This paper focuses primarily on the means by which biochemical information can be used to identify enzymes which, upon inhibition, produce lethal phenotypes and the enzyme inhibitor design strategies that have the highest probability of not only inhibiting the enzyme but also translating that inhibition into herbicidal efficacy. The identification of an exquisitely lethal target site is the key initial component to this approach and has often been one of the most difficult steps because the attributes of a lethal site have, at best, been ill-defined. An examination of the characteristics of known targets provides some insight as to the definition of a lethal target. Recently, antisense RNA suppression of enzyme translation has been used to determine the extent of inhibition required for toxicity and offers potential as a strategy for identifying lethal target sites. After identification of a lethal target, detailed knowledge of the enzyme's chemical and kinetic mechanism as well as the protein's structure may be used to design potent inhibitors. Various types of inhibitors may be designed for a given enzyme. The advantages and disadvantages of a given type with respect to in vivo efficacy as well as the probability of herbicide resistance development will be discussed.

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5′-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit β-globin, and human interferon-γ (IFN-γ). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility.