Directed Evolution of Nucleotide-Based Libraries Using Lambda Exonuclease

Lim, Bee Nar; Choong, Yee Siew; Ismail, Asma; Glökler, Jörn; Konthur, Zoltán; Lim, Theam Soon

doi:10.2144/000113964

Cited by 17 publications

(12 citation statements)

References 53 publications

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“…Transfer tubes/plate to thermal cycler and heat at 95°C for 30 sec, then immediately transfer to ice for 1 min. (Lim et al, 2012), or if template or primer is biotinylated, XNA can be isolated using streptavidin-coated magnetic beads (see Taylor & Holliger, 2015b); in this scenario, synthesis can be primed using RNA, which can subsequently be removed by alkaline hydrolysis].…”

Section: Methodsmentioning

confidence: 99%

Selecting Fully‐Modified XNA Aptamers Using Synthetic Genetics

Taylor

Holliger

2018

CP Chemical Biology

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This unit describes the application of "synthetic genetics," i.e., the replication of xeno nucleic acids (XNAs), artificial analogs of DNA and RNA bearing alternative backbone or sugar congeners, to the directed evolution of synthetic oligonucleotide ligands (XNA aptamers) specific for target proteins or nucleic acid motifs, using a cross-chemistry selective exponential enrichment (X-SELEX) approach. Protocols are described for synthesis of diverse-sequence XNA repertoires (typically 10 molecules) using DNA templates, isolation and panning for functional XNA sequences using targets immobilized on solid phase or gel shift induced by target binding in solution, and XNA reverse transcription to allow cDNA amplification or sequencing. The method may be generally applied to select fully-modified XNA aptamers specific for a wide range of target molecules. © 2018 by John Wiley & Sons, Inc.

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Section: Methodsmentioning

confidence: 99%

Selecting Fully‐Modified XNA Aptamers Using Synthetic Genetics

Taylor

Holliger

2018

CP Chemical Biology

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“…Afterwards, Klenow fragment is employed to create dsDNA. In this manner, site-specific mutagenesis can be performed using primers that contain degenerate bases [246].…”

Section: Enzyme-based Approachesmentioning

confidence: 99%

Evolving a Peptide: Library Platforms and Diversification Strategies

Bozovičar

Bratkovič

2019

IJMS

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Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.

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“…Apart from conventional directed evolution that relies on digestion and ligation, other enzymes with different functions can be utilized for mutagenesis. Lim et al reported the ability to carry out DNA shuffling using an ssDNA template and lambda exonuclease [186]. In nature, lambda exonuclease assists in DNA recombination of viral DNA.…”

Section: In Vitro Affinity Maturation Strategiesmentioning

confidence: 99%

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

Lim

Choong

Lim

2019

IJMS

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Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some ‘fine tuning’ may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.

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Directed Evolution of Nucleotide-Based Libraries Using Lambda Exonuclease

Cited by 17 publications

References 53 publications

Selecting Fully‐Modified XNA Aptamers Using Synthetic Genetics

Selecting Fully‐Modified XNA Aptamers Using Synthetic Genetics

Evolving a Peptide: Library Platforms and Diversification Strategies

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

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