Indirect selection

Natural proteins have been selected not only to fold into stable structures, but to do so under various conditions and in reasonable time. More obviously, they have been selected for particular functions that often require hydrophobic surface groups and nonregular secondary structures. Finally, most extant proteins have been indirectly selected to be insensitive to most mutations. We note that those early proteins that could not accept numerous mutations without loss of function or structure would not adapt to changing conditions or functional requirements. All of these factors mean that natural proteins are unlikely to fit any single optimization concept. 

In vitro selection strategies can be sub-divided into two types direct and indirect selections. These two types of selection experiments directed at the isolation of synthetic catalytic nucleic acids differ mainly by their technical concept, their design and their outcome. 

While indirect selections work quite well for antibodies they have been less successful in the case of catalytic nucleic acids. There are only three examples which prove that it is possible in principle to obtain a ribo- or deoxyribozyme by selecting an aptamer that binds to a TSA A rotamase ribozyme [7], a ribozyme capable of catalyzing the metallation of a porphyrin derivative [92], and one catalytic DNA of the same function [93]. Another study reported the selection of a population of RNA-aptamers which bind to a TSA for a Diels-Alder reaction but the subsequent screen for catalytic activity was negative for all individual RNAs tested [94]. The attempt to isolate a transesterase ribozyme using the indirect approach also failed [95]. 

The second example of a catalytic RNA obtained by the indirect selection approach is the isolation of a 35 nucleotide RNA molecule which binds meso-porphyrin IX and catalyzes the insertion of Cu into the porphyrin with a value of kcat/Kivi of 2100 [92]. Remarkably, the k at/KM achieved by the RNA was... 

The only indirect selection that led to a catalytic DNA is a deoxyribozyme that catalyzes the same class of porphyrin metallation as the aforementioned ribozyme. The ssDNA oligonucleotide showed a k at of 13 h" for the insertion of into mesoporphyrin IX [93, 96-99]. This corresponds to a rate enhancement of 1400 compared to the uncatalyzed reaction which is as good as a catalytic antibody for the same reaction. 

With Arthrobacter ilicis the time required for the appearance of L-ara-bitol-utilizing papillae is dramatically reduced for rhamnose-utilizing or ribitol-utilizing mutants, compared with the wild-type strain (Fig. 2). Indirect selection need not be a multistep process, however. When 110 L-arabi-tol mutants of Arthrobacter ilicis were tested, 42 could also utilize D-ribose, and 18 of the 42 could also utilize L-rhamnose. Similarly, 16 of 60 Lac+ mutants could also utilize L-arabinose. 

Other effects Internal Direct Direct and indirect Selective ingestion of less recalcitrant compounds leads to the enrichment of recalcitrant SOM Degradation of more labile compounds in the gut gradually increases the average recalcitrance of nonassimilated carbon... 

External Direct Indirect Selective translocation of SOM leads to enrichment either of less or of more recalcitrant SOM Rapid depletion of nutrients and labile carbon compounds shortly after defecation leads to an increase in recalcitrant components... 

All ribozymes described so far were isolated by the method of direct selection, whereby the partly or completely randomized pool of nucleic acids is subjected to a competitive situation in which only those molecules survive that can catalyze a particular reaction. A different strategy by which catalysis can be achieved is the indirect selection for binding to transition state analogs (TSAs), a tech-... 

There are two general steps to evolving a catalytic RNA. First, from a pool of sequence variants there must be a screening protocol that separates active RNA with the desired trait from others without it. Second, the genome of the survivors must be copied and amplified via DNA intermediates prior to the next round of selection. The process is iterative, and heavily reliant on the ease of nucleic acid copying by PCR amplification. Thereafter, there are two methods of selection, direct or indirect. In a direct selection, there is chemical transformation of the catalytic RNA during the selection step. In an indirect selection, an RNA aptamer approach is taken but RNA aptamers are identified and evolved to bind to a transition state in the reaction that is desired to be catalysed (this indirect method is similar to the strategy used to develop catalytic antibodies - see Section 10.5). 

The selection set is a presentation of the items from which the user makes choices. The elements in the selection set correspond to the elements of a specific activity output. Selection sets may have written letters, words and sentences, symbols used to represent ideas, computer icons, or line draw-ings/pictures. They may be presented in visual (e.g., letters on keys), tactile (e.g., Braille), or auditory (e.g., voice synthesis) form. We can define two selection methods through which the user makes selections using the control interface direct selection and indirect selection (Cook and Hussey, 2(X)2). For any particular application, the three elements of the human-technology interface will be chosen based on the best match to the consumer s skills (motor, sensory, linguistic, and cognitive) (Cook and Hussey, 2002). 

Indirect methods of selection use a single switch or an anay of switches. Cook and Hussey (2002) describe a number of interfaces that are used for indirect selection. 

The system in Fig. 30.1 may be modified to include remote control over TV or VCR functions such as volume control, channel selection, play, fast forward, and reverse. In this case, the level (for volume) or number (for channel) is under the control of the human user. Often these functions are incoiporated into EADLs by modifying standard TV or VCR remote controls. This may be done merely by adding a single switch to the remote control or by more elaborate adaptations that allow indirect selection. Sometimes univeisal remote controls that can learn the signal for a particular TV or VCR are used. This allows several tqqtliances to be controlled from the same EADL. Cook and Hussey (2002) describe several conunercial approaches to this type of EADL. 

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