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

Abstract

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...

Analysis of intermolecular base pair formation of prohead RNA of the phage ø29 DNA packaging motor using NMR spectroscopy

“…In addition, the 2D 1 H- 1 H NOESY spectra of the 19-mer/35 -uucg heterodimer are the same as those of the 19-mer/35-mer heterodimer, except for the signals derived from residues within the UUCG tetraloop (data not shown). Whereas previous mutagenesis studies have shown that the E-loop is important for the DNA packaging activity ( 17 , 43 ) and chemical modification show that it becomes protected during oligomerization ( 45 ), our current studies suggest that the E-loop is not directly involved in the intermolecular interaction of pRNA in solution.…”

“…The result of this modified HNN-COSY experiment was also negative as we did not observe any signal indicating intermolecular A–U base pairing using the U-labeled 19-mer/A-labeled 35 -uucg heterodimer (data not shown). These results indicated that only two G–C base pairs are stably formed between the D-loop and the CE-loop, in contrast to the hypothesis that four base pairs are formed between these loops ( 5 , 6 , 43 , 44 ).…”

RNA Nanotechnology: Engineering, Assembly and Applications in Detection, Gene Delivery and Therapy

“…75 98-101 SELEX allows screening for co-variation of several nucleotides and can be used to reveal noncanonical interaction which is difficult to prove by classic genetic and biochemical approaches. 75 98-101 For example, SELEX has been used to select pRNA sequences that bind procapsids 102 and proved that pRNA can bind ATP. …”

In Vitro Selection of Protein-Binding DNA Aptamers as Ligands for Biosensing Applications

“…If the protein does not contain such a tag, separation of bound and unbound DNA molecules can be carried out by using nitrocellulose filters (Millipore HWAP), which will retain the protein−DNA library complex while letting the unbound DNA pass through. Other methods for separating ligands without tags include capillary electrophoresis (12), gel electrophoresis (13), sedimentation centrifugation (14), and flow cytometry (15 3. During PCR amplification of selected DNA, it is important not to over-amplify the sequences.…”