Library · Biology & lab methods
What Site-Directed Mutagenesis got wrong, from 37 theses
Summary written by a model from the records below: Site-directed mutagenesis attempts often fail when engineered point substitutions eliminate essential catalytic activity or severely destabilize protein folding. Furthermore, researchers frequently encounter negative epistasis when combining mutations, insensitivity caused by interface redundancy, and technical barriers during mutant cloning or propagation. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 9 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
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Substitutions at active sites and binding interfaces eliminate catalytic function or target recognition
Introducing point mutations into catalytic pockets or critical contact interfaces frequently abolishes enzymatic activity, substrate cleavage, or target binding rather than fine-tuning function. Conserved active-site residues and direct paratope contacts proved strictly intolerant to substitution, completely inactivating enzymes or disrupting receptor surface expression.
Tried and failed
ancestral sequence reconstruction guided point mutation applied to protein redox active site engineering. Reason: mutation completely inhibited radical oxidation instead of reversing hydrogen-bonding network directionality
Bioinformatic and bioenergetic studies on the evolution of photosystem II · Imperial · PhD
Tried and failed
Site-directed mutagenesis of conserved active-site residues applied to nuclease catalytic activity preservation. Outcome: worse than baseline. Reason: Non-polar, charged, and polar substitutions all abolished enzymatic exonuclease function
Structural and dynamics studies of the Mre11-Rad50 DNA damage response complex · Texas Tech · PhD
Tried and failed
cysteine substitution site-directed mutagenesis applied to recombinant therapeutic enzyme engineering. Outcome: worse than baseline. Reason: substitutions impaired catalytic activity or completely inactivated the enzyme
Enzyme-mediated methylthioadenosine depletion as a novel immune checkpoint therapy · UT Austin · PhD
Tried and failed
site-saturation mutagenesis of paratope contact residues applied to antibody affinity maturation. Outcome: worse than baseline. Reason: direct contact residues were strictly intolerant to substitution, drastically reducing antigen binding affinity
DISCOVERY AND ENGINEERING OF ANTIBODIES TARGETING GLYCAN ANTIGENS · Cornell · PhD
Tried and failed
introducing targeted point mutations into engineered scaffold applied to engineered antibody Fc receptor binding variant. Outcome: worse than baseline. Reason: substitutions failed to improve binding selectivity beyond the parent engineered clone
Identification of the effector phenotypes triggered by engagement of FcγRIIb in B cells · UT Austin · PhD
Tried and failed
point mutation of signalling domain binding motif applied to chimeric antigen receptor costimulatory domains. Outcome: worse than baseline. Reason: disrupting the adapter-binding motif impaired receptor surface expression and subsequent activation marker expression
A Comparison of Co-stimulatory HVEM Domains in Second Generation CAR-T Cells · Harvard · Master's
Tried and failed
catalytic site point mutation applied to in vitro enzymatic nucleotide synthesis. Outcome: no signal. Reason: mutation abolished the catalytic activity required to synthesize the target product
Nucleotide Sponges and Evasion of Antiviral Immunity · Harvard · PhD
Tried and failed
site-directed point mutagenesis of helicase coupling residues applied to DNA fork duplex unwinding activity. Outcome: no signal. Reason: mutation abolished duplex separation without disrupting stable binding to the substrate fork
Probing mechanisms of genome maintenance using single molecule methods · Johns Hopkins · PhD
Tried and failed
site-directed mutagenesis of catalytic pocket arginine residue applied to mismatch-specific DNA glycosylase activity. Outcome: no signal. Reason: substitutions of the critical arginine completely abolished base excision catalytic activity
Mutagenicity and repair of small DNA lesions · UT Austin · PhD
Tried and failed
rational point mutation of binding interface residues applied to altering endonuclease sequence recognition specificity. Outcome: no signal. Reason: engineered interface substitutions abrogated catalytic cleavage activity completely
Design of custom CRISPR-Cas9 PAM variant enzymes via scalable engineering and machine learning · Harvard · PhD
Tried and failed
site-directed mutagenesis of putative binding residues applied to protein-nucleic acid complex binding interface. Outcome: no signal. Reason: disrupting key positively charged or loop residues abolished double-stranded DNA binding activity
Structures of Class 1 CRISPR-Cas surveillance complexes · UT Austin · PhD
Engineered substitutions disrupt protein folding and stability to cause aggregation or expression loss
Point mutations targeting core hydrophobic networks, surface patches, or transmembrane regions frequently destabilize native tertiary structure and trigger protein aggregation or insolubility. These structural perturbations often prevent soluble protein expression, induce subunit dissociation during purification, or cause local disorder that impedes structural characterization.
Tried and failed
conserved residue interaction network guided site-directed mutagenesis applied to enzyme catalytic site modification. Reason: double mutations disrupted structural stability leading to poor expression, aberrant chromatography, and complete loss of activity
Residue Interaction Network Approach to Conformational Dynamics and Enzyme Evolution in β-Lactamases and Protein Tyrosine Phosphatases · Georgia Tech · PhD
Tried and failed
site-directed mutagenesis of a conserved glycine residue applied to recombinant glycoprotein expression and secretion. Reason: substitutions caused complete protein insolubility and loss of secretion in mammalian and bacterial hosts
Deorphanizing Enzymes: Characterization of NXPE1 and Its Role in Mucin Glycosylation and Ulcerative Colitis Pathogenesis · Harvard · PhD
Tried and failed
single-point targeted site mutagenesis applied to engineered protein-protein interaction interface. Reason: substitutions caused complete loss of soluble protein expression, likely disrupting structural stability
Tried and failed
combinatorial alanine mutagenesis of core hydrophobic residues applied to full-length pro-apoptotic protein expression. Outcome: unstable. Reason: disrupting multiple core hydrophobic network residues destabilised protein folding, preventing expression or recovery in solution
Mechanistic Insights into the Conformational Regulation of Pro-Apoptotic BAX · Harvard · PhD
Tried and failed
Site-directed mutagenesis of surface residues to tryptophan applied to recombinant protein expression and purification. Outcome: unstable. Reason: mutations in surface patches and bulky hydrophobic substitutions caused protein insolubility and aggregation
INVESTIGATING THE EVOLUTION OF NUCLEOTIDE SPECIFICITY AND ORGANIZATION OF DNA BINDING IN MCRBC COMPLEXES · Cornell · PhD
Tried and failed
point mutation for catalytic disruption applied to heteromeric protein complex activity. Outcome: unstable. Reason: mutation induced subunit dissociation during purification rather than directly inhibiting catalytic function
Unifying regulatory mechanism of bacterial cell wall synthesis · Harvard · PhD
Considered and rejected
Considered and rejected: Restricted scanning mutagenesis of TMEM127 exclusively to cytoplasmic loops/tails, avoiding transmembrane domains because prior pheochromocytoma missense mutations caused gross mislocalisation into diffuse cytoplasmic aggregates.
Molecular mechanism of suppression of T cell responses by Salmonella effector SteD · Imperial · PhD
Considered and rejected
Considered and rejected: Rejected mini-protein scaffolds stabilized by zinc-finger motifs or disulfide bonds because combinatorial mutagenesis could disrupt native folds and disulfide pairing.
Development and preclinical evaluation of theranostic agents targeting muc16/ca125 and nectin-4 expression in breast, ovarian and pancreatic cancers · Saskatchewan · PhD
Single point mutations fail to modulate function due to interface redundancy or structural buffering
Alanine scanning and individual residue substitutions frequently fail to disrupt protein interactions, alter DNA binding specificity, or switch substrate recognition. These attempts were buffered by interface redundancy and demonstrated that functional shifts often require broader structural context or combinatorial mutations.
Tried and failed
alanine scanning mutagenesis of interface residues applied to disrupting antibody-antigen binding affinity. Outcome: no signal. Reason: most single point mutations had minimal impact on overall binding due to interface redundancy
On Epitope-Paratope Interactions of Emerging to Endemic Viruses · MIT · PhD
Tried and failed
single site-directed mutagenesis in recognition domains applied to transcription factor DNA-binding specificity. Outcome: no signal. Reason: single point mutations in recognition helices were insufficient to significantly alter binding specificity without combinatorial substitutions
Engineering Synthetic Allosteric Transcription Factors · Georgia Tech · PhD
Tried and failed
single point mutagenesis to switch substrate specificity applied to CRISPR-associated transposon target recognition. Outcome: no signal. Reason: single residue substitutions were insufficient to alter PAM recognition preferences without broader structural context changes
MOLECULAR MECHANISM OF CRISPR-ASSOCIATED TRANSPOSONS · Cornell · PhD
Tried and failed
single-point alanine scanning mutagenesis applied to enzyme active site loop and helix. Outcome: no signal. Reason: single point mutations were insufficient to disrupt interactions or yield detectable adduct formation
LSD1-mediated Grob-like fragmentation as a novel drug resistance mechanism · Harvard · PhD
Tried and failed
active-site point mutation and loop swapping applied to enzyme activity engineering. Outcome: no signal. Reason: isolated active-site mutations and loop swaps failed to confer neofunctionalized catalytic activity
Mechanisms of Enzyme Neofunctionalization in Plant Specialized Metabolism · MIT · PhD
Tried and failed
Single-site alanine scanning mutagenesis applied to bacterial toxin active domain. Outcome: no signal. Reason: No individual residue substitution was sufficient to disrupt antibacterial toxicity in inhibition assays.
Exploration of natural and engineered small antimicrobial proteins · UT Austin · PhD
Tried and failed
site-saturation mutagenesis guided by sequence alignment applied to chimeric transcription factor allosteric positions. Outcome: no signal. Reason: primary sequence alignment was insufficient to identify allosteric mutations conferring the desired phenotype
DEVELOPING A TRANSCRIPTIONAL PROGRAMMING EDIFICE USING SYSTEMS OF ENGINEERED TRANSCRIPTION FACTORS · Georgia Tech · PhD
Tried and failed
conserved-site saturation mutagenesis for phenotype conversion applied to homologous transcriptional allosteric repressor proteins. Outcome: no signal. Reason: mutation at the corresponding position did not produce super-repressor phenotypes across different family homolog scaffolds
Engineering Systems of Anti-Repressors for Next-Generation Transcriptional Programming · Georgia Tech · PhD
Tried and failed
individual phosphorylation site alanine mutagenesis applied to protein-kinase binding interaction. Reason: single-site mutations only partially disrupted binding, indicating redundant multi-site interaction motifs
Separase cleaves the kinetochore protein Meikin to direct the meiosis I/II transition · MIT · PhD
Technical cloning barriers and host toxicity prevent successful mutant isolation and propagation
Mutagenesis protocols regularly fail during primer annealing and second-strand synthesis on large vectors or through corruption of essential sequence elements. In other cases, severe plasmid burden, cellular lethality, or loss of autonomous replication prevents the recovery of viable transformed clones or recombinant viral vectors.
Tried and failed
lowering incubation temperature during transformation applied to toxic or burdensome plasmid mutagenesis. Reason: failed to rescue low transformation and cloning efficiency
Engineered plasmids : uncovering lost histories and improving annotation · UT Austin · PhD
Tried and failed
site-directed mutagenesis to correct plasmid mutations applied to bacterial expression plasmid construct. Reason: primers failed to produce the intact desired construct across all screened colonies
EVALUATION OF BACTERIAL BIODEGRADATION AS A MEANS TO REDUCE POLYETHYLENE POLLUTION · Johns Hopkins · Master's
Tried and failed
Site-directed mutagenesis for viral culture applied to retroviral envelope escape variants. Reason: The engineered point mutation abolished viral replication competence and prevented culturing
Longitudinal Evolutionary Dynamics of HIV Escape from Broadly Neutralizing Antibodies · Harvard · PhD
Tried and failed
Site-directed mutagenesis of viral plasmids applied to rescuing viable recombinant viruses. Reason: Inadvertent corruption or loss of essential terminal palindromic repeats during standard cloning
EVOLUTION OF FELINE AND CANINE PARVOVIRUSES: UNDERSTANDING THE CAPSID STRUCTURE, THE NATURAL VARIATION, AND THE ANTIBODY SELECTION · Cornell · PhD
Tried and failed
cytoplasmic tail mutagenesis for pseudotype optimization applied to chimeric viral vector propagation. Outcome: worse than baseline. Reason: tail modifications impaired autonomous viral replication in host cells
Viral and host determinants of cell entry of emerging and neglected enveloped viruses · Harvard · PhD
Combining individually beneficial mutations causes negative epistasis that degrades performance
Combining multiple point mutations that were individually beneficial often triggers negative non-additivity that reduces binding affinity or completely abolishes signaling activity. These epistatic interactions can also impair transient expression yields and cause multi-site variants to perform significantly worse than baseline single mutations.
Tried and failed
combining beneficial single point mutations applied to protein thermal stability and signaling. Outcome: no signal. Reason: negative epistasis causing complete loss of signaling activity across all temperatures
Evolution and engineering of protein-protein interactions · MIT · PhD
Tried and failed
Combining individually beneficial single-point mutations applied to protein directed evolution. Outcome: worse than baseline. Reason: Negative epistasis caused the combination of mutations to perform worse than the best single mutation.
Harnessing biological diversity and machine learning to build a cell engineering toolbox · MIT · PhD
Tried and failed
combining single point mutations for affinity maturation applied to peptide-protein interface binding. Outcome: worse than baseline. Reason: negative non-additivity between simultaneous mutations significantly reduced target binding affinity
Affinity Maturation of Peptides to Bind Protein-Protein Interfaces · MIT · PhD
Tried and failed
structure-guided CDR liability removal mutagenesis applied to therapeutic antibody light chain engineering. Outcome: worse than baseline. Reason: specific combination of light chain mutations significantly impaired transient protein expression yields
The integration of antibody engineering with biomolecular interaction visualization tools for the reduction of sequence liabilities in the generation of therapeutic antibodies · Harvard · Master's
Tried and failed
expanding multi-site saturation mutagenesis library size applied to protein binding domain engineering. Outcome: worse than baseline. Reason: simultaneously mutating an additional critical contact residue severely abrogated enzymatic catalytic activity
Design of custom CRISPR-Cas9 PAM variant enzymes via scalable engineering and machine learning · Harvard · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Test F32 DHFR mutations (F32I, F32V, F32A, F32W, F32Y) for thermostability increases and design Met20 loop/archaeal hinge mutants. Blocker: Requires wet lab molecular biology techniques including site-directed mutagenesis, protein expression in mutant E. coli strains, and thermostability assays.
Evolutionary motif swapping of human dihydrofolate reductase rewires the enzymatic cycle · Harvard · PhD
Left open
Perform rational design and directed evolution on enzymes to recognize AQS as a cofactor and enhance stability in alkaline environments. Blocker: Requires wet lab facilities for directed evolution, protein expression, mutagenesis, and biochemical assays.
Rapid High-Throughput Screening Methods for Monitoring Electron Transfer Reactions in Biological Systems and Microalgae Phenotyping · Virginia Tech · Doctorate
Left open
Perform experimental mutagenesis to validate tuning directional interactions relative to CH-pi stacking to modulate carbohydrate-binding specificity or enzyme activity. Blocker: Requires a wet biology lab for site-directed mutagenesis, protein expression, and binding/activity assays
The Role of CH–π Interactions in Protein-Carbohydrate Binding · MIT · PhD
Left open
Determine the exact residue location of radical propagation in W191Y ZnCcP using site-directed mutagenesis on distal versus proximal Trp residues. Blocker: Requires a biochemistry wet lab, protein expression, site-directed mutagenesis, and spectroscopic analysis apparatus.
Left open
Experimentally verify the proposed PSII-APC supercomplex structure and excitation injection pathways using site-directed mutagenesis. Blocker: Requires wet-lab experimental facilities, biological samples, and site-directed mutagenesis capabilities.
Structural studies of chlorophyll f-based photosynthesis · Imperial · PhD
Left open
Normalize oligo melting temperatures and optimize thermocycling protocols to eliminate uneven mutation frequencies in Random Site Saturation Mutagenesis. Blocker: Requires wet-lab experimental access to synthesize oligos, run mutagenesis thermocycling, and sequence resulting libraries.
Emulsions and Their Applications in High-Throughput Screening of Proteins · Harvard · PhD
Left open
Perform site-directed mutagenesis to elucidate the physical and functional properties and wavelength assignments of individual Chl f sites in FR-PSI. Blocker: Requires a wet lab and biological experimental setup for site-directed mutagenesis and spectroscopy.
Structural studies of chlorophyll f-based photosynthesis · Imperial · PhD
Left open
Perform affinity measurements and PCR-directed site mutagenesis affinity maturation of humanized LO1 antibody fragments targeting MDA-LDL. Blocker: Requires wet-lab molecular biology facilities, PCR reagents, antibody expression systems, and binding assay equipment.
The humanisation and generation of antibody fragments against oxidised low density lipoprotein for the optical molecular imaging of atherosclerosis · Imperial · PhD
Left open
Perform affinity maturation on the conformation-selective anti-EGFR nanobody HD01 to enhance affinity and therapeutic window. Blocker: Requires wet-lab molecular biology experiments (mutagenesis libraries, yeast display, FACS screening, EGFR binding assays)
Discovering conformation selective anti-EGFR nanobodies · Harvard · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Giovanni Consoli · Imperial · PhD · 2025
Structural studies of chlorophyll f-based photosynthesis - Rachel Anne Silverstein · Harvard · PhD · 2025
Design of custom CRISPR-Cas9 PAM variant enzymes via scalable engineering and machine learning - Renee Bridget Chang · Harvard · PhD · 2026
Nucleotide Sponges and Evasion of Antiviral Immunity - Dariia Yehorova · Georgia Tech · PhD · 2026
Residue Interaction Network Approach to Conformational Dynamics and Enzyme Evolution in β-Lactamases and Protein Tyrosine Phosphatases - Zachary Kirchner · Johns Hopkins · Master's · 2026
EVALUATION OF BACTERIAL BIODEGRADATION AS A MEANS TO REDUCE POLYETHYLENE POLLUTION - Weiyao Wang · Cornell · PhD · 2025
DISCOVERY AND ENGINEERING OF ANTIBODIES TARGETING GLYCAN ANTIGENS - Rebecca Zawistowski · Cornell · PhD · 2025
INTRA- AND INTER-PROTEIN ELECTRON TRANSFER MECHANISMS IN CYTOCHROME C AND CYTOCHROME C PEROXIDASE UNDER EXTREME CHEMICAL AND PHYSICAL PERTURBATIONS - Allison M. Keys · MIT · PhD · 2025
The Role of CH–π Interactions in Protein-Carbohydrate Binding - Sean Michael Watson · Harvard · PhD · 2025
Discovering conformation selective anti-EGFR nanobodies - Dennis E. Brookner · Harvard · PhD · 2025
Evolutionary motif swapping of human dihydrofolate reductase rewires the enzymatic cycle - Kaiyi Jiang · MIT · PhD · 2025
Harnessing biological diversity and machine learning to build a cell engineering toolbox - Ashley N. Hersey · Georgia Tech · PhD · 2025
Engineering Synthetic Allosteric Transcription Factors
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