Site Saturation Scanning for Every Lab: Supercharging Site- Directed Mutagenesis

Transcription

1 Site Saturation Scanning for Every Lab: Supercharging Site- Directed Mutagenesis Holly Hogrefe, Ph.D. R&D Manager Genomics Solutions Division Agilent Technologies 1

2 New Product Introduction: QuikChange HT Protein Engineering System Create rationally-designed complex mutant libraries targeting: Entire proteins, up to 1000 codons per kit Custom oligonucleotide libraries used to achieve: An expanded menu of mutagenesis strategies, not possible with standard or degenerate oligos: Alanine and site-saturation scanning Rational combinatorial mutagenesis High-quality libraries, lacking the biases and unwanted mutations associated with other mutagenesis techniques 2

3 Definitions 3

4 Random mutagenesis: Tool for identifying nucleotide or amino acid changes that alter function Error-prone PCR conditions used to introduce single bp changes at random locations in a gene or promoter of interest Strategy most commonly used when the location of beneficial changes is unknown Advantages over rational (non-random) approaches: No prior knowledge of structure required Survey large mutational target regions, up to several kbp in size Agilent Product: GeneMorph Random Mutagenesis kit Limitations: Screen large libraries to identify useful mutations Changes limited to single base substitutions; a large fraction of protein sequence space is not accessible since most amino acid changes require 2-3 point mutations in the same codon With single base mutations, only 5.7 amino acid substitutions on average are accessible from any given amino acid residue (Miyazaki and Arnold (99) J Mol Evol 49: ) Page 4

5 Site-Directed Mutagenesis: Process of creating one or more nucleotide or codon changes in a gene or regulatory sequence Rational (non-random) approach that employs DNA polymerase activity to incorporate mutagenic oligos into wild-type DNA sequences Strategy used when location of desired modifications is known; potential target sites can be identified by analyzing protein structures, amino acid sequence homologies, or prior mutagenesis data Used to determine structure-function relationships in proteins or promoters, identify post-translational modifications, define protein-protein interactions, modify enzyme activity, optimize expression, add restriction sites, and correct unwanted mutations Agilent Products: QuikChange Site-Directed Mutagenesis kits Limitations: Throughput; 1 mutant per reaction Structural information is lacking in many cases Page 5

6 The QuikChange Method Features a simple 3-step protocol Creates single base changes, codon replacements, insertions and deletions with >80% mutation efficiency MIX Denature plasmid and annealing primers containing desired mutation X CYCLE Temperature cycle to extend and incorporate mutation primers resulting in nicked circular strands DIGEST Digest parental DNA template Gene in plasmid with mutation target site Mutagenic primers Employs high-fidelity Pfu polymerase and linear amplification to avoid unintended mutations TRANSFORM Transform the resulting annealed double-stranded nicked DNA molecules Referenced in tens of thousands of publications After transformation, XL-1Blue E. coli cell repairs nicks in plasmid Page 6

7 Definitions cont d Site-saturation mutagenesis: Process of determining the impact of every amino acid replacement at a single codon Most practical method uses degenerate oligos (referred to as codon-saturation ); oligos synthesized with an NNN codon comprise 64 unique sequences encoding 20 different amino acids at one position Site-saturation libraries can be screened to identify the best side chain replacements, and required structural features (size, charge, etc.) for optimal activity Site-saturation with degenerate codons is less-than-ideal: Bias toward amino acid changes encoded by redundant codons Frequency of unwanted termination and rare codons Limited scalability or target size: No. sequences required No. sites to saturate: Rational design ,000 Degenerate codons ,144 16,777,216 Scalability limited by transformation efficiency of E. coli and quality of degenerate codon libraries, as codon redundancy and amino acid bias increase exponentially as the number of mutation sites is increased; No. oligos & rxns 4 160,000 1 N = nt position synthesized with equimolar mixture of 25% AGCT) Agilent Product: QuikChange Multi Site-Directed Mutagenesis kit* Page 7

8 Example of Site-Saturation Mutagenesis JDF-3 P410L DNA polymerase (CCT CTT) Yield from random selection of 30 QuikChange mutants: Random mutagenesis: Random mutant library of Thermococcus sp. JDF-3 Pol B was expressed in E. coli and screened for radioactive-ddntp uptake Arezi et al (02) J. Mol. Biol. 322: Rare in EPPCR library (2 nt changes) pjdf3 13% McElhinny et al (09) in Molecular Themes in DNA Replication (Cox L.S., ed) Hogrefe (10) in In Vitro Mutagenesis Protocols (Bramam, J. ed) pg Page 8

9 Definitions cont d Alanine (or single mutation) scanning: Systematic process for evaluating the functionality of a group of amino acids A collection of site-directed mutants is constructed, where each mutant contains an alanine (or other neutral amino acid) substitution at a unique location; after measuring impact to function, the information is used collectively to map essential positions Limitations: While powerful, alanine scanning is generally limited to peptides or small domains due to the cost and amount of labor needed to construct multiple single-site mutants (one QC reaction and primer pair per codon) 100% 10% 0% Alanine scanning of the HIV gp41 loop Jacobs et al (05) JBC 280: gp41 alanine mutants were constructed using the QuikChange kit with 30 primer pairs; viral entry was measured using a Luciferase reporter assay Page 9

10 Definitions cont d Site-saturation scanning: Combination of site-saturation and scanning mutagenesis strategies When feasible, provides comprehensive structure-function analysis by surveying the functional consequences of every side-chain replacement at each location Hypothetical sequence-function map for a 25 amino acid portion of a protein. The color of each square in the heat map illustrates the functional consequence of individual side chain substitutions (single-letter abbreviation) at each position. Deep mutational scanning: a new style of protein science. Fowler & Fields (2014) Nature Methods 11:801-7 By enabling the impact of mutations to be examined in an unbiased fashion, deep mutational scanning can reveal the unexpected. Significant limitations: Requires a high-throughput cost-effective method for constructing site-directed mutants, in addition to substantial screening and sequencing Agilent Product: QuikChange HT Protein Engineering System Page 10

11 QuikChange HT Protein Engineering System 11

12 Game-Changing Mutagenesis Technology + Agilent s SurePrint Technology QuikChange Lightning Site-Directed Mutagenesis QuikChange HT Protein Engineering System Quick, accurate, and cost-effective method for constructing complex rationally-designed mutant protein libraries 12

13 QuikChange HT Protein Engineering System Rationally-designed oligo libraries provide: An expanded menu of mutagenesis strategies, not available using standard oligos Single-site (Alanine or other) scanning Site-saturation scanning Targeted combinatorial mutagenesis Comprehensive custom content Up to 120,000 user-specified sequences Easy-to-use oligo library design tool Flexible design options, one or more proteins Better quality libraries, more information content User-specified codon usage for optimal expression No unwanted codons; avoid biases associated with degenerate codons Reduced screening requirement Constructing oligo libraries with QuikChange offers: Fast and easy one-day workflow for in-house library construction High incorporation efficiency with minimal polymerase-induced mutations Ultra-high transformation efficiencies for maximum library size 13

14 X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C X B C QuikChange HT - Workflow X B C X B C X B C X B C QC HT Step 1: Design mutagenic library specific to sequence and application with earray design software. QC HT Step 2: Library is synthesized per design. A set of mutant oligos is designed for each target region and contains identical 5 and 3 ends which serve as PCR primer binding sites. Oligo sets are isolated from the oligo library by PCR. SurePrint Inkjet Oligo Library Synthesis Oligo library cleaved PCR & Purify oligo sets 1,2, E.coli library Amplified OLS incorporation QC HT Step 3: Perform QuikChange mutagenesis with each oligo set, move onto mutant screening in less than a day Nat gene V 1 V 2 Screening Sequencing Identify and sequence selected clones V 3 V 4 V 5 V 6 V 7 V N V1,0 01 Dpn I digestion Nick repair (E. coli) Site Directed Mutagenesis Incorporate oligo sets separately into plasmid DNA with QuikChange, followed by Dpn I enrichment & transformation Page 14

15 earray Mutagenesis Workspace Design Workflow FREE design tool: Supports codon-based design of mutant protein libraries Design one pair of PCR primers and one set of mutagenic oligos ( oligo set ) for each target region; QuikChange HT kits configured to construct up to 10 or 20 sub-libraries (10 or 20 site kits) For each oligo set, selections include: Mutagenesis strategy Oligo length / Target size (17 codons with 100mers; 50 codons with 200mers) Codon usage 15

16 QuikChange HT Mutagenesis Strategies Mutagenic oligos Number unique per oligo set* QuikScan-1: Separately replace each amino acid in the wild-type mutational region with a selected amino acid. Ala replacements ( alanine scanning ) are commonly used to identify key functional amino acids. Target sequence Oligo set 1 Mutation (same codon) Mutation region 1 PCR primer annealing sites 1 mutation type per codon; 1 codon change per oligo QuikScan-19: Sitesaturation scanning systematically replaces each amino acid in the wild type mutational region with all 19 other amino acids, resulting in 19 mutagenic oligos for each amino acid position. For deep mutational scanning. QuikCombine: Combine multiple mutations in groups of 1-4 positions with define variation at each site. Create up to 120,000 variants per library, or combine a few identified mutations and validate functional relevance. 19 mutation types per codon; Each oligo Oligo library can contain up to 120,000 unique sequences set 2 Mutation (19 separate codon changes) 1 codon change per create 120,000 different combinations of mutations within a single oligo domain, or target up to 20 regions in the same protein or different proteins 50AA x 19 mut = QC HT reaction With 20 sets of oligos, 200nt in length, you can target up to Mutation region 3 Target 1000 codons per QC HT library. Any of the specified Variable sequence mutations mutations; 1-4 codon changes per Oligo set 3 oligo PCR primer binding sites *17-50 codon targets per oligo depending on length ( nt); #Up to 10 or 20 oligo sets per library; 20 oligo sets X 50 codon targets/set = 1000 possible codon targets (~100kDa protein) per QC HT library Total per library: 120,000 # Page 16

17 QuikChange HT Workflow 1) Order QuikChange HT kit; includes one custom oligo library and one pair of PCR primers for each oligo set 2) Amplify the oligo sets separately, and purify using the supplied spin cups: no need to convert amplicon to ssdna or remove primer binding sites 3-5) Set up one mutagenesis reaction per amplified oligo set. Follow the standard QuikChange protocol, substituting PCR amplicon for the pair of mutant oligos After transformation and overnight growth, mutant libraries are ready for sub-cloning into a suitable expression strain (as needed) for screening or downstream analysis 5 Sub-library 1 Sub-library 2 17

18 Test Cases 18

19 Single Mutation Scanning Before initiating comprehensive site-saturation scanning, we assessed the mutability of the fingers domain of Pfu DNA polymerase QuikScan-1 library containing 51 unique sequences was designed to introduce a Met (ATG) substitution at each codon in the fingers domain Heat-treated bacterial cultures were prepared from 467 # random clones and screened for DNA polymerase activity Results show the fingers domain is highly mutable; Pfu DNA polymerase retains some level of detectable activity when mutated at 34 positions in the fingers domain # 95% confidence level; takes into account number of unique sequences and oligo synthesis error rate QuikScan-1 Library WT FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 450 LMTSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 451 FIMSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 452 FIPMILGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 453 FIPRMMGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 454 FIPSLMRHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 455 FIPSLLMNLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 456 FIPSLLGMLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 457 FIPSLLGQMIEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 458 FIPSLLGHLMEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 459 FIPSLLGHLLMKRQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 460 FIPSLLGHLLEMRQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 461 FIPSLLGHLLEEMKKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 462 FIPSLLGHLLEERMKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 463 FIPSLLGHLLEERQMIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 464 FIPSLLGHLLEERQKMKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 465 FIPSLLGHLLEERQKIMTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 466 FIPSLLGHLLEERQKIKMKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 467 FIPSLLGHLLEERQKIKTMMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 468 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA 469 FIPSLLGHLLEERQKIKTKIMKTQDPIEKILLDYRQKAIKLLANSFYGYYGYA 470 FIPSLLGHLLEERQKIKTKMKMTQDPIEKILLDYRQKAIKLLANSFYGYYGYA 471 FIPSLLGHLLEERQKIKTKMKEMKDPIEKILLDYRQKAIKLLANSFYGYYGYA 472 FIPSLLGHLLEERQKIKTKMKETMNPIEKILLDYRQKAIKLLANSFYGYYGYA 473 FIPSLLGHLLEERQKIKTKMKETQMTIEKILLDYRQKAIKLLANSFYGYYGYA 474 FIPSLLGHLLEERQKIKTKMKETQEMIEKILLDYRQKAIKLLANSFYGYYGYA 475 FIPSLLGHLLEERQKIKTKMKETQDPMKKILLDYRQKAIKLLANSFYGYYGYA 476 FIPSLLGHLLEERQKIKTKMKETQDPIMKILLDYRQKAIKLLANSFYGYYGYA 477 FIPSLLGHLLEERQKIKTKMKETQDPIEMILLDYRQKAIKLLANSFYGYYGYA 478 FIPSLLGHLLEERQKIKTKMKETQDPIEKMILDYRQKAIKLLANSFYGYYGYA 479 FIPSLLGHLLEERQKIKTKMKETQDPIEKIMIDYRQKAIKLLANSFYGYYGYA 480 FIPSLLGHLLEERQKIKTKMKETQDPIEKILMNYRQKAIKLLANSFYGYYGYA 481 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLMNRQKAIKLLANSFYGYYGYA 482 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLEMRQKAIKLLANSFYGYYGYA 483 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLD(-)MKKAIKLLANSFYGYYGYA 484 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRMKAIKLLANSFYGYYGYA 485 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQMTIKLLANSFYGYYGYA 486 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKMIKLLANSFYGYYGYA 487 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAMKLLANSFYGYYGYA 488 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIMILANSFYGYYGYA 489 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKMIANSFYGYYGYA 490 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLMTNSFYGYYGYA 491 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLMNSFYGYYGYA 492 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLAMTFYGYYGYA 493 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLAKMIYGYYGYA 494 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSMNGYYGYA 495 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSLMRYYGYA 496 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSF(-)MNYGYA 497 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGMNGYA 498 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYG(-)MSYA 499 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGY(-)MNA 500 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGMT 5 4 # active hits* Pfu fingers domain consists of 2 anti-parallel helices ( O-P) that make numerous contacts with an incoming nucleotide. Mutations in this domain have been shown to increase incorporation of modified nucleotides IPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGY *library properties: <10% parental DNA background; total size: 1.7x10 5 cfus Page 19

20 -barrel structure E120G Site-Saturation Scanning of GFP chromophore Humanized Renilla reniformis GFP mutant expressed in a range of mammalian cell types by transfection with Vitality II phrgfp II-C vector Renilla reniformis GFP E120G: Previously identified Gly substitution at position 120 that improves fluorescence intensity of Rre GFP in both E. coli and mammalian cells Isolated by random mutagenesis of humanized Rre gfp sequence and screening the resulting libraries for increased brightness in E. coli Nucleotide substitution (GAG GGG) was subsequently incorporated into Agilent s Vitality II (hrgfp II) mammalian expression vectors Resides in 5-6 loop, pointing away from the chromophore Enhances solubility and protein folding Page 20

21 GFP Site-Saturation Scan Library Design (expression & screening in E. coli) nt nt nt 50AA x 19 mut = 950 oligos Oligo set 1 Strategy: QuikScan-19 Target: nt / 28-77aa Oligo length: 200nt Codon usage: E. coli, R. reniformis 50AA x 19 mut = 950 oligos Oligo set 2 Strategy: QuikScan-19 Target: nt / aa Oligo length: 189nt Codon usage: E. coli, R. reniformis 50AA x 19 mut = 950 oligos Oligo set 3 Strategy: QuikScan-19 Target: nt / aa Oligo length: 198nt Codon usage: E. coli, R. reniformis Cloning: Incorporated amplified oligo sets into pmal-gfp -1 using QC HT kit; pmal-gfp -1 vectors contain frameshift mutations in each target region; oligos were designed to correct frameshift (1bp insertion) while introducing desired codon replacement Screening: After IPTG induction, fluorescent colonies were visualized using a light source and appropriate excitation and emission filters. Clones displaying higher fluorescence intensity compared to GFP or GFP E120G transformants were isolated, and plasmid DNA was prepared. Positives were subject to 1-2 additional rounds of transformation and screening to verify isolation of the brightest GFP mutants. Page 21

22 Screening Results: Domain 1 Transformation & Screening (E. coli) AA: OS * OS OS Library size (x10 5 ) % fluorescent clones # colonies screened (x10 4 ) No. clones brighter than wild-type No. clones brighter than E120G *70-72aa chromophore N28G Isolated only one mutation in domain 1 that confers increased fluorescence Domain 1 appears more sensitive to mutations (0.5% fluorescent) than domains 2 and 3, presumably due to proximity of the chromophore Page 22

23 Amino acid side chain Screening Results: Domain 2 Mutations: Wild-type codon: Causal- Brighter than E120G Comparable to E120G Putative- (multi-site mutants) Key G * A V 2 Domain 2 sequence: L I M 2 F P S 1 T Changes to 6 can modulate fluorescence C 1 N Q 1 Y W D 1 1 E H K 1 R Page 23

24 Amino acid side chain Screening Results: Domain 3 Mutations: Wild-type codon: Causal- Brighter than E120G Comparable to E120G Putative- (multi-site mutants) Key Domain 3 sequence: G A V L I M F P 1 P S 2 S T 1 T C N Q Y 1 W D E 1 H K 1 R 1 G G A A V V L L I I hotspot in M F C N 1 Q Y W D E H K R M F P S T C N Q Y W D 2 E H K R Page 24

25 Comparison of Mutagenesis Techniques Kit Strategy GFP Target QuikChange HT QuikScan ; ; (60%) GeneMorph II PCR under low-mid-high mutation rates; EZClone & pool libraries 5x (100%) 5x10 5 Observations: Most but not all mutants isolated from QuikScan-19 libraries were single-site mutants; allowing the identification of 17 causal mutations (11 sites) in one round of screening 10 mutations at 7 sites were brighter than E120G 7 mutations at 6 sites were comparable to E120G 5 putative sites identified in QuikScan-19 clones containing secondary mutations; non-specified mutations were attributed to oligo synthesis errors (1 per >300nt) Confirmed, isolated as a single mutation Relative fluorescence of QuikScan-19 mutants: > wild-type; = GFP E120G > GFP 120G Page 25

26 Observations cont d In contrast, majority of clones isolated from EP-PCR libraries were multi-site mutants; 4 causal mutations were identified in single-point mutants; E120G was the brightest 16 putative sites were identified in multi-site mutants; additional mutagenesis to distinguish causal from neutral mutations was not performed (none were brighter than E120G) E120 mutations were common to both screens (fluorescence emission was comparable for GFP E120D and E120G) 2 of 4 mutations identified by EP-PCR were located in a region not covered by site-saturation scanning Key: Mutagenesis method: QS-19 EP-PCR Relative fluorescence: > wild-type = GFP E120G > GFP 120G Page 26

27 Potential Next Steps What s the goal? Complete structure-function map of Rre GFP??? OR Improve fluorescence of mammalian hrgfp expression vectors??? Page 27

28 Potential Next Steps Goal: Complete structure-function map of Rre GFP QuikScan-19 on remaining domains; site-saturation scanning of the entire GFP protein could be accomplished with 5 oligo sets (~4500 sequences) Assay and sequence a statistically significant number of random clones to identify not only the brightest mutants, but mutations that eliminate, reduce or have no impact on fluorescence Goal: Improve fluorescence of mammalian hrgfp expression vectors QuikCombine to create all combinations of brightest mutations (E. coli) 2 oligo sets Domain 2: N116E, E120D, M121E/Q, V123E, Y124V/C, R125HDK, K129H, R131HS, V154M Domain 3: L184RKSTE, M185Y, F194P, G213N, G229D Move to mammalian screening system Perform in parallel with above using staggered oligo sets Apply site-saturation scanning & rational combinatorial mutagenesis to 6 and 9 domains, using codon changes that optimize mammalian expression Page 28

29 Agilent SurePrint Oligo Library Synthesis 29

30 Optimized synthesis chemistry produces highquality long oligos Depurination side reaction 1) Coupling Long length synthesis is achieved by improved coupling efficiency & lower depurination rates* Injet 3) Deblock Flood 2) Oxidation HO O N i O O P O O RO O O P O RO *Synthesis of High-Quality Libraries of Long (150mer) Oligonucleotides by a Novel Depurination Controlled Process. LeProust, E. et al (10) NAR 38: Repeat n times N 2 O O O P O RO N 1 PCR isolates full-length oligos, generating sufficient material for QC HT mutagenesis 150mer complex library oligo set PCR PCR amplified 30

31 Long Oligo Advantage Mutagenize the longest stretch of sequence possible per QC HT reaction Up to 50 codons with 200mers Up to 17 codons with 100mers Trade-off between target length and mutation frequency; longer the oligo, the greater the likelihood of an unintended synthesis error due to less than perfect coupling efficiency; simply adjust the number of clones you screen to take advantage of the benefits of long oligos Frequency of unspecified mutations is 1 error per ~300 bases; errors are predominantly deletions (single, multiple) Fidelity of QC HT # Oligo length 200nt 163nt 112nt Number clones (bp) sequenced 196 (28.8kb) 198 (21.2kb) 58 (3.5kb) % correct 63% 70% 81% % artifacts + wild-type 4% 3% 0% Number unspecified mutations Point mutations 4% 8% 0% Single nt deletions 54% 53% 58% Deletions of >1nt 42% 25% 34% Insertion 0% 13% 8% Oligo error rate 0.29% (1 per 343nt) 0.29% (1 per 342) 0.34% (1 per 294) #PCR errors expected to be <0.2% based on an error rate of 4x10-7 MF/bp/d for PfuUltra II DNA polymerase QC HT Library: unique oligos 5 oligo sets (gfp libraries) 3 oligos: 112, 163, 200nt 2 features per array Page 31

32 Oligo Quality Verified with QC HT Kit Control Adapted from standard QuikChange kit control; processed in parallel with custom oligo sets 100nt lacz sequence printed alongside custom oligos 10 features per 244K array WT lacz sequence reverts stop codon in pwhitescript, restoring -gal activity Oligo synthesis fidelity verified by scoring percent blue colonies after plating control QC HT transformations on X-gal/IPTG agar plates Agilent verifies the quality of each custom oligo library Researchers can use the QC HT kit control to: Confirm oligo library quality Troubleshoot issues with protocol (PCR, mutagenesis, transformation) or reagents (comp cell storage conditions, agar plates) Page 32

33 Quality-controlled oligo libraries QC HT lacz kit control Custom Oligo Library Mean % blue Std Dev Passing spec: 10 l of control transformation must yield >100 colonies, with at least 80% blue* *corresponds to <1 error per 300nt Page 33

34 General Guidelines for Screening QuikChange HT Libraries 34

35 Theoretical Screening Requirements - Assay sufficient number of clones to ensure that most variants have been sampled at least once - Level of oversampling required is determined by the total library diversity If the oligo pool contains V variants, the size of library L required to have probability P of observing a particular variant is given by L1 = -V ln(1-p) The size of library required to have probability P that all variants are represented is L2 = -V ln (-ln(p)/v) Variants Probability L1 L Ala scan (50 variants) Site saturation scan (950 variants) 120K Multi-site (120,000 variants) - A random sampling of 3-10,000 clones should contain at least the majority of variants in site-saturation scanning library constructed with 200mers; - Practical number of clones to screen - Not limited by transformation efficiency of bacteria (~ transformants per QuikChange reaction), ensuring fully-representative sub-libraries Page 35

36 General Screening Strategies Sub-cloning: Purify plasmid DNA library and transform suitable expression host (BL21, etc.) Availability and type of a mutant screening assay largely determines mutagenesis strategy Low throughput Alanine scan Medium throughput Site-Saturation scan High throughput Multiple site-saturation scanning libraries, complex combinatorial libraries Plate appropriate number of clones Transfer to 96- (deep) well plate; culture & induce protein expression Affinity tag protein purification Low-throughput screen ( ): Purified protein needed (e.g., specific activity changes, activity is undetectable in extracts, etc.) Affinity protein purification; process 96 colonies per plate Measure protein concentration and functional activity to detect changes in specific activity or expression level Sequence change-of-function mutants to identify useful mutations; optionally, sequence mutants with wild-type activity for deeper structure-function mapping Measure specific activity Sequence to identify essential/nonessential codons Page 36

37 Mid-throughput screening assays ( ) Screen recombinant proteins in host cell background, as extracts (96 well plates) or filter-lifts (plaques, lysed cells) Assay activity: Directly: radiolabelled or colorimetric substrate (e.g., polymerases, -gal) fluorescence (e.g., GFP) Indirectly: GFP or -gal fusions Monitor stability/solubility changes; expression-level changes in codon optimization studies Selection methods (>10 6 ): Viability of recombinant bacteria under selective metabolic pressure Binding under stringent conditions (M13 phage display; e.g., IgG) Fitness under selective PCR condition (CSR) Colony-based filter lift Plate desired number clones Replica plate onto filters Lysis Incubate with radio-labeled substrate Example (T.sp. jdf-3 pol B): Arezi et al (02) J. Mol. Biol. 322: Extract screen 42 C 55 C wt wt Mutant polymerase library Compartmentalized Self-Replication Selection (GoF) Examples (PCR enzymes): Hansen et al (11) Nucleic Acids Res. 39: ; Arezi et al (14) Frontiers in Microbiology 5: 408 Example (MMLV RT): Arezi et al (09) Nucleic Acids Res., 37: Isolate clone from master plate and sequence Identify beneficial mutations Qualitative (GoF) Thermal activity assay: Colonies were cultured in deep-well plates overnight. Cells extracts were prepared and spotted onto filter paper. Filters were incubated at elevated temperature with primed DNA and radioactive nucleotides. Mutations that confer thermal resistance were identified by DNA sequencing. Semi-quantitative G/LoF Adapted from Ghadessy (01) PNAS 98: Page 37

38 Summary 38

39 Evolution of QuikChange Kits Parallel improvements in High-Fidelity PCR & Long-Oligo Synthesis QuikChange Site-Directed Mutagenesis System QuikChange Multi Site-Directed Mutagenesis System QuikChange II & II XL Site-Directed Mutagenesis System QuikChange Lightning Multi Site-Directed Mutagenesis System QuikChange Lightning Site-Directed Mutagenesis System QuikChange HT Protein Engineering System Pyrococcus furiosus isolated PfuTurbo First of 6 QuikChange patents issue Accuracy (x10 5 ) fusion SurePrint Oligo Libraries (expanded target regions; up to 50 codons per reaction) Native Pfu Cloned Pfu Standard oligos (1 pair per mutant) PfuUltra Pfu Phusion DV Vent KOD Pfx Taq PfuUltra Standard or Degenerate oligos (2-5 per reaction) PfuUltra II Scanning & targetedcombinatorial libraries (up to 122,000 variants) Page 39 Double-to-pentuple mutants & codonsaturation libraries

40 Summary QuikChange HT provides fast, efficient, and cost-effective construction of rationally-designed libraries, enabling comprehensive mutagenesis with flexible scaling and no prior knowledge of structure-function relationships Fast & efficient: 1-day protocol for 3-step cloning with >80% incorporation efficiency Cost-effective: In-house method provides lower cost-per-mutant and faster turn-around-times compared to commercial gene variant libraries Use SurePrint inkjet technology to synthesize high-quality oligos up to 200nt in length Enables the construction of complex plasmid DNA libraries with targeted mutations spanning amino acids Rational design: High-quality information content minimizes amount of screening required User-specified codon usage eliminates issues with degenerate codon primers; libraries lack termination codons and biases associated with codon redundancy, while utilizing host-preferred codons for maximum expression 40

41 Key Features cont d Comprehensive; only commercial kit that enables: Scanning for deep structure-function mapping with no prior knowledge Targeted combinatorial mutagenesis for protein engineering Flexible scaling: Easy-to-use mutagenesis design software supports multiple design options Construct up to 20 libraries in parallel, targeting one or more proteins Choose design strategy that fits experimental and screening requirements, from alanine scanning to site-saturation scanning and rational combinatorial mutagenesis Future Offerings (potential collaboration and beta-testing opportunities): - Nucleotide-based oligo library design (promoter structure-function studies) - Kits for incorporating more than one oligo set per QC HT reaction, to increase library diversity and mutational target size Contact info: marc.valer@agilent.com 41

42 Ordering Information Scope PN Description US List Price Commercial G5900A QuikChange HT Protein Engineering System 150nt, 10 sites 10, Academic/ government G5900B QuikChange HT Protein Engineering System 150nt, 20 sites 15, G5901A QuikChange HT Protein Engineering System 200nt 10 sites 12, G5901B QuikChange HT Protein Engineering System 200nt 20 sites 19, G5902A QuikChange HT Protein Engineering System 150nt-Academic, 10 sites 6, G5902B QuikChange HT Protein Engineering System 150nt-Academic, 20 sites 9, G5903A QuikChange HT Protein Engineering System 200nt-Academic, 10 sites 7, G5903B QuikChange HT Protein Engineering System 200nt-Academic, 20 sites 11, All Inclusive: Mutagenic Primer Library + Library Amplification and Cleanup + QuikChange Lightning cloning (fast protocol) + Competent Cells + Controls 1. QuikChange HT Mutagenesis Library (one tube) 2. QuikChange HT Mutagenesis Sub-Library Primers (2-40 tubes) 3. QuikChange HT Mutagenesis Reagents 4. QuikChange HT DNA Cleanup Kit Room Temperature (bind & wash buffers + spin cups) 5. SoloPack Gold Supercompetent Cells (15 tubes + 1pUC18) 42