In Vitro (and Bacterial In Vivo) Binding Assays

Recent advances in biotechnology have spurred the growth of many different in vitro techniques for high-throughput measurements of protein-DNA, protein-RNA, and microRNA-mRNA binding. Below are brief descriptions of the main binding assays that are used to generate the binding data which is used by the ADB to build affinity models.

 

Protein-DNA Binding

1. Protein Binding Microarray (PBM)

PBMs use microarrays with double-stranded DNA probes to measure the fluorescence of alphaGST-tagged proteins bound to their sequence-specific binding sites on the probes. There are quite a few different PBM designs – with the UPBM, ME, and HK being the most popular. All three of these designs have roughly 44K DNA probe sequences that are engineered using deBruijn sequences to cover (nearly) all overlapping 10-mers at least once.

Advantages: Fast, relatively inexpensive, and provides real-valued measurements of binding.

Disadvantages (2015): Most array designs only cover all 10-mers, making it difficult to model binding sites larger than 10 base pairs. Contains a strong artifact of preferential binding at binding positions near the free-ends of the probes.

2. SELEX-seq/HT-SELEX

The SELEX-seq (also called HT-SELEX) method combines classical protein-DNA SELEX (Systematic Evolution of Ligands by EXponential enrichment) assays with massively parallel sequencing. Before the first round of SELEX, an oligonucleotide containing a randomized region that is flanked by defined primer docking sites is used to bind the protein complex of interest. DNA bound by the complex is then separated from unbound DNA and the bound DNA is then amplified by PCR and used for subsequent rounds of DNA binding and selection. SELEX-seq leverages the depth of next generation sequencing to characterize millions of selected DNA molecules at each round of selection. For each round, SELEX-seq produces integer-valued, poisson-distributed sequence read counts for all the selected reads.

Advantages: No limit to the size of the binding site. Can capture binding measurements of large protein complexes. Quantity of binding data is limited only by the depth of sequencing.

Disadvantages (2015): Relatively expensive. The initial pool always contains strong sequence biases and additional biases are introduced during the many rounds of PCR amplification. Provides integer-valued, poisson-distributed sequence read counts.

3. HiTS-FLIP

HiTS-FLIP (High-Throughput Sequencing – Fluorescent Ligand Interaction Profiling) combines the PBM protocol with a high-throughput sequencer to measure the fluorescence of tagged proteins bound to their sequence-specific binding sites in the millions of colonies on the flow cell of the sequencer. Measurements are taken at different protein concentrations to build binding curves for each protein-bound sequence read.

Advantages: No limit to the size of the binding site. Can capture binding measurements of large protein complexes. Provides real-valued measurements of binding at different protein concentrations. Deep sequencing is not required. Thus, in a way HiTS-FLIP combines the best qualities of PBM and SELEX-seq.

Disadvantages (2015): Relatively high initial start-up cost. Relatively expensive per sequencing run. Cannot select for a particular protein complex.

4. MITOMI 2.0

MITOMI (Mechanically Induced Trapping of Molecular Interactions) 2.0 uses microfluidics and microarrays to simultaneously construct binding curves and relative affinities for thousands of dsDNA 8-mer oligomers. Each MITOMI device contains thousands of microcells and each microcell contains a DNA chamber and a protein chamber controlled by micromechanical valves.

Advantages: Can individually measure the binding activity of each binding site.

Disadvantages (2015): Relatively high initial start-up cost. Can only measure binding to sites of length 8 base pairs.

5. Bacterial 1Hybrid (B1H, B1H-seq)

In the B1H binding assay, the protein is expressed as a fusion to a subunit of RNA polymerase. In addition, a library of randomized oligonucleotides are cloned into a separate vector containing the selectable genes HIS3 and URA3. When the protein (bait) binds to a DNA site (prey) in vivo in E. coli, it recruits RNA polymerase to the promoter and activates transcription of the reporter genes in that clone. The two reporter genes allow for positive and negative selections and the positive clones are sequenced.

Advantages: Relatively inexpensive and low tech. Does not require antibodies. Requires only one round of selection. Fewer protein purification steps.

Disadvantages (2015): Limitation on the size of the binding site. Cannot select for protein-DNA complexes. Some eukaryotic factors do not express or fold efficiently in the bacterial system.