates.     

Total Stepwise Solid-Phase Peptide-Oligonucleotide Conjugate Synthesis on Macroporous Polystyrene

Abstract

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2′-deoxynucleoside or 2′-O-methylribonucleoside phosphodiesters, or gapmers containing 2′-deoxyphosphorothioate sequences flanked by 2′-O-methyl wings.     

Evaluation of different chemical strategies for conjugation of oligonucleotides to peptides

We developed novel assays for high-throughput detection of one or many kinases or proteases. The assays use hundreds of different peptide substrates, each covalently linked to an oligonucleotide tag. After incubation with sample, the pool of substrates is hybridized to a microarray containing oligonucleotides complementary to the tag sequences. We screened several specific chemistries for the conjugation based on the following criteria: easy derivatization of oligonucleotides and peptides; high efficiency of the conjugation reaction; good stability of the conjugates; and satisfactory conjugate performance in our assays. We have validated selected method during the successful generation of thousands oligonucleotide-peptide conjugates.     

Strand invasion by DNA-peptide conjugates and peptide nucleic acids.

Peptide nucleic acids (PNAs) and conjugates between oligonucleotides and cationic peptides possess superior potential for strand invasion at complementary sequences. We discovered that oligonucleotide-peptide conjugates and PNAs fall into three classes based on their hybridization efficiency; i) those complementary to inverted repeats within AT-rich region hybridize with highest efficiency; ii) those complementary to areas adjacent to inverted repeats or near AT-rich regions hybridize with moderate efficiency; and iii) those complementary to other regions do not detectably hybridize. The correlations between oligomer chemistry, DNA target sequence, and hybridization efficiency that we report here have important implications for the recognition of duplex DNA.     

肽缀合反义寡核苷酸的合成和性能

反义寡核苷酸可作为基因表达的抑制剂和潜在的治疗药物,但多种类型的寡核苷酸为聚阴离子化合物,难以跨过细胞膜.已知包括融合肽、信号肽在内的多种生物活性多肽具有跨膜与核定位能力.讨论了反义寡核苷酸-肽缀合物的合成和生物活性.     

Synthesis of oligonucleotide derivatives using ChemMatrix supports

The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.     

Synthesis and binding properties of oligonucleotides carrying nuclear localization sequences

The synthesis of oligonucleotides carrying nuclear localization peptide sequences is described using two strategies: first, oligonucleotides carrying a thiol group at the 5' end were reacted with maleimido peptides; second, peptide and oligonucleotide were prepared stepwise on the same support, yielding oligonucleotide-3'-peptide conjugates. This second approach was thoroughly studied. Using amino acids and small peptides as model compounds, some side reactions were analyzed, detected, and minimized. Oligonucleotides complementary to Ha-ras gene and carrying nuclear localization peptides at the 3' and 5' ends were prepared. Melting temperature studies showed that duplexes containing nuclear localization peptides were more stable than duplexes with unmodified oligonucleotides. Moreover, oligonucleotide-peptide conjugates maintain a good mismatch discrimination when they bind to their target RNA.     

Synthesis of oligonucleotide 2'-conjugates via amide bond formation in solution

An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide.     

Affinity Modification of the Restriction Endonuclease <Emphasis Type="Italic">Sso</Emphasis>II by 2′-Aldehyde-Containing Double Stranded DNAs

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.     

Synthesis of antisense oligonucleotide-peptide conjugate targeting to GLUT-1 in HepG-2 and MCF-7 Cells

A simple procedure for the preparation of oligonucleotide-peptide conjugate was developed. p-Hydroxy-benzoic acid was used as a linker for the connection of the fragments of peptide and oligonucleotide. It was found that such formed linkage was stable under the conditions of conjugate synthesis. The designed conjugate targeting to GLUT-1 showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Comparing to the results from the expressed antisense RNA in cancer cells, it was proposed that the conjugate of signal peptide mimic and antisense oligonucleotide could improve the permeability of antisense oligonucleotide through cell membrane.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells. A double immunocytochemical study

Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP anti-body producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin (mu or gamma chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin anti-body-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.     

Transformation from block-type to graft-type oligonucleotide-glycopolymer conjugates by self-organization with half-sliding complementary oligonucleotides and their lectin recognition

Block-type oligonucleotide-glycopolymer conjugates bearing alpha-mannosides and beta-galactosides were prepared by coupling 5'-thiol-modified oligonucleotides with iodoacetamidated glycopolymers that were synthesized by telomerization. The conjugates minimally affected the DNA conformation and melting behavior of the duplex. Their self-organization via hybridization with the half-sliding complementary oligonucleotides produced graft-type conjugates or macromolecular gapped DNA duplexes grafted with glycopolymers at regular intervals, which was confirmed using size exclusion chromatography and electrophoresis. The binding affinity of block-type and self-organized graft-type conjugates to lectins was investigated using fluorometry. The affinity of the graft-type duplex assembly bearing mannosides to Con A was approximately 2 times stronger than that of block-type single-stranded or double-stranded conjugates with full complementary oligonucleotides. The organization strategy of DNA-glycopolymer conjugates might be useful for constructing novel glyco-clusters and also for developing a new methodology for gene therapy.