4.4: SELEX (Selective Evolution of Ligands by Exponential Enrichment)

Tuerk and Gold, Science (1990) 249:505-510

Phage T4 genome encodes a DNA polymerase (T4 DNA pol)

The mRNA for T4 DNA pol contains a sequence which has binding affinity for T4 DNA pol. This sequence overlaps the Shine/Dalgarno sequence and binding of T4 DNA pol prevents the mRNA from being transcribed (and producing more T4 DNA pol). Thus, the expression of T4 DNA pol is self-regulating (autogenous regulation). The structure of the mRNA in this region forms a stem-loop structure

Figure 4.4.1: T4 DNA polymerase mRNA

The experimental design:

In simple terms, generate a library of mRNA molecules with a random nucleotide sequence in the loop region. This library will have a size of 4⁸ or 65,536 uniquely different sequences (if only the loop region is randomized). Identify those sequences with high affinity for T4 DNA pol and determine their sequence.

Template Construction

A "synthetic gene" duplex DNA template is constructed from five primers:

Figure 4.4.2: Template construction

• The synthetic gene is 110 nucleotides in length. It is composed of oligonucleotides #3, #4 and #5. Oligonucleotides #1 and #2 can be thought of as "bridging" oligos which allow #3, #4 and #5 to be ligated
• Oligonucleotide #4 contains the 5' end of the T4 DNA Pol gene corresponding to the mRNA Shine/Dalgarno sequence and the T4 DNA pol binding step/loop. It is synthesized to contain random bases at the 8 base long loop structure of the T4 DNA pol binding domain.
• Oligonucleotide #1 contains the sequence for T7 RNA polymerase promoter region. Transcription can be driven from this promoter by addition of T7 RNA polymerase and ribonucleotides.

in vitro transcription

• The 110 nucleotide gene (double-stranded DNA form) can be transcribed in vitro by T7 RNA pol
• Transcription produces a 92 nucleotide long RNA molecule. There will be a library of potentially 65,536 unique RNA molecules

Figure 4.4.3: In vitro transcription

Selection by binding to T4 DNA pol

Those RNA transcripts with a sequence which confers binding specificity for T4 DNA pol are selected for by incubating the RNA library with T4 DNA pol which has been immobilized on nitrocellulose filters. The filters are washed to remove non-specifically bound RNA molecules. Tight (specific) binding RNA molecules are eluted from the T4 DNA pol protein and are collected

Figure 4.4.4: Specific binding

cDNA construction

The eluted RNA molecules are converted to single strand cDNA using oligonucleotide #5 as a primer, and adding Reverse transcriptase and dNTP's. Duplex DNA is produced from the single strand cDNA by PCR using oligonucleotides #1 and #5.

Figure 4.4.5: cDNA Construction

In vitro transcription

• In vitro transcription produces a (smaller) library of RNA molecules enriched for sequences with binding affinity for T4 DNA pol
• the process of selection and enrichment is continued until a collection of high affinity binding sequences has been produced

Experimental results

1. Library construction

• The original "synthetic gene" library was sequenced en mass
• A "ladder" of bases at the random position indicated that the library started out being more or less a completely random collection of sequences in this region (i.e. there did not appear to be any sequence bias in the collection of molecules)

2. Samples from the sequential rounds of selection and amplification were saved for sequencing analysis

• The results indicated that as the selection and amplification process continued certain bases were observed at specific positions in the 8 base mutagenized region

Figure 4.4.6: Mutant sequence

The wild type sequence could be found within this heterogenous mixture of tight binding sequences. 20 clones from this final library were sequenced.

Figure 4.4.7: Mix of tight binding sequences

• There were essentially only two different sequences present: the known wild type sequence and another different sequence (which varied at four positions)
• Both had similar tight binding characteristics (the minor sequences identified exhibited weaker binding)
• This alternative sequence may have a different stem-loop structure than the wild-type

Figure 4.4.8: Change in stem loop structure

Conclusions

1. The SELEX method successfully identified the wild type sequence and an alternative (previously unknown) sequence with high affinity T4 DNA pol binding properties
2. The results suggest that the sequence of the wild type translational operator is pretty much optimized for binding (unable to improve further?)
3. The SELEX method should simplify the study of interactions between
   • transcriptional activators and repressors and transcriptional complexes at promoter sites
   • replication accessory proteins and DNA pols at origins of replication
   • ribosomes and translational repressors at ribosome binding sites
4. Results may be relevant to DNA/protein binding interactions in certain cases (i.e. if interactions do not involve 2' hydroxyl group and if a structure which DNA can adopt is involved)
5. Instead of beginning with all possible sequences, start with a smaller library and combine replication with error prone polymerases to generate additional sequences