GeneArt Combinatorial DNA Libraries

A combinatorial DNA library is a collection of DNA sequences where multiple nucleotide positions are mutated within the sequence, allowing for various combinations of mutations at different positions. This process enables the exploration of a wide range of genetic variations. Based on true rational design, combinatorial DNA libraries can achieve a maximum diversity to screen for and identify synergistic beneficial mutations, such as affinity maturation via phage display libraries.

Applications of combinatorial DNA library

Combinatorial DNA libraries are utilized in various fields such as drug discovery, protein engineering, and gene function studies, enabling researchers to explore genetic variations and identify optimal sequences for various applications, including:

• Optimizing antibody affinity: Facilitate antibody affinity optimization through affinity maturation
• Humanizing antibodies: Modify antibodies to be more human-like to reduce immune responses
• Modifying antibody specificity: Alter the specificity of antibodies to target different antigens
• Improving protein properties: Help enhance physiochemical properties such as solubility, heat stability, and activity in industrial environments
• Enhancing function: Help improve promoter activity or enzymatic properties
• Prolonging therapeutic half-life: Help extend the half-life of therapeutics for in vivo applications
• Improving enzyme function: Help optimize enzyme function for industrial use
• Modifying enantioselectivity: Change the enantioselectivity of enzymes
• Altering patented sequences: Induce changes to modify proteins from patented sequences

All GeneArt Combinatorial Library products are sequenced and statistically analyzed to help ensure that they meet the following quality benchmarks:

• Sequencing of up to 96 individual transformants to verify that customer’s specifications regarding amino acid content and distribution have been met, and to verify sequence integrity of unmutated portions of the construct
• Bulk sequencing to verify that nondegenerate portions of the construct have the correct sequence and randomized portions meet customer’s specifications
• Real-time PCR prior to amplification to verify that library diversity objectives have been met
• Optional next-generation sequencing quality control (additional fee applies)

Please download the GeneArt Combinatorial Libraries Custom Service Requirements Form to submit project information. For secure data transfer, please register at our customer portal.

Libraries made with conventional technology by the incorporation of degenerate codons (NNS, VNS, etc.) are also available. Please use the above-mentioned questionnaire for both technologies.

For further information regarding this service or the ordering process, please contact geneartsupport@thermofisher.com.

Besides randomization via degenerate codons (e.g., NNS), the sequences of GeneArt Combinatorial DNA Libraries can also be diversified using preassembled trinucleotide building blocks (trinucleotide mutagenesis (TRIM) technology) within the chemical synthesis process, to achieve significantly higher quality than by using conventional technologies. This allows for complete customization of the amino acid composition at randomized sites and thus avoids the occurrence of unwanted stop codons or amino acids. Achieve an extra level of QC by requesting optional next-generation sequencing of your combinatorial DNA libraries using the Thermo Scientific Ion Torrent sequencing platform (see case study below).

• Rational diversity: Control important features of the library using TRIM technology to create rational diversity where it is likely to have the most impact
• Screening efficiency: Helps significantly reduce screening efforts compared to conventional mutagenesis methods, thus helping to save time and budget
• Success rate: Customized design allows for the inclusion of only substitutions known to contribute to a certain property, which increases the likelihood of screening success
• Flexibility: Even the ratio of allowed amino acids can be accurately controlled, for example, with ratios reflecting exactly the ones found in humans
• Accuracy: TRIM significantly reduces frameshift mutations and "abolishes" the occurrence of stop codons which makes the screening of libraries more efficient. Ancillary mutation rates achieved are typically in a correctness range of >82%
• Achievable library diversity: Combinatorial DNA libraries can achieve diversities of up to 10¹²
• Speed: Our capacities allow for fast production times that are project-specific

Highlights:

• Accurate: Determination of library correctness, amino acid distribution, and uniqueness of clones
• Comprehensive: Delivers millions of reads for analysis
• Reliable: Sequence at clonal level with a proprietary technology and software solution

A GeneArt Combinatorial DNA Library from our production workflow was characterized using our current standard quality control protocol (“CE-sequenced”) and our novel next-generation sequencing (NGS)-based quality control for combinatorial DNA libraries (“NGS-sequenced”). The expected amino acid distribution is shown in the right-most panel (“expected”); in this case cysteines were not desired in the randomized positions. Our current standard QC protocol entails CE sequencing of at least 96 library clones and subsequent statistical analysis of the amino acid distribution at randomized positions. In this case, 304 variant clones were analyzed. In contrast, our novel NGS-based quality control for combinatorial DNA libraries on the Ion PGM (Personal Genome Machine) Sequencer allows for the analysis of tens of thousands of library clones. In this case, 41,625 clones were analyzed.

The table shows the percentage at which each amino acid appears in that position for 4 codons (X1 – X4). Note that in the CE-sequenced peer group the codons for two amino acids were not found, although they are present in the library, as shown by NGS sequencing (red and green circles).

The research leading to results regarding NGS quality control has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 613931.