Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis

BackgroundCombinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges.
Methodology/Principal FindingsWe gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at >1,000-fold faster rate, and reduce costs ∼250-fold for generating 106 ligand sequences.
Conclusions/SignificanceOur analyses demonstrates that whereas this approach correlates with the traditional colony-counting, it is also capable of a much larger sampling, allowing a faster, less expensive, more accurate and consistent analysis of phage enrichment. Overall, qPhage plus pyrosequencing is superior to TU-counting plus Sanger sequencing and is proposed as the method of choice over a broad range of phage display applications in vitro, in cells, and in vivo.

研究背景
过去20年间，组合噬菌体展示（combinatorial phage display）已被用于蛋白质-配体与蛋白质-蛋白质相互作用的鉴定，揭示了诸多具有重要意义的分子识别事件。组合噬菌体展示文库筛选的两大限速步骤分别为：(i) 转导单位（transducing units, TU）计数；(ii) 对编码的展示配体进行测序。本研究针对上述挑战，对新兴基因组技术进行了适配优化。

研究方法与主要结果
我们通过联用实时荧光定量PCR（real-time PCR）实现快速定量，从而无需培养细菌即可完成噬菌体展示文库筛选，并结合噬菌体DNA下一代测序（next-generation sequencing）技术开展大规模配体分析，由此构建了一套完整集成的高通量定量与分析工具体系。该方法的劳动强度大幅降低，且可实现精准定量；在医学应用场景中，例如患者体内的筛选流程，本方法还可实现对文库给药后更长时间窗口内，患者活检或尸检样本中数十万靶向颗粒的定量分布分析与配体鉴定。相较于现有技术手段，本方法还具备灵敏度更高、变异性更低、线性范围更优、可扩展性更强以及成本大幅降低的多重优势。通过qPhage联用焦磷酸测序（pyrosequencing）获得的序列数据，与传统桑格测序（Sanger sequencing）转导单位法得到的数据集高度相似，且不存在GC含量、密码子使用偏好、氨基酸或肽段频率相关的测序偏差。本工具可将噬菌体展示筛选与配体分析的速度提升1000倍以上，并在生成10^6条配体序列时将成本降低约250倍。

研究结论与意义
本研究分析表明，尽管该方法与传统菌落计数法具有良好相关性，但其可实现更大规模的样本采样，从而实现噬菌体富集过程的更快、更经济、更精准且一致性更强的分析。总体而言，qPhage联用焦磷酸测序技术优于转导单位计数联用桑格测序法，可作为体外、细胞内及体内多种噬菌体展示应用场景的首选方法。