[19] Affinity selection-amplification from randomized ribooligonucleotide pools

Ciesiołka, Jerzy; Illangasekare, Mali; Majerfeld, Irene; Nickles, Tim; Welch, Mark; Yarus, Michael; Zinnen, Shawn

doi:10.1016/s0076-6879(96)67021-9

Cited by 80 publications

(95 citation statements)

References 38 publications

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“…Fifty-microliter fractions were collected, and the first two fractions containing the leading edge of the liposome peak (detected by OD 320 ) were pooled. Liposomes were disrupted by 5-min incubation at room temperature with 0.1% Triton X-100, and RNA was precipitated with ethanol and processed as described (7). Triton X-100 was used because the chloroform extraction used previously (2) leads to loss of RNA into the interphase.…”

Section: Methodsmentioning

confidence: 99%

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes

Vlassov

Khvorova²,

Yarus³

2001

Proc. Natl. Acad. Sci. U.S.A.

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121

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In an RNA world, RNAs would have regulated traffic through normally impermeable bilayer membranes. Using selection-amplification we previously found RNAs that bind stably and increase the ionic conductance of phospholipid membranes at high Mg 2؉ and Ca 2؉ concentrations. Now selection in reduced divalents yields RNAs that bind phosphatidylcholine liposomes under conditions closer to physiological. Such affinity for phospholipid membranes requires interactions between RNAs. In fact, we detected no functional monomeric membrane-binding RNAs. A membraneactive end-to-end heterotrimer consisting of 2 RNA 9 and 1 RNA 10 is defined by nucleotide protection, oligonucleotide competition, and mutant analysis. Oligomers of the heterotrimer bind stably, cause release of liposome-encapsulated solutes, and disrupt model black membranes. Individual RNA molecules do not show any of these activities. This novel mechanism of RNA binding to lipid membranes may not only regulate membrane permeability, but suggests that arrays of catalytic or structural RNAs on membranes are plausible. Finally, a selection met only by RNA complexes evokes new possibilities for selection-amplification itself.T he RNA world hypothesis (1) suggests that ancestral RNAs played roles presently taken by both nucleic acids and proteins. To show that RNA might serve essential membrane functions, we used selection-amplification to isolate RNA molecules with affinity for phospholipid liposomes (2). Some RNAs bound stably and increased the ionic permeability of liposomes, as well as the plasma membranes of cultured human cells. Analysis of individual isolated RNAs suggested that specific RNA sequences and folds are needed for membrane activity. In fact, one RNA appeared to bind in only one of two prevalent conformers. Earlier reports detected weaker, nonspecific interactions between membranes and nucleic acids (3), but these selected RNAs were the first stably bound nucleic acids with measurable membrane effects.The goal of the present study was to elucidate the mechanism of RNA binding to lipid membranes at moderate concentrations of divalent cations, closer to those found in cells and tissues. We have selected (4, 5) RNAs capable of efficiently binding to pure phosphatidyl choline liposomes, and we investigated RNA structure and interactions with liposomes and black membranes. Surprisingly, binding to the liposome surface requires formation of complexes between RNAs. Active multimer formation occurs via pairing of varied selected complementary sequences in weakly structured terminal regions, including ''kissing loops'' (6). Experimental ProceduresSelection. Incubation of internally 32 P-labeled RNA [1,000 pmol (cycles 1-4) and 100 pmol (cycles 5-11), 30 l] with liposomes (20 l, 20 mg͞ml) was performed in 50 mM Hepes (pH 7.0), 50 mM NaCl, 5 mM MgCl 2 , and 2 mM CaCl 2 at room temperature for 5 min, followed by gel filtration on a Sephacryl S-1000 (Amersham Pharmacia) 1-ml column. To decrease nonspecific sorption, the column was presaturated with liposomes (1...

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Section: Methodsmentioning

confidence: 99%

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes

Vlassov

Khvorova²,

Yarus³

2001

Proc. Natl. Acad. Sci. U.S.A.

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121

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“…Bound RNAs were eluted with 1 mL of L-isoleucine containing buffer after washing with 5-7.5 column volumes of selection buffer. The number of template DNA sequences implies a mean of ∼ 60,000 copies of each 16-mer sequence and 19 copies of each possible 22-mer transcribed, so there is a low probability, ∼ 10 −9 (Poisson probability of zero copies, P(0) = e −19 ≈ 6 × 10 −9 ; Ciesiolka et al 1996) that any contiguous 22-mer sequence was absent. Therefore, the design of the experiments implies that any simpler 16-mer or 22-mer isoleucine site, if it exists, should be present at the start of these selections, perhaps many times over.…”

Section: Selections With Limited Numbers Of Randomized Nucleotidesmentioning

confidence: 99%

“…The selection of diverse activities from randomized RNA is relevant to the hypothesis of an RNA World (Gilbert 1986), in which it has been suggested that selection from varied, mostly nonfunctional sequences may have been a principal mode of biological evolution (Yarus 2002). This idea makes the simplest active RNA for all reactions of particular evolutionary interest, as the site requiring the fewest nucleotides should be most numerous in initial pools of randomized sequence (Ciesiolka et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Selection of the simplest RNA that binds isoleucine

Lozupone¹,

Changayil²,

Majerfeld³

et al. 2003

RNA

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114

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We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA-amino acid binding structures.

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“…RNA sequences bound to human eRF1 were isolated by filter binding using mixed cellulose ester MF-Millipore filters (catalog #HAWP02500)+ RNA pools were prepared for selection by passage through two filters in the absence of protein to eliminate filter-binding RNAs+ Then each round of selection comprised three binding reactions containing 0, 15, or 60 pmol eRF1 and 750 pmol RNA incubated at 37 8C for 4 min in 200 mL binding buffer (20 mM Tris, pH 7+5, 2 mM MgCl 2 , 40 mM NH 4 Cl, 10 mM KCl)+ MF-Millipore filters in a Millipore 1225 sampling manifold were prewashed with 1 mL of binding buffer, the binding reaction was applied, and the filters were washed with 2 mL binding buffer+ RNA retained on the filter was detected by Cerenkov scintillation+ The filter with the greatest ratio of bound radioactivity to a control with no protein was carried forward+ RNA was extracted from this filter with 1 mL of a 2:1 mixture of phenol (equilibrated to pH 8):8 M urea at room temperature for 30 min+ Water (400 mL) was added and the aqueous phase was ethanol precipitated and RNA was resuspended in water+ Reverse transcription and PCR amplification were performed by standard methods (e+g+, Ciesiolka et al+, 1996)+ After nine rounds of selection, cDNAs of the selected pool of RNAs were digested with BamHI and HindIII, cloned into the matching sites of pGEM3Zfϩ (Promega), and sequenced (Fig+ 1A)+…”

Section: Selection For Aptamers Binding To Erf1mentioning

confidence: 99%

Suppression of eukaryotic translation termination by selected RNAs

Carnes

Frolova

Zinnen³

et al. 2000

RNA

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Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1•eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1•eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

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[19] Affinity selection-amplification from randomized ribooligonucleotide pools

Cited by 80 publications

References 38 publications

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes

Binding and disruption of phospholipid bilayers by supramolecular RNA complexes

Selection of the simplest RNA that binds isoleucine

Suppression of eukaryotic translation termination by selected RNAs

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