A quantitative method for evaluating the stabilities of nucleic acids

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed “reference” duplex (usually a Watson-Crick duplex) and a related “test” duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.

Redirecting the specificity of ubiquitination by modifying ubiquitin-conjugating enzymes.

Depletion of specific cellular proteins is a powerful tool in biological research and has many medical and agricultural benefits. In contrast to genetic methods currently available to attenuate protein levels, we describe an alternative approach that redirects the ubiquitin-dependent proteolytic pathway to facilitate specific proteolytic removal. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target protein. This attachment is accomplished, in part, by a family of enzymes designated E2s (or ubiquitin-conjugating enzymes), some of which use domains near their C termini for target recognition. Here, we demonstrate that E2 target recognition can be redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. In five dissimilar examples, chimeric E2s were created that recognized and ubiquitinated their respective binding partners with high specificity. We also show that ubiquitination of one protein targeted by this method led to its ATP-dependent degradation in vitro. Thus, by exploiting interacting domains derived from natural and synthetic ligands, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins.