Function Of Loop Length Research Articles

Counterion and Polythymidine Loop-Length-Dependent Folding and Thermodynamic Stability of DNA Hairpins Reveal the Unusual Counterion-Dependent Stability of Tetraloop Hairpins

Stem-loop DNA hairpins containing a 5-base-pair (bp) stem and single-stranded polythymidine loop were investigated using thermodynamic melting analysis and stopped-flow kinetics. These studies revealed the thermodynamic stability and folding kinetics as a function of loop length and counterion concentration. Our results show the unusually high thermodynamic stability for tetraloop or 4 poly(dT) loop hairpin as compared with longer loop length hairpins. Furthermore, this exceptional stability is highly counterion-dependent. For example, in the higher counterion concentration regime of 50 mM NaCl and above, the tetraloop hairpin displays enhanced stability as compared with longer loop length hairpins. However, at lower counterion concentration of 25 mM NaCl and below, the thermal stability of tetraloop hairpin is consistent with the longer loop hairpins. The enhanced stability of tetraloop hairpins at higher counterion concentration can be explained on the basis of the combined entropic effect of loop closure as well as base stacking in the loop regions. The stability of longer loop length hairpins at all counterion concentrations as well as tetraloop hairpin at lower counterion concentration can be explained on the basis of entropic effect of loop closure alone. The thermodynamic parameters at lower and higher counterion concentrations were determined to quantify the enhanced stability of base-stacking effects occurring at higher counterion concentrations. For example, for 100 mM NaCl, excess Gibbs energy and enthalpy due to base stacking within the tetraloops were measured to be -1.2 ± 0.14 and -3.28 ± 0.32 kcal/mol, respectively, whereas, no excess of Gibbs energy and enthalpy was observed for 0, 5, 10, and 25 mM NaCl. These findings suggest significant base-stacking interactions occurring in the loop region of the tetraloop hairpins at higher counterion concentration and less significant base-stacking interactions in the lower counterion concentration regime. We suggest that at higher counterion concentrations, hydrophobic collapse of the nucleotides in the loop may be enhanced due to the increased polarity of the solvent, thereby enhancing base-stacking interactions that contribute to unusually high stability.

HuCAL PLATINUM, a Synthetic Fab Library Optimized for Sequence Diversity and Superior Performance in Mammalian Expression Systems

This article describes the design of HuCAL ( human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production. Gene optimization has been used to improve expression of the antibody master genes in the library context. As a result, full-length IgGs derived from the library show both significant improvements in expression levels and less undesirable glycosylation sites when compared to the previous HuCAL GOLD library. Additionally, in-depth analysis of sequences from public databases revealed that diversity of CDR-H3 is a function of loop length. Based upon this analysis, the relatively uniform diversification strategy used in the CDR-H3s of the previous HuCAL libraries was changed to a length-dependent design, which replicates the natural amino acid distribution of CDR-H3 in the human repertoire. In a side-by-side comparison of HuCAL GOLD and HuCAL PLATINUM, the new library concept led to isolation of about fourfold more unique sequences and to a higher number of high-affinity antibodies. In the majority of HuCAL PLATINUM projects, 100–300 antibodies each having different CDR-H3s are obtained against each antigen. This increased diversity pool has been shown to significantly benefit functional antibody profiling and screening for superior biophysical properties.