In vitro selection of RNA against kanamycin B.

Aminoglycosides are well-known antibiotics that function by interacting with ribosomal RNA in bacteria. In order to understand the molecular details between RNA and the drug, RNA aptamer was selected against kanamycin B. After 12 cycles of selection, RNA was cloned and sequenced. Among 9 clones, sequences of three clones were identical, suggesting the selected RNA was enriched. Among the cloned RNA molecules, the triplicated RNA was the maximum binding RNA. It showed a 180 nM affinity (KD) to the cognate aminoglycoside, as measured by a surface plasmon resonance, and a competition assay using a fluorescence anisotropy technique. The affinity of the maximum binding RNA to a similar aminoglycoside, tobramycin, was much stronger than 12 nM of KD. The binding site of the aminoglycoside in the maximum binding RNA was a stem loop located at the end of the 5' region. A stem loop structural motif, found in this study, was similar to those previously reported, even though the sequences of the RNA were totally different from the known sequences of the aminoglycoside binding site of other aptamers. The present study suggests that the aminoglycoside-binding region in RNA does not have a sequence specificity, but has a shape-specific bulged stem loop, even though it has a nanomolar affinity.     

In vitro selection of molecular beacons

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.     

In vitro selection of signaling aptamers

Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 microM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general "switch" for transducing molecular recognition to optical signals.     

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.     

In vitro selection of deoxyribozymes with DNA capping activity.

In vitro selection was used to isolate a series of deoxyribozymes from a pool of random-sequence DNAs that catalyze an ATP-dependent self-capping reaction. Each deoxyribozyme catalyzes the transfer of the nucleoside and alpha-phosphate moieties of ATP to the phosphate group located at its 5' terminus, thereby creating a 5',5'-pyrophosphate cap. This same pyrophosphate cap structure is formed by T4 DNA ligase during the classical process of DNA ligation. These DNA capping enzymes representative of a collection of self-processing deoxyribozymes that can be used for the directed modification of DNA.     

In vitro selection and extended culture of antigen-specific T lymphocytes. II. Mechanisms of selection.

Functional selection of antigen-specific T lymphocytes can be achieved by culturing thymus-dependent (T) lymphocytes from immunized guinea pigs on "monolayers" of antigen-pulsed adherent peritoneal exudate cells (PEC) from nonimmune syngeneic donors. Several aspects of the in vitro selection of T lymphocyte-rich peritoneal exudate lymphocytes (PEL) were studied. It was shown that irradiated adherent PEC were equivalent to nonirradiated adherent PEC in supporting selection cultures, indicating that the lymphocytes harvested at the end of the selection culture derive from the immune donors of the PEL and not from the nonimmune donor of the adherent PEC. The relative importance of specific adherence and specific proliferation for achieving selection was determined by comparing the degree of selection obtained when nonadherent cells were discarded at 24 hr with that noted when the discard step was omitted. It was found that omitting the discard step markedly diminished the degree of selection. On the other hand, blocking proliferation with specific alloantisera after the discard step did not diminish the degree of selection, although it did diminish the cell yield. Thus, specific adherence to antigen-pulsed PEC appeared to be critical in the selection culture procedure. An estimate of the degree of enrichment obtained by the selection culture procedure was obtained by culturing selected cells in an excess of nonprimed PEL, so that auxiliary cells would not be limiting. Under these conditions, it appeared that selected cells were enrichied from 4- to 10-fold in antigen-responsive cells with respect to the initial cell population.     

In vitro selection of hemin-binding catalytic RNA

Catalytic RNAs with peroxidase activity were obtained by the in vitro selection of RNA aptamer-binding hemin. One of the RNA aptamers selected showed binding affinity to hemin with a dissociation constant of 0.8 μM and exhibited high peroxidase activity by forming a complex with hemin. The catalytic efficiency of the RNA–hemin complex was 10-fold higher than that of hemin alone.     

In vitro selection of adenine-dependent hairpin ribozymes

Adenine-dependent hairpin ribozymes were isolated by in vitro selection from a degenerated hairpin ribozyme population. Two new adenine-dependent ribozymes catalyze their own reversible cleavage in the presence of free adenine. Both aptamers have Mg(2+) requirements for adenine-assisted cleavage similar to the wild-type hairpin ribozyme. Cleavage kinetics studies in the presence of various other small molecules were compared. The data suggest that adenine does not induce RNA self-cleavage in the same manner for both aptamers. In addition, investigations of pH effects on catalytic rates show that both adenine-dependent aptamers are more active in basic conditions, suggesting that they use new acid/base catalytic strategies in which adenine could be involved directly. The discovery of hairpin ribozymes dependent on adenine for their reversible self-cleavage presents considerable biochemical and evolutionary interests because we show that RNA is able to use exogenous reactive molecules to enhance its own catalytic activity. Such a mechanism may have been a means by which the ribozymes of the RNA world enlarged their chemical repertoire.     

In vitro selection of RNAs with increased tertiary structure stability.

An in vitro selection system was devised to select RNAs based on their tertiary structural stability, independent of RNA activity. Selection studies were conducted on the P4-P6 domain from the Tetrahymena thermophila group I intron, an autonomous self-folding unit that contains several important tertiary folding motifs including the tetraloop receptor and the A-rich bulge. Partially randomized P4-P6 molecules were selected based on their ability to fold into compact structures using native gel electrophoresis in the presence of decreasing concentrations of MgCl2. After 10 rounds of the selection process, a number of sequence alterations were identified that stabilized the P4-P6 RNA. One of these, a single base deletion of C209 within the P4 helix, significantly stabilized the P4-P6 molecule and would not have been identified by an activity-based selection because of its essential role for ribozyme function. Additionally, the sequence analysis provided evidence that stabilization of secondary structure may contribute to overall tertiary stability for RNAs. This system for probing RNA structure irrespective of RNA activity allows analysis of RNA structure/function relationships by identifying nucleotides or motifs important for folding and then comparing them with RNA sequences required for function.     

In vitro selection for sense codon suppression

The universal genetic code links the 20 naturally occurring amino acids to the 61 sense codons. Previously, the UAG amber stop codon (a nonsense codon) has been used as a blank in the code to insert natural and unnatural amino acids via nonsense suppression. We have developed a selection methodology to investigate whether the unnatural amino acid biocytin could be incorporated into an mRNA display library at sense codons. In these experiments we probed a single randomized NNN codon with a library of 16 orthogonal, biocytin-acylated tRNAs. In vitro selection for efficient incorporation of the unnatural amino acid resulted in templates containing the GUA codon at the randomized position. This sense suppression occurs via Watson-Crick pairing with similar efficiency to UAG-mediated nonsense suppression. These experiments suggest that sense codon suppression is a viable means to expand the chemical and functional diversity of the genetic code.     

In vitro selection of drug resistant Schistosoma mansoni

Schistosomules of Schistosoma mansoni were cultured for 3 days in the presence of schistosomicides and then inoculated intraperitoneally into mice. Drug concentrations killing greater than 99.8% of schistosomules were amoscanate 0.1 p.p.m., oltipraz, 0.5 p.p.m., oxamniquine 240 p.p.m., praziquantel 8 p.p.m. Comparison of drug response of the unselected and selected strains as adult worms in mice showed an increase in tolerance to amoscanate, oltipraz and oxamniquine, but not praziquantel. The oxamniquine tolerant strain did not respond to oxamniquine at 500 mg kg⁻¹. The unselected strain increased in tolerance to three drugs during routine passage in the laboratory. Greater numbers of schistosomules derived from snails exposed to ethyl methane sulfonate appeared to survive culture in metrifonate, suggesting that it may be possible to produce drug resistant schistosomes by mutation and selection.     

In vitro selection of functional nucleic acid sequences

The power of in vitro selection methods for the isolation of nucleic acids that display a desired property derives from the enormous number of sequence variants that can be surveyed with relative ease using controlled in vitro biochemistry. This methodology has found a variety of applications, ranging from the study of nucleic acid-protein interactions and natural ribozymes to the isolation of nucleic acids with potential as diagnostic or therapeutic reagents or with new catalytic activities. The number of reported applications is growing exponentially, and each application presents new variables and challenges. The goal of this article is to guide prospective users through the myriad decisions that must be made in the design and execution of a successful in vitro selection experiment.     

In vitro selection of kinase and ligase deoxyribozymes

Exploration of the limits of biocatalysis has led to the discovery that DNA has significant potential for enzymatic function. This makes possible the construction of DNA enzymes or "deoxyribozymes" for catalyzing various chemical reactions that could be used to address fundamental questions in biocatalysis or that could find unique applications in biotechnology. Of significant interest are self-modification reactions, given the fundamental role that DNA serves in modern living systems. Recently, in vitro selection strategies have been used to isolate prototypical ATP-dependent deoxyribozymes from random-sequence populations of DNA that catalyze DNA phosphorylation and others that catalyze DNA adenylation. In nature, protein enzymes such as T4 DNA kinase and T4 DNA ligase catalyze identical chemical reactions. These findings suggest that DNA constructs could be engineered to efficiently catalyze other self-modifying reactions, including ATP-dependent DNA ligation. This article provides a detailed overview of the methods used to isolate deoxyribozymes that promote ATP-dependent DNA ligation.