We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.

Molecular Beacons: Probes that Fluoresce upon Hybridization

This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and the oligonucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Expression monitoring by hybridization to high density oligonucleotide arrays

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report here that bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides induce murine B cells to proliferate and secrete immunoglobulin in vitro and in vivo. This activation is enhanced by simultaneous signals delivered through the antigen receptor. Optimal B-cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5' purines and two 3' pyrimidines. Oligodeoxynucleotides containing this CpG motif induce more than 95% of all spleen B cells to enter the cell cycle. These data suggest a possible evolutionary link between immune defence based on the recognition of microbial DNA and the phenomenon of 'CpG suppression' in vertebrates. The potent immune activation by CpG oligonucleotides has implications for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.

Cpg motifs in bacterial dna trigger direct b-cell activation

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

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

Nuclease-resistant phosphorothioate analogs of certain oligodeoxynucleotides have been tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Phosphorothioate analogs complementary to HIV sequences, as well as noncomplementary analogs including homooligomers, exhibited potent antiviral activity. The antiviral activity was related to the base composition of the analogs, and longer phosphorothioates were more effective than shorter ones. A 28-mer phosphorothioate oligodeoxycytidine (S-dC28) at a concentration of 1 microM exhibited potent antiviral activity and inhibited de novo viral DNA synthesis as shown by Southern blot analysis. However, S-dC28 failed to inhibit gag expression in chronically infected T cells assessed by immunofluorescent assay at concentrations up to 25 microM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanism of action is likely to be complex.

https://www.pnas.org/content/pnas/84/21/7706.full.pdf

Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.

Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells

Oligonucleotides specifically bind to complementary sequences of either genomic DNA or genomic RNA through hydrogen bonding of base pairs. In principle, relatively short oligomers (<20 bases) can specifically hybridize with DNA or RNA and thus be used for novel drug design strategies involving targeted interference of genetic expression at the level of transcription or translation. Conceivable chemotherapeutic applications predicated on sequence-specific hybridization (“antisense” inhibition) require oligonucleotide analogues that are resistant to in vivo degradation by enzymes such as nucleases. Nuclease-resistant analogues having modified internucleoside linkages (e.g., methylphosphonates or phosphorothioates) or modified nucleosides (e.g., 2′-0-methylribose or α-anomers) are now readily available by means of automated synthesis, and there are various classes of pendant groups (e.g., alkylating or intercalating agents) that can be attached to increase the efficacy of these analogues. The present account reviews this area of research by classifying structures and mechanisms of action, with comments on stereochemistry. Biological studies are briefly summarized, and pharmaceutically related topics of interest are noted.

Oligonucleotide Analogues as Potential Chemotherapeutic Agents

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2886%2981539-3

Gerald Zon

Papers

Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus.

Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells

Oligonucleotide Analogues as Potential Chemotherapeutic Agents

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy

Automated solid-phase synthesis, separation, and stereochemistry of phosphorothioate analogs of oligodeoxyribonucleotides