Random sequence pool

The process of selecting an RNA catalyst with a particular function begins with a large pool of random sequences. This pool can be acquired by producing random single-stranded DNA on a nucleic acid synthesizer or by in vitro mutagenesis of an existing nucleic acid library [18,19]. Random sequence pools produced by chemical... 

The Rev protein of HIV-1 facilitates the nuclear export of incompletely spliced viral mRNAs and plays, therefore, an important role in the production of viral structural proteins. Rev specifically binds to a responsive element which folds into a stem-internal loop-stem secondary structure, the Rev-binding element (RBE) located into the rev gene. In vitro selection has been used to determine interactions between Rev and the RBE. RNA motifs which could bind Rev up to ten-fold better than the wild-type sequence have been isolated either from an RNA library constituted of partly randomized RBE (Bartel et al., 1991) or from completely random sequence pools, based on the RBE secondary structure (Giver et al., 1993 Tuerk and MacDougal-Waugh, 1993). Novel RNA sequences and secondary structural motifs have been selected. In particular, a wild-type G G pair is frequently replaced by an A A or even by a C A pair which are isosteric (Giver etal., 1993). 

Nucleic acids that can perform a wide variety of binding reactions have been selected from random sequence pools by affinity immobilization. Oliphant et al. [2] selected DNA molecules that could bind to the yeast transcriptional activator GCN4 from a random-sequence DNA pool that spanned nine positions. Since then, aptamers (nucleic acid ligands) have been selected against a variety of protein targets that naturally bind to nucleic acids, such as EF-Tu, ribosomal proteins, QP replicase, and reverse transcriptase (reviewed in Ref. 3). In addition, aptamers have been selected against intracellular and... 

Finally, novel nucleic acid catalysts have also been selected from random sequence pools (reviewed in Ref. 19). Joyce and co-workers have manipulated the function of the Group I self-splicing ribozyme, selecting variants that can utilize calcium or cleave DNA from partially randomized pools [20,21], Lorsch and Szostak [22] selected a polynucleotide kinase ribozyme from a completely random sequence pool that flanked a previously selected ATP binding site. Many of the novel ribozymes can catalyze reactions that are relevant to protein biosynthesis, bolstering arguments that translation may have arisen in a putative RNA world. For example, Lohse and Szostak [23] have selected ribozymes that can carry out an acyl transfer reaction, while Illangasekare et al. [24] have isolated a... 

How does the length of the random sequence pool affect the landscape ... 

Aptamer Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. DNA or RNA aptamers consist of (usually short) strands of oligonucleotides, while peptide aptamers consist of a short variable peptide domain, attached at both ends to a protein scaffold. 

PCR amplification of the single-stranded (ss) DNApool will result in multiple copies of a double-stranded DNA pool. Chemical lesions occurring during the chemical synthesis of the ss DNA pool, and the possibility that some sequences are more amplified than others during PCR procedures, may result in a limited pool size and predominance of sequence motifs in the random sequence (see below). Therefore it is necessary to sequence and analyze a small number of individual sequences of the random pool (2). 

Use a word processing program to analyze random sequences for equal occurrence of AA, AC, AG, and AT motifs in the random regions (repeat for C, G, and T). If one or more of these motifs predominate or the base composition is not random in the random sequences, the pool may not contain enough different sequences for a successful SELEX experiment. 

SELEX is a widely used technique for screening of aptamers which are nucleic acid ligands. According to this method, a pool of DNA with a random sequence region attached to a constant chain is constituted by amplification then transcribed to RNA. RNA pool is separated according to the affinity of RNA molecules to a target protein. DNA molecules obtained by reverse transcription from retarded RNA molecules are amplified and the cycle is repeated. 

Bartel, D.P. Szostak, J. W. (1993). Isolation of a new ribozyme from a large pool of random sequences. Science 261, 1411-1418. 

Tian, H. and Kole, R. (1995) Selection of novel exon recognition elements from a pool of random sequences. Mol. Cell Biol., 15, 6291-6298. 

Deoxyribozymes (also called DNA enzymes or DNAzymes) are specific sequences of DNA that have catalytic activity. All currently known deoxyribozymes have been identified by in vitro selection from large random-sequence DNA pools (Joyce, 2004 Silverman, 2009). The catalytic range of DNA encompasses both oligonucleotide and nonoligonucleotide substrates (Baum and Silverman, 2008 Silverman, 2008). This report focuses on deoxyribozymes that are useful for reactions of RNA substrates, especially to assist studies of RNA structure, folding, and catalysis. 

Fig. 8. Consensus sequences of arginine aptamers. (a) Sequence derived from a pool containing 25 random sequence positions Connell et al. [38], (b) This sequence binds to both arginine and guanosine [39], (c) 30 positions Tao and Frankel [40], (d) 74 random positions, partially random pool Famulok et al. [41]. (e) 113 random positions Geiger et al. [42].

The amount of double-stranded DNA fragments required depends on the number of randomized nucleotides and on the expectation of how many times a unique sequence combination should be represented in the pool. Each extension of the randomized sequence by one nucleotide requires a fourfold increase of nucleic acid molecules [61]. Another important parameter is the transformation efficiency of the system used. 

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