Mutant prohead RNAs in the in Vitro packaging of bacteriophage φ29 DNA-gp3

Wichitwechkarn, Jesdawan; Johnson, D. A.; Anderson, Dwight L.

doi:10.1016/0022-2836(92)90257-k

Cited by 34 publications

(27 citation statements)

References 20 publications

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“…Previously, the highly conserved residues U 54 and A 56 were changed to A and C, respectively. Modest decreases were observed in prohead binding competitor activity and a 4-fold and 2-fold drop, respectively, in capacity to reconstitute proheads and support phage assembly in prohead-defective extracts (15). The SELEX I results suggested that the least important residues of the E loop in prohead binding were G 57 and U 58 , in which change was quite frequent (Table I).…”

Section: Fig 4 Competition Filter Binding With Rna Pools Of Selex IImentioning

confidence: 94%

“…The competition activities, which normalize the [ 32 P]pRNA fraction bound with wild-type pRNA competitor (100%) and no competitor (0%) (8, 9), were as follows: 5S rRNA, 2%; pRNA, 100%; cycle 0, 4%; cycle 1, 12%; cycle 2, 16%; cycle 3, 46%; cycle 4, 83%; cycle 5, 97%; and cycle 6, 91%. changes in the pRNA sequence can produce drastic alterations in the predicted foldings (15). Studies on pRNA mutants with limited numbers of G to A or C to U changes generated by bisulfite mutagenesis showed that the foldings of pRNA have prognostic value.…”

Section: Fig 4 Competition Filter Binding With Rna Pools Of Selex IImentioning

confidence: 99%

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In Vitro Selection of Bacteriophage φ29 Prohead RNA Aptamers for Prohead Binding

Zhang¹,

Anderson²

1998

Journal of Biological Chemistry

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Prohead RNA (pRNA) of the Bacillus subtilis bacteriophage 29 is needed for in vitro packaging of DNA-gene product 3 (DNA-gp3). Residues 22-84 of the 174-base pRNA bind the portal vertex of the prohead, the site of DNA packaging. To define the nucleotides of pRNA needed for prohead binding and DNA-gp3 packaging and to seek biologically active variants of pRNA, segments of pRNA were randomized to obtain vast repertoires of RNA molecules. RNA aptamers, ligands best suited for prohead binding, were obtained by multiple rounds of in vitro selection. Evolution of pRNA aptamers was followed by a competition binding assay and nucleotide sequencing, and mutants were tested for DNA-gp3 packaging. Aptamers selected following randomization of the E stem and loop and a part of the C-E loop that were active in DNA-gp3 packaging were invariably wildtype. DNA-gp3 packaging activity also required nucleotides G 82 and G 83 that form base pairs intermolecularly with C 47 and C 48 to produce a novel hexameric oligomer of pRNA. The only mutant aptamers that retained full DNA-gp3 packaging activity showed changes of the U residues at positions 81, 84, and 85 of the D loop. Thus, the in vitro selections essentially recapitulated the natural evolution of pRNA.The double-stranded DNA-gene product 3 (DNA-gp3) 1 complex of the Bacillus subtilis bacteriophage 29 is packaged efficiently into a prohead with the aid of the ATPase gp16 and ATP hydrolysis in a completely defined in vitro system (1). A unique 174-base 29-encoded RNA, termed prohead RNA (pRNA), is present on the portal vertex (head-tail connector) of the prohead and is an essential constituent of the DNA packaging machine (2). pRNA is hypothesized to bind a supercoiled DNA-gp3-gp16 complex to link the DNA and prohead (3), recognize the left end of the DNA-gp3, which is packaged first (4), and unite with gp16 to form the DNA translocating ATPase (5).The secondary structure of the pRNA was established by a phylogenetic analysis (6). pRNA binding to proheads is specific, rapid, and irreversible in the presence of 10 mM Mg 2ϩ . Proheads protect nucleotides 22-84, 5Ј to 3Ј, of pRNA from ribonuclease attack, and the use of site-directed mutants of pRNA has identified elements and sequences of pRNA that are required for prohead binding and DNA-gp3 packaging (7-9). The mutant studies also identified a pseudoknot involving a ninemembered bulge loop and a five-base hairpin loop in pRNA that is essential in DNA-gp3 packaging (9). Recently, the pseudoknot has been shown to be an intermolecular interaction, requiring just two base pairs, that links six identical molecules of pRNA into a structure that is positioned on the portal vertex of the prohead. The structure of pRNA as it interacts with the proteins of the packaging machine and DNA-gp3 is integral to understanding the mechanism of DNA packaging. To better define the sequence and structural elements of pRNA essential for prohead binding, a 62-base segment of the prohead binding domain (residues 30 -91) was partially randomized, and pRNA...

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Section: Fig 4 Competition Filter Binding With Rna Pools Of Selex IImentioning

confidence: 94%

Section: Fig 4 Competition Filter Binding With Rna Pools Of Selex IImentioning

confidence: 99%

In Vitro Selection of Bacteriophage φ29 Prohead RNA Aptamers for Prohead Binding

Zhang¹,

Anderson²

1998

Journal of Biological Chemistry

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“…For Northern blot, 20 mg of denatured RNA was resolved in a 0.6 M formaldehyde-1% agarose gel and transferred onto Hybond N+ nylon membrane (Amersham). Probes were prepared by random priming with the 1.8 kb XbaI fragment of HBV (adr) from plasmid p3.6 II and [a- 32 P] dATP according to the supplier (Promega). After hybridization with HBV probe, the blot was stripped and rehybridized with a probe of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that served as an internal control for normalizing the level of total cell RNA.…”

Section: Analysis Of Hbv Viral Rna Transcriptionmentioning

confidence: 99%

“…21 This RNA is termed packaging RNA or 'pRNA'. Computer models of the three-dimensional (3D) structure of a pRNA monomer, dimer and hexamer has been constructed 22 based on experimental data derived from photoaffinity crosslinking, 23,24 chemical modification and chemical modification interference, [25][26][27] complementary modification, [28][29][30][31][32] nuclease probing, 24,33 oligo targeting, 34 competition assays, 35,36 and cryo-atomic force microscopy. 25,27,37 pRNA hexamer docking with the connector crystal structure reveals a very impressive match with available biochemical, genetic, and physical data concerning the 3D structure of pRNA.…”

Section: Introductionmentioning

confidence: 99%

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

et al. 2003

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The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5 0 /3 0 ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems -exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5 0 /3 0 ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

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“…Cloning and production of RNA in bacteria with high yield has been reported (Wichitwechkarn et al, 1992;Ponchon and Dardel, 2007;Ponchon et al, 2009;Delebecque et al, 2011;Ponchon and Dardel, 2011). Bacteria fermentation is the direction for industry production, but currently, the bacteria high yield production of RNA nanoparticles with therapeutic functionality has not been reported.…”

Section: Low Yield and High Production Costsmentioning

confidence: 99%

Uniqueness, Advantages, Challenges, Solutions, and Perspectives in Therapeutics Applying RNA Nanotechnology

Guo¹,

Haque²,

Hallahan³

et al. 2013

RNA Nanotechnology and Therapeutics

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The field of RNA nanotechnology is rapidly emerging. RNA can be manipulated with the simplicity characteristic of DNA to produce nanoparticles with a diversity of quaternary structures by self-assembly. Additionally RNA is tremendously versatile in its function and some RNA molecules display catalytic activities much like proteins. Thus, RNA has the advantage of both worlds. However, the instability of RNA has made many scientists flinch away from RNA nanotechnology. Other concerns that have deterred the progress of RNA therapeutics include the induction of interferons, stimulation of cytokines, and activation of other immune systems, as well as short pharmacokinetic profiles in vivo. This review will provide some solutions and perspectives on the chemical and thermodynamic stability, in vivo half-life and biodistribution, yield and production cost, in vivo toxicity and side effect, specific delivery and targeting, as well as endosomal trapping and escape.

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Mutant prohead RNAs in the in Vitro packaging of bacteriophage φ29 DNA-gp3

Cited by 34 publications

References 20 publications

In Vitro Selection of Bacteriophage φ29 Prohead RNA Aptamers for Prohead Binding

In Vitro Selection of Bacteriophage φ29 Prohead RNA Aptamers for Prohead Binding

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Uniqueness, Advantages, Challenges, Solutions, and Perspectives in Therapeutics Applying RNA Nanotechnology

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