Unmethylated CpG motifs are prevalent in bacterial but not vertebrate genomic DNAs. Oligodeoxynucleotides (ODN) containing CpG motifs activate host defense mechanisms leading to innate and acquired immune responses. The recognition of CpG motifs requires Toll-like receptor (TLR) 9, which triggers alterations in cellular redox balance and the induction of cell signaling pathways including the mitogen activated protein kinases (MAPKs) and NF kappa B. Cells that express TLR-9, which include plasmacytoid dendritic cells (PDCs) and B cells, produce Th1-like proinflammatory cytokines, interferons, and chemokines. Certain CpG motifs (CpG-A) are especially potent at activating NK cells and inducing IFN-alpha production by PDCs, while other motifs (CpG-B) are especially potent B cell activators. CpG-induced activation of innate immunity protects against lethal challenge with a wide variety of pathogens, and has therapeutic activity in murine models of cancer and allergy. CpG ODN also enhance the development of acquired immune responses for prophylactic and therapeutic vaccination.

CpG Motifs in Bacterial DNA and Their Immune Effects

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.

III. Foldamer Research 3910 A. Overview 3910 B. Motivation 3910 C. Methods 3910 D. General Scope 3912 IV. Peptidomimetic Foldamers 3912 A. The R-Peptide Family 3913 1. Peptoids 3913 2. N,N-Linked Oligoureas 3914 3. Oligopyrrolinones 3915 4. Oxazolidin-2-ones 3916 5. Azatides and Azapeptides 3916 B. The â-Peptide Family 3917 1. â-Peptide Foldamers 3917 2. R-Aminoxy Acids 3937 3. Sulfur-Containing â-Peptide Analogues 3937 4. Hydrazino Peptides 3938 C. The γ-Peptide Family 3938 1. γ-Peptide Foldamers 3938 2. Other Members of the γ-Peptide Family 3941 D. The δ-Peptide Family 3941 1. Alkene-Based δ-Amino Acids 3941 2. Carbopeptoids 3941 V. Single-Stranded Abiotic Foldamers 3944 A. Overview 3944 B. Backbones Utilizing Bipyridine Segments 3944 1. Pyridine−Pyrimidines 3944 2. Pyridine−Pyrimidines with Hydrazal Linkers 3945

A field guide to foldamers.

The partial amino-acid sequence of purified human transforming growth factor-beta (TGF-beta) was used to identify a series of cDNA clones encoding the protein. The cDNA sequence indicates that the 112-amino acid monomeric form of the natural TGF-beta homodimer is derived proteolytically from a much longer precursor polypeptide which may be secreted. TGF-beta messenger RNA is synthesized in various normal and transformed cells.

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Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells.

The conversion of 3',5'-disilylated 2'-O-(methylthiomethyl)ribonucleosides to 2'-O-(phthalimidooxymethyl)ribonucleosides is achieved in yields of 66% to 94%. Desilylation and dephtalimidation of these ribonucleosides by treatment with NH(4)F in MeOH produce 2'-O-aminooxymethylated ribonucleosides, which are efficient in producing stable and yet reversible 2'-conjugates upon reaction with 1-pyrenecarboxaldehyde. Exposure of 2'-pyrenylated ribonucleosides to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF or DMSO results in the cleavage of their iminoether functions to give the native ribonucleosides along with an innocuous nitrile side product. Conversely, the reaction of 2'-O-(aminooxymethyl)uridine with 5-cholesten-3-one leads to a permanent uridine 2'-conjugate, which is left unreacted when treated with TBAF. The versatility and uniqueness of 2'-O-(aminooxymethyl)ribonucleosides is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence. Furthermore, the conjugation of 2'-O-(aminooxymethyl)ribonucleosides with various aldehydes, including those generated from their acetals, is also presented. The preparation of 5'-O-(aminooxymethyl)thymidine is also achieved, albeit in modest yields, from the conversion of 5'-O-methylthiomethyl-3'-O-(levulinyl)thymidine to 5'-O-phthalimidooxymethyl-3'-O-(levuliny)lthymidine followed by hydrazinolysis of both 5'-phthalimido and 3'-levulinyl groups. Pyrenylation of the 5'-O-(aminooxymethyl)deoxyribonucleoside also provides a reversible 5'-conjugate that is sensitive to TBAF, thereby further demonstrating the usefulness of 5'-O-(aminooxymethyl)deoxyribonucleosides for permanent or reversible modification of DNA sequences. Curr. Protoc. Nucleic Acid Chem. 50:4.52.1-4.52.36. © 2012 by John Wiley & Sons, Inc.

Convenient and efficient approach to the permanent or reversible conjugation of RNA and DNA sequences with functional groups.

Deoxyribonucleoside phosphoramidites 1a-d have been regenerated, in a stepwise manner, during solid-phase oligonucleotide synthesis and have been efficiently reutilized in the solid-phase synthesis of polydeoxyribonucleotides. The activator 1H-tetrazole has also been recovered and recycled

Stepwise Regeneration and Recovery of Deoxyribonucleoside Phosphoramidite Monomers During Solid‐Phase Oligonucleotide Synthesis.

An improved process for the release of synthetic DNA sequences from a solid-phase capture support

The reaction of 2'-O-aminooxymethylribonucleosides with 2-cyano-2-methyl propanal leads to the formation of stable and yet reversible 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides in post-purification yields of 54% to 82% Phenoxyacetylation of the exocyclic amino functions of these ribonucleosides proceeds in yields of 74% to 89%, and subsequent 5'-O-dimethoxytritylation and 3'-O-phosphitylation of the corresponding N-phenoxyacetylated ribonucleosides provide the fully protected ribonucleoside phosphoramidite monomers in isolated yields of 69% to 88% These ribonucleoside phosphoramidites are employed in solid-phase synthesis of three chimeric RNA sequences, each differing in purine/pyrimidine content The stepwise coupling efficiency of the ribonucleoside phosphoramidites (as 015 M solutions in acetonitrile) averages 99% over a coupling time of 180 s when 5-benzylthio-1H-tetrazole is used as an activator Upon completion of RNA chain assembly, removal of the nucleobase- and phosphate-protecting groups and release of sequences from the solid support are carried out under standard basic conditions Finally, the 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl) protective groups are cleaved from the RNA sequences by treatment with 05 M tetra-n-butylammonium fluoride in dry DMSO for 24 to 48 hr at 55°C without releasing RNA-alkylating side-products Characterization of the fully deprotected RNA sequences by PAGE, enzymatic hydrolysis, and MALDI-TOF mass spectrometry confirms the identity and high quality of these sequences

2′‐Hydroxy Protection of Ribonucleosides as 2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl Ethers in Solid‐Phase Synthesis of RNA Sequences

B ecause of the significant progress achieved in the chemical synthesis of oligonucleotides during the past four decades, synthetic oligonucleotides have become readily available and have fueled the biotechnology revolution that has irreversibly changed biomedical research and the pharmaceutical industry. For example, without the ability to rapidly and efficiently synthesize DNA oligonucleotides, the development of the polymerase chain reaction (PCR) and its multiple applications would have been difficult, if not impossible, because this technology is completely dependent upon the use of DNA primers. Similarly, the availability of synthetic oligonucleotides has been instrumental in the development of automated DNA sequencing, which is also dependent on the use of DNA primers. An important biological application of synthetic oligonucleotides relates to site-specific mutagenesis of genes. Mutagenesis of this type has been utilized to study protein structure-function relationships, and to alter the therapeutic spectrum of pharmaceutically active proteins. Synthetic oligonucleotides have also found application as diagnostic hybridization probes and, more recently, in the use of DNA chips for highthroughput detection of genetic diseases and identification of genomic single-nucleotide polymorphisms. Furthermore, the availability of synthetic RNA oligonucleotides has led to the preparation of ribozymes, which are RNAs with catalytic activity. In this regard, it is being speculated that RNA may have been the primary self-replicating molecule from which life originated. Collectively, the multiple biomedical applications of synthetic DNA and RNA oligonucleotides are a direct measure of the colossal impact the methods for rapid and efficient preparation of these biomolecules have had on the life sciences.

Serge L. Beaucage

Papers

Convenient and efficient approach to the permanent or reversible conjugation of RNA and DNA sequences with functional groups.

Stepwise Regeneration and Recovery of Deoxyribonucleoside Phosphoramidite Monomers During Solid‐Phase Oligonucleotide Synthesis.

An improved process for the release of synthetic DNA sequences from a solid-phase capture support

2′‐Hydroxy Protection of Ribonucleosides as 2‐Cyano‐2,2‐Dimethylethanimine‐N‐Oxymethyl Ethers in Solid‐Phase Synthesis of RNA Sequences

Synthesis of Unmodified Oligonucleotides