Craig Tuerk

Selexion: Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis

Recently, novel technologies for isolation of nucleic acid molecules with specific biological activities have been reported. In each case, the enrichment process involves repeated rounds of selection from complex mixtures of nucleic acid sequences, followed by polymerase chain reaction (PCR) amplification of ligand sequences that function in the desired manner. Particular variations in experimental conditions can dramatically alter the outcome of these processes. In this study, we use mathematical analysis and computer simulation to predict which variations have the greatest impact and to develop strategies and guidelines for enhanced effectiveness. First, we perform reconstruction tests to demonstrate that a mathematical description based on equilibrium binding is sufficient to explain the high levels of enrichment attained in the laboratory after just a few rounds. Then, we show the expected enrichment for an extensive range of conditions; and, finally, we determine the optimum protein and nucleic acid concentrations to use for maximum enrichment, while also ensuring a high likelihood of recovering even the rare molecule that binds well. The strategies and guidelines for enhanced effectiveness are generally applicable to processes for systematic enrichment of DNA, RNA or peptide ligands and have been implemented in an interactive simulation program for integrated non-linear optimization of enrichment using any target of interest.

Selection of high affinity RNA ligands to the bacteriophage R17 coat protein.

RNA ligands with high affinity for the bacteriophage R17 coat protein were isolated from a pool of random RNA molecules using SELEX. Of the 38 ligands isolated, 36 were found to contain a hairpin very similar to the naturally occurring coat protein binding site in the R17 genome. The common features of these 36 sequences provide a consensus binding site and predict components of a hairpin that promote favorable interaction with the coat protein. These include a tetraloop of primary sequence AUCA and a variable-length stem with a bulged adenosine residue at a specific stem position. The predicted consensus agrees well with the highest-affinity RNA binding site of the R17 coat protein, identified through classical but laborious techniques. These results demonstrate the value of SELEX as a tool for isolating high affinity RNA ligands to a specific target protein, and the further value of those ligands to point the researcher toward natural sequences for that target protein.

In vitro evolution of functional nucleic acids: high-affinity RNA ligands of HIV-1 proteins.

SELEX (Systematic Evolution of Ligands by EXponential enrichment) is a protocol for isolating, from a pool of variant nucleic acid sequences, high-affinity ligands to a target protein [Tuerk and Gold, Science 249 (1990) 505-510]. This procedure involves cycles of affinity selection by a target molecule from a heterogeneous population of nucleic acids, replication of the bound species (the ligands), and in vitro transcription to generate an enriched pool of RNA. We have used the SELEX procedure to obtain high-affinity RNA ligands against the reverse transcriptase and the Rev and Tat proteins of human immunodeficiency virus 1 (HIV-1). Through sequence comparisons within the collection of ligands isolated for each of these target proteins, we derive consensus descriptions of what secondary structure and primary sequences are required for binding. These descriptions serve as the starting point for the ultimate development of compounds intended to alter the course of HIV-1 infection.

Autogenous translational operator recognized by bacteriophage T4 DNA polymerase

The synthesis of the DNA polymerase of bacteriophage T4 is autogenously regulated. This protein (gp43), the product of gene 43, binds to a segment of its mRNA that overlaps its ribosome binding site, and thereby blocks translation. We have determined the Kd of the gp43-operator interaction to be 1.0 x 10(-9) M. The minimum operator sequence to which gp43 binds consists of 36 nucleotides that include a hairpin (containing a 5 base-pair helix and an 8 nucleotide loop) and a single-stranded segment that contains the Shine-Dalgarno sequence of the ribosome binding site. In the distantly related bacteriophage RB69 there is a remarkable conservation of this hairpin and loop sequence at the ribosome binding site of its DNA polymerase gene. We have constructed phage operator mutants that overproduce gp43 in vivo, yet are unchanged for in vivo replication rates and phage yield. We present data that show that the replicative and autoregulatory functions are mutually exclusive activities of this polymerase, and suggest a model for gp43 synthesis that links autoregulation to replicative demand.

19 RNA: The Shape of Things to Come

This enormous book and the literature behind it establish a new conventional wisdom. Today it is common to assert that an RNA world once existed and flowed into the present biosphere. Introductory texts and review articles softly (or staunchly [Watson et al. 1987]) proclaim that RNA comprises the collection of genetic and catalytic molecules that began the biosphere and then gracefully stepped into the background as a richer life unfolded. In this volume are papers that define the position RNA might have held. Having read many such papers, we note that by the scientific rules of evidence, the concept of an RNA world may stand only as an attractive idea, an idea that never can be tested adequately. One problem is that known RNA catalysts are few, and thus we have no way to assess the catalytic breadth of RNA by normal observation and experimentation. Methods now exist to inspect the catalytic and binding properties of small RNA molecules without recourse to the identification of RNAs in the present biosphere. In this paper, we illustrate the chemical and likely catalytic potential of RNA by reference to work done by us using these new methods. The plausibility and limitations of an early RNA world are addressed by the work. We have come to an astonishing and unexpected conclusion. RNA apparently has a huge distribution of sizes and shapes, and a collection of RNA molecules, even a collection of rather short RNA molecules, could have been used for an enormous variety of...