ISSN:1050-5261    

DOI:10.1089/ard.1993.3.1

年代:1993

数据来源: MAL

摘要:

We have analyzed the implications of a simple two-compartment mathematical model (Hargrove and Schmidt, 1989) to anticipate the limits of antisense oligodeoxyribonucleotide action within single cells. The steady-state equations are derived for four special cases representing the following mechanisms: (i) ribosome blockage, (ii) mRNA cleavage by RNase H, (iii) concurrent ribosome exclusion and RNase H action, and (iv) decreased delivery of mature mRNA to the cytoplasm due to transciptional blockage, interference with nucleocytopiasmic transport, or splicing. Dose-response relationships have been derived for these mechanisms under ideal conditions. Our results indicate that frequently translated mRNAs producing stable proteins are the most attractive antisense targets because these protein levels are sensitive to the changes in the mRNA levels that can be effected using antisense oligodeoxyribonucleotides. The nonsteady-state solutions show that both mRNA and protein half-life can determine the kinetics of antisense oligonucleotide action. A rapid onset of effect will be observed when the mRNA is rapidly degraded and slowly translated and when the translated protein is rapidly degraded. When the protein is slowly degraded, the kinetics of effect are limited by protein half-life. When the translational rate constant is large compared to the absolute difference between the mRNA and protein degradation rate constants, the kinetics of antisense action are determined by both degradation rate constants but are limited by the slower of the two degradative processes. We also show that the steady-state and nonsteady-state solutions may be used to design experiments that discriminate among mechanisms of antisense action.

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.3

年代:1993

数据来源: MAL

摘要:

Although having variability in primary sequence, the v3 loop of gp120 in pathogenic strains of human immunodeficiency virus type-1 (HIV-1) is positively charged and known to interact with sulfated polysaccharides. Because the interaction of sulfated polysaccharides with the v3 loop inhibits HIV infectionin vitro, we investigated the interaction of the v3 loop with phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos). In a solid-phase ELISA assay, a PS 28-mer homopolymer of cytidine, SdC28, blocked the binding of the v3 loop-specific monoclonal antibody (mAb) 9284 to rgp120 more potently than did dextran sulfate. In addition, like dextran sulfate, SdC28 appeared to bind specifically to the v3 loop, because neither compound inhibited the binding of other anti-gp120 mAbs. In contrast to PS oligos, PO oligos did not inhibit mAb 9284 binding. The length dependence of the interaction of PS oligos with the v3 loop was studied by using a series of PS oligos. A discrete loss of inhibiting activity occurred as a function of decreasing PS oligo length, which was most marked between PS oligos of 18-mer and 12-mer in length. We further probed the chemical nature of the interaction of oligos with gp120 by measuring the gp120 binding affinities of PS and PO oligos of various lengths. We employed a 5′-32P-labeled alkylating oligo, CIRNH32P-OdT15, and determined that the Kmof gp120 binding is 4 μM. We also determined values of competition constant (Kc) for PS competitors of CIRNH32P-OdT15 binding. The binding constant (= 1/Kc) for PS oligos showed a discrete increase in gp120 binding for PS oligos>12- to 18-mer in length, with no further increment beyond an 18-mer. Given the important role of the v3 loop in HIV-1 pathogenicity, these data suggest that therapeutic trials of PS oligos should be consider

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.19

年代:1993

数据来源: MAL

摘要:

A 16-mer oligodeoxynucleotide (ODN) which specifically recognizes the polypurine tract (PPT) located upstream of the 3′ long terminal repeat (LTR) of human immunodeficiency virus (HIV) proviral DNAviatriplex formation is shown to have a dramatic effect onin vitrotranscription from the HIV-LTR promoter. In the presence of HeLa cell extracts, a shorter RNA transcript is obtained in the presence of the 16-mer ODN. This truncated RNA lacks about 200 nucleotides from its 3′ region. The PPT sequence is not responsible for this effect. Instead, this process involves a purine-rich sequence in thegagmRNA located around position +400. The imperfect hybrid formed between the 16-mer ODN and mRNA is precisely cleaved by RNase H contained in HeLa cell extracts. These data show that sophisticated control experiments must be designed before any conclusion can be drawn on the effect of oligonucleotides usedin vitroand in cell cultu

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.33

年代:1993

数据来源: MAL

摘要:

For the biological and therapeutic application of antisense nucleic acids, there is a need to identify effective local target regions of given cellular target mRNAs or viral single-stranded nucleic acids. One critical parameter for the effectiveness of antisense nucleic acids could be the potential of intramolecular folding of a given sequence element of the target strand and the antisense strand, respectively. The folding potential of such subsequences was calculated by using an established secondary structure prediction algorithm. For the genomic RNA and the complementary RNA strand of the human immunodeficiency virus type 1 (HIV-1), an energy profile was calculated that monitors the local folding potential of each sequence position surrounded by a window of given length ranging from 50 to 400 nucleotides. The resulting energy profile was compared to the effectiveness of HIV-1-directed antisense RNAs. It was found that significant minima of the local folding potential (high ΔGvalues) correlated with antisense RNA target regions involved in strong inhibition of HIV-1 replication that had been measured independently in two earlier studies by using different experimental approaches. Conversely, antisense RNAs directed against local subregions with a high folding potential (low ΔGvalues) showed weak or no antiviral effect in human cells. The results indicate that analyses of the local folding potential of a given target RNA can support the selection of effective target sequences for antisense RN

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.45

年代:1993

数据来源: MAL

摘要:

The effects of phosphorothioate (S-oligonucleotide) or terminal phosphorothioate-phosphodiester (S-O-oligonucleotides) or methylphosphonate-phosphodiester (MP-O-oligonucleotides) modifications on mouse spleen cell surface binding, uptake, and degradation were studied using fluorescein (FITC)-conjugated oligonucleotides.S-oligonucleotides had the highest cell binding and uptake, followed byS-O-,O-, and MP-O-oligonucleotides. Competition studies indicated thatS-oligonucleotides have an increased affinity for cell membrane oligonucleotide binding sites, because they could completely blockO-oligonucleotide binding at a molar ratio of just 0.1. Uptake of all oligonucleotides was higher in B cells than T cells and was increased by stimulation with the B-cell mitogen, lipopolysaccharide. Although our cells had been purified using conventional techniques to eliminate dead cells, there remained about 5% of cells that were dead or dying, as determined by flow cytometry using propidium iodide staining. Of note, oligonucleotide association with dead cells was approximately 50-fold greater than that with living cells. Confocal microscopy confirmed that the oligonucleotides in living cells were intracellular, and indicated little nuclear uptake by 4 h. While extensive degradation of intracellularO-oligonucleotides was apparent by 4 h, there was no detectable degradation ofS-,S-O, or MP-O-oligonucleotides.

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.53

年代:1993

数据来源: MAL

摘要:

We have compared the effects of a number of different oligonucleotides on the growth and morphology of normal finite life span and immortally transformed human mammary epithelial cells. The oligonucleotide sequences chosen initially for study were based on that of the NB-1 gene, which encodes a calmodulin-like protein of unknown function. We found that certain thioated oligonucleotides 15-20 residues in length altered the morphology and decreased the growth rate of the normal cells in a concentration-dependent manner. These effects were rapid, occurring within 24-48 h of oligonucleotide addition. The effects, which occurred without an accompanying detectable decrease in the levels of NB-1 mRNA or protein, were most pronounced in the normal epithelial cells, less apparent in the immortalized epithelial cells, and unobserved in normal breast fibroblasts. Identical sequences having mixed phosphodiester and phosphorothioate backbones, or phosphodiester backbones alone, had little or no effect on normal epithelial cell morphology or growth. Two out of seven additional thioated oligonucleotides which were not complementary to NB-1 mRNA, also affected normal epithelial cell morphology and growth when used at similar concentrations (10 μM). Taken together, the observed effects on normal epithelial cells indicate that certain thioated oligonucleotides may have pharmacological consequences that do not depend on strict complementarity of their sequences to known mRNAs

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.67

年代:1993

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.79

年代:1993

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.83

年代:1993

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1993.3.87

年代:1993

数据来源: MAL