Photomodulating RNA cleavage using photolabile circular antisense oligodeoxynucleotides
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Abstract:
Caged antisense oligodeoxynucleotides (asODNs) are synthesized by linking two ends of linear oligodeoxynucleotides using a photocleavable linker. Two of them (H30 and H40) have hairpin-like structures which show a large difference in thermal stability (Delta T(m) = 17.5 degrees C and 11.6 degrees C) comparing to uncaged ones. The other three (C20, C30 and C40) without stable secondary structures have the middle 20 deoxynucleotides complementary to 40-mer RNA. All caged asODNs have restricted opening which provides control over RNA/asODN interaction. RNase H assay results showed that 40-mer RNA digestion could be photo-modulated 2- to 3-fold upon light-activation with H30, H40, C30 and C40, while with C20, RNA digestion was almost not detectable; however, photo-activation triggered >20-fold increase of RNA digestion. And gel shift assays showed that it needed >0.04 microM H40 and 0.5 microM H30 to completely bind 0.02 microM 40-mer RNA, and for C40 and C30, it needed >0.2 microM and 0.5 microM for 0.02 microM 40-mer RNA binding. However, even 4 microM C20 was not able to fully bind the same concentration of 40-mer RNA. By simple adjustment of ring size of caged asODNs, we could successfully photoregulate their hybridization with mRNA and target RNA hydrolysis by RNase H with light activation.Keywords:
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Nuclease protection assay
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Cleavage (geology)
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In an attempt to develop a lead for the application of 2–5A-antisense to the targeted destruction of human immunodeficiency virus (HIV) RNA, specific target sequences within the HIV mRNAs were identified by analysis of the theoretical secondary structure. 2-5A-antisense chimeras were chosen against a total of 11 different sequences: three in the gag mRNA, three in the rev mRNA and five in the tat mRNA. 2-5A-antisense chimera synthesis was accomplished using solid-phase phosphoramidite chemistry. These chimeras were evaluated for their activity in a cell-free assay system using purified recombinant human RNase L to effect cleavage of 32 P-labelled RNA transcripts of plasmids derived from HIV NL4-3. This screening revealed that of the three 2-5A-antisense chimeras targeted against gag mRNA, only one had significant HIV RNA cleavage activity, approximately10-fold-reduced compared to the parent 2-5A tetramer and comparable to that reported for the prototypical 2-5A-anti-PKR chimera, targeted against PKR mRNA. The cleavage activity of this chimera was specific, since a scrambled antisense domain chimera and a chimera without the key 5′-monophosphate moiety were both inactive. The 10 other 2-5A-antisense chimeras against tat and rev had significantly less activity. These results imply that HIV gag RNA, like PKR RNA and a model HIV tat-oligoA- vif RNA, can be cleaved using the 2-5A-antisense approach. The results further imply that not all regions of a potential RNA target are accessible to the 2-5A-antisense approach.
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ABSTRACT The par stability determinant, encoded by the Enterococcus faecalis plasmid pAD1, is the only antisense RNA regulated postsegregational killing system identified in gram-positive bacteria. Because of the unique organization of the par locus, the par antisense RNA, RNA II, binds to its target, RNA I, at relatively small, interspersed regions of complementarity. The results of this study suggest that, rather than targeting the antisense bound message for rapid degradation, as occurs in most other antisense RNA regulated systems, RNA I and RNA II form a relatively stable, presumably translationally inactive complex. The stability of the RNA I-RNA II complex would allow RNA I to persist in an untranslated state unless or until the encoding plasmid was lost. After plasmid loss, RNA II would be removed from the complex, allowing translational activation of RNA I. The mechanism of RNA I activation in vivo is unknown, but in vitro dissociation experiments suggest that active removal of RNA II, for example by a cellular RNase, may be required.
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RNA-induced transcriptional silencing
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Our previous study identified approximately 6,000 abiotic stress-responsive noncoding transcripts existing on the antisense strand of protein-coding genes and implied that a type of antisense RNA was synthesized from a sense RNA template by RNA-dependent RNA polymerase (RDR). Expression analyses revealed that the expression of novel abiotic stress-induced antisense RNA on 1,136 gene loci was reduced in the rdr1/2/6 mutants. RNase protection indicated that the RD29A antisense RNA and other RDR1/2/6-dependent antisense RNAs are involved in the formation of dsRNA. The accumulation of stress-inducible antisense RNA was decreased and increased in dcp5 and xrn4, respectively, but not changed in dcl2/3/4, nrpd1a and nrpd1b RNA-seq analyses revealed that the majority of the RDR1/2/6-dependent antisense RNA loci did not overlap with RDR1/2/6-dependent 20-30 nt RNA loci. Additionally, rdr1/2/6 mutants decreased the degradation rate of the sense RNA and exhibited arrested root growth during the recovery stage following a drought stress, whereas dcl2/3/4 mutants did not. Collectively, these results indicate that RDRs have stress-inducible antisense RNA synthesis activity and a novel biological function that is different from the known endogenous small RNA pathways from protein-coding genes. These data reveal a novel mechanism of RNA regulation during abiotic stress response that involves complex RNA degradation pathways.
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Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.
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Antisense therapy
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A mutant of ribonuclease T1 (RNase T1), denoted RNase Tα, that is designed to recognize double-stranded ribonucleic acid was created. RNase Tα carries the structure of RNase T1 except for a part of its loop L3 domain, which has been swapped for a corresponding domain from α-sarcin. The RNase Tα maintains the pleated β-sheet structure and retains the guanyl-specific ribonuclease activity of the wild-type RNase T1. A steady-state kinetic study on the RNase Tα-catalyzed transesterification of GpU dinucleoside phosphates reveals a slightly reduced Km value of 6.94×10−7 M. When the stranded specificity is examined, RNase Tα catalyzes the hydrolysis of guanine base not only of single-stranded but also, as by design, of double-stranded RNA. The change of stranded specificity suggests the feasibility of using domain swapping to make a substrate-specific ribonuclease. This study suggests that the loop L3 in RNase T1 can be used as a ‘cassette player’ for inserting a functional domain to make ribonuclease of various specificities.
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RNase H
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Antisense therapy
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RNase H
Ribonucleotide
Ribonuclease III
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RNA digestion by RNase H, which is responsible for the antisense effect, was efficiently photoregulated by use of the duplex of azobenzene-tethered sense DNA and native antisense DNA. In the dark, RNA digestion was suppressed because antisense DNA was strongly hybridized with azobenzene-tethered sense DNA, and accordingly RNA was isolated. On UV irradiation, antisense DNA was released from the azobenzene-tethered DNA due to the trans-to-cis isomerization and hybridized with RNA, which was digested by RNase H.
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Through the use of two internal controls, we have developed an improved method of quantitating ribonuclease protection assay (RPA) results. A truncated sense RNA fragment and an antisense RNA fragment for the gene of interest were transcribed from PCR fragments containing T7 bacterial promoters. An 18S ribosomal RNA fragment was also used. When radiolabeled antisense and 18S probes, along with sense fragment and sample RNA, were hybridized, digested with RNase A/T1 and gel-electrophoresed, three distinct bands resulted. The antisense RNA fragment bound to the sense RNA fragment confirmed the integrity of the reaction. The antisense RNA fragment bound to endogenous mRNA measured the amount of specific gene expression in the sample. The 18S RNA fragment bound to endogenous mRNA determined the actual amount of sample added to the gel. Using the specific activities of the antisense and 18S transcripts, and scintillation counts of the protected fragments, we calculated the amounts of message and total RNA on the gel, determining picogram of message per microgram of total RNA. Final results were not based on assumed original amounts of RNA placed in the assay nor were they biased by lane-to-lane variations. Through the described adaptations, we have developed a well-controlled RPA that accurately and reproducibly quantifies gene expression.
Nuclease protection assay
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