From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops

Abstract

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?

Figure 1

The sequences and reasonable secondary structures of two RNAs that bind with identical affinities to bacteriophage T4 DNA polymerase are shown. The green nucleotides in the major variant are the positions of difference between the two RNAs.

Figure 2

The conserved AACs of two RNAs that bind bacteriophage T4 DNA polymerase are shown. The AACs are in red in Fig. 1. The NMR structures and discussion are reproduced with permission from refs. and (Copyright 1996, Am. Chem. Soc.).

Figure 3

A perfect library generated by random priming should contain a complete set of genomic inserts starting (and ending) at each nucleotide of the genome. The diagram above shows a hypothetical set of library fragments with genomic inserts 36 bases long and a hypothetical binding site of 26 bases. These and only these fragments will survive to the final round of SELEX. The primer sequences (underlined) allow PCR amplification, while the T7 promoter (indicated with asterisks) is used when RNA is the target.

Figure 4

We developed a technique to examine the distribution of endpoints of genomic inserts that contain any short unique sequence (36). We isolated a set of library fragments that contain the Saccharomyces cerevisiae NDC1 gene sequence underlined above. Each bar above a nucleotide in the adjacent sequence means that nucleotide was adjacent to the T7 containing promoter in one of the 43 fragments examined. This limited analysis revealed 25 out of 29 possible endpoints! In our examination of five genes in four libraries, the largest “gap” that we found (i.e., stretch of nucleotides for which no endpoints were observed) is 9 nt. Assuming judicious choice of insert size, we are confident that every nucleic acid binding site for every protein is to be found in every library constructed by this method.