Bind-n-Seq: high-throughput analysis of in vitro protein-DNA interactions using massively parallel sequencing - PubMed (original) (raw)
Bind-n-Seq: high-throughput analysis of in vitro protein-DNA interactions using massively parallel sequencing
Artem Zykovich et al. Nucleic Acids Res. 2009 Dec.
Abstract
Transcription factor-DNA interactions are some of the most important processes in biology because they directly control hereditary information. The targets of most transcription factor are unknown. In this report, we introduce Bind-n-Seq, a new high-throughput method for analyzing protein-DNA interactions in vitro, with several advantages over current methods. The procedure has three steps (i) binding proteins to randomized oligonucleotide DNA targets, (ii) sequencing the bound oligonucleotide with massively parallel technology and (iii) finding motifs among the sequences. De novo binding motifs determined by this method for the DNA-binding domains of two well-characterized zinc-finger proteins were similar to those described previously. Furthermore, calculations of the relative affinity of the proteins for specific DNA sequences correlated significantly with previous studies (R(2 )= 0.9). These results present Bind-n-Seq as a highly rapid and parallel method for determining in vitro binding sites and relative affinities.
Figures
Figure 1.
Bind-n-Seq overview. The Bind-n-Seq substrate is an oligo containing constant regions (Primer A and Primer B) a 3-nucleotide bar code (BC) and 21 bp random region. Bar coded oligonucleotides are mixed with various proteins, washed to remove unbound DNA, pooled and sequenced with short read technology. Reads are sorted by their bar codes and processed through several bioinformatics procedures that result in motifs corresponding to the DNA binding sites of each protein.
Figure 2.
Motif enrichment. The fold-enrichment of known motifs in various binding reactions is shown for Zif268 (blue) and Aart (red). Y-axis: fold-enrichment of a motif in a binding reaction over control (no-protein). Reaction conditions: z, Zif268; a, Aart; protein concentration is shown in nM, salt (KCl) concentration is shown in mM, gs, gel shift; lw, long wash; +r, extra round of selection; f, ficoll. Error bars show the range of values for replicated experiments.
Figure 3.
Comparison of CAST and Bind-n-Seq motifs. Intermediate motifs are the result of several non-overlapping sets of reads. Final motifs use reads matching intermediate motifs.
Figure 4.
Comparison of relative affinities determined by Bind-n-Seq and QuMFRA. Bind-n-Seq relative affinity is calculated as the fold-enrichment of the 15 sequences (10-mer) (
Supplementary Table S2
) compared to a no-protein control. All reactions are 5 nm protein and 100 mM salt. Squares are run 1, circles are run 2, triangles are long wash. The relative affinity for the same 15 sequences (10-mer) assessed by QuMFRA is taken from Liu et al., 2005.
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