Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biotinylation of phosphodiester oligodeoxynucleotides (PO-ODN) allows for conjugation to avidin-based transcellular delivery systems. In addition, biotinylation of PO-ODN at the 3'-terminus provides complete protection against serum 3'-exonuclease degradation. The present study was undertaken to determine if antisense 3'-biotinylated PO-ODN-avidin constructs are able to recognize and inactivate the target mRNA through RNase H-mediated degradation. A 21-mer antisense PO-ODN complementary to the tat gene encompassing nucleotides 5402-5422 of the HIV-1 genome was synthesized with biotin conjugated to the 3'-terminus (bio-tat). Gel mobility assays using [5'-32P]-labeled bio-tat ODN and avidin showed that the bio-tat ODN was fully monobiotinylated. Aliquots of [32P]-labeled sense or antisense tat RNA (337 and 351 nucleotides, respectively) were prepared from transcription plasmids and were preincubated with an excess of bio-tat ODN with or without avidin constructs and digested with RNase H. Products were resolved with sequencing gel and analyzed by autoradiography. Complete conversion to predicted RNA fragments resulting from RNase H digestion of the RNA-ODN duplex (53 and 263 nucleotides) was observed when [32P]-tat sense RNA was incubated with antisense bio-tat ODN or conjugated to avidin or an avidin-cationized human serum albumin (cHSA) complex. Conversely, no degradation of [32P]-tat-antisense RNA was observed after incubation with antisense bio-tat ODN and RNase H. In addition, the avidin-cHSA complex significantly increased (84-fold) the uptake of [32P]-internally labeled bio-tat ODN and its stability against cellular nuclease degradation in peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Complete inactivation of target mRNA by biotinylated antisense oligodeoxynucleotide-avidin conjugates. 784 69

Efforts have been made to reduce the disadvantages associated with the natural oligonucleotides (all-PO) for antisense application by introducing phosphorothioate (PS) linkages into the molecule. A series of such oligodeoxynucleotide copolymers (17-mers) complementary to the coding region of the rabbit beta-globin mRNA, and containing different proportions and arrangements of PO and PS bonds, were synthesized and tested for their protein-binding properties, nuclease stability in vitro, hybridizing ability with the complementary DNA (cDNA), ability to form RNase H-sensitive substrates and antisense activity in cell-free systems. The melting temperatures (Tm) of the co-polymers were reduced by up to 6 degrees C relative to the all-PO oligo, compared to 11 degrees C for the all-PS compound, indicating intermediate hybridizing abilities of the co-polymers. The protein-binding studies with human serum albumin exhibited a linear correlation with the percentage of PS linkage present in the molecule. Nuclease susceptibilities of the co-polymers were also improved, but the number and position of the PS linkages played a significant role in such improvement. Translation inhibition by these oligonucleotides was only found in wheat germ agglutinin (WGA) extract, but not in rabbit reticulocyte lysate (RRL) cell-free system, suggesting the involvement of RNase H in their antisense activities. Provided they have > or = 50% PS linkages, the co-polymers produced almost the same increased inhibition in the WGA system as that of the all-PS oligo. The translation arrest in WGA extract is in good agreement with the in vitro cleavage found for rabbit globin mRNA in the oligo:mRNA duplex by RNase H alone. It is concluded that a copolymer of PO and PS might be preferable to either all-PO or all-PS for antisense applications.
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PMID:Phosphorothioate-phosphodiester oligonucleotide co-polymers: assessment for antisense application. 838 13

Dense, ultrathin networks of isocyanate terminated star-shaped poly(ethylene oxide) (PEO) molecules, cross-linked at their chain ends via urea groups, were shown to be extremely resistant to unspecific adsorption of proteins while at the same time suitable for easy biocompatible modification. Application by spin coating offers a simple procedure for the preparation of minimally interacting surfaces that are functionalized by suitable linker groups to immobilize proteins in their native conformations. These coatings form a versatile basis for biofunctional and biomimetic surfaces. We have demonstrated their advantageous properties by using single-molecule fluorescence microscopy to study immobilized proteins under destabilizing conditions. Biotinylated ribonuclease H (RNase H) was labeled with a fluorescence resonance energy transfer (FRET) pair of fluorescent dyes and attached to the surface by a biotin-streptavidin linkage. FRET analysis demonstrated completely reversible denaturation/renaturation behavior upon exposure of the surface-immobilized proteins to 6 M guanidinium chloride (GdmCl) followed by washing in buffer. A comparison with bovine serum albumin (BSA) coated surfaces and linear PEO brush surfaces yielded superior performance in terms of chemical stability, inertness and noninteracting nature of the star-polymer derived films.
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PMID:Biofunctionalized, ultrathin coatings of cross-linked star-shaped poly(ethylene oxide) allow reversible folding of immobilized proteins. 1505 12