Jay Hesselberth

Simultaneous detection of diverse analytes with an aptazyme ligase array

Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.

Optimization and optimality of a short ribozyme ligase that joins non-Watson–Crick base pairings

A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.

Combinatorial methods: aptamers and aptazymes

Combinatorial methods have been used to generate nucleic acid molecules with specific characteristics. Aptamers are nucleic acid binding species, and can be modified to directly transduce molecular recognition to optical signals. Aptazymes are allosteric or effector-activated ribyzymes. We have designed or selected aptazymes that are responsive to a variety of ligands. In particular, we have selected a ribozyme ligase that is activated 10,000-fold in the presence of an oligonucleotide effector, and have designed ligases that are up to 1,600-fold dependent on small molecule effectors. Even in those instances where designed constructs were initially unresponsive, we have been able to use selection to optimize their response characteristics.

Switching nucleic acids for antibodies

Can ribozymes replace antibodies in future array formats for proteomics and metabolomics?