Dear Visitor, we can set up here your advertisements upon your wish. Please get in touch with us.
  Login:    Sitemap:    Search:

Ribozyme-based Molecular Circuit as Programmable Nanodevices

An important feature of molecular logic gates is the ability to generate signals that can control the activity of other molecular switches. The production of a diversity of highly-responsive oligonucleotide-sensing ribozymes would expand the complexity and efficiency of engineered nucleic acid circuits like those demonstrated previously.

To demonstrate a molecular cascading circuit, we employed construct YES-1 and a variation of construct YES-2 to create a simple molecular circuit (Fig. 1a). The sequence and length of YES-2 was altered in stem I to create a YES-2 variant that generates a new 21-nt 3` fragment on self-cleavage (Fig. 1a). This RNA fragment is complementary to the oligonucleotide binding site of YES-1, and therefore, it should activate the second ribozyme upon cleavage and dissociation from the first ribozyme in the circuit. This simple molecular signaling pathway was demonstrated using radiolabeled YES-1 with various combinations of unlabeled YES-2 and the oligonucleotides DNA-1 and DNA-2. Although DNA-1 triggers YES-1 cleavage as demonstrated here, no cleavage is observed when both ribozymes are simultaneously incubated in the absence of DNA effector (Fig. 1b). This demonstrates that the YES-1 ribozyme is not activated when its RNA-1 signal oligonucleotide remains attached to the YES-2 variant ribozyme. In contrast, the addition of DNA-2 induces YES-1 cleavage only when the YES-2 variant ribozyme is present. Furthermore, the kinetics of YES-1 function as part of the complete signaling pathway indicate a lag phase that we interpret to be caused by the time required to release RNA-1 from the YES-2 variant upon activation by DNA-2 (Fig. 1c). These findings are consistent with the design of the ribozyme signaling pathway wherein an activator of the first ribozyme in the series triggers the release of an activator of the second ribozyme. Although simple in design, our findings suggest that a variety of more complex molecular circuitry could be constructed wherein oligonucleotide triggers could be used to carry out a variety of logic-based ribozyme functions.

Figure 1. Ribozyme-based molecular cascading circuit using YES-1 and a variant of YES-2. (A) Nucleotides of the YES-2 variant RNA that differ from YES-2 are depicted. Upon activation of YES-2 variant by effector DNA-2, the 3` cleavaged fragment (RNA-1) is released and serves as an effector for YES-1 activation. Schematic presentations of the logic circuit of the two YES gates are depicted. (B) Assay depicting function of a ribozyme signaling pathway. YES-1 RNAs are radiolabeled in all lanes. (C) Kinetic analysis of YES-1 self-cleavage in the presence of YES-2 variant and its effector DNA-2. Details are as described in B.

References:
1. Robert Penchovsky & Ronald R. Breaker - Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes – 2005, Nature Biotechnology, 10870156, Q1 (Biochemistry, Genetics and Molecular Biology), IF – 43,5.

2. Robert Penchovsky - Engineering integrated digital circuits with allosteric ribozymes for scaling up molecular computation and diagnostics – 2012, ACS Synth Biol, 21615063, Q1 (Biochemistry, Genetics and Molecular Biology), IF – 5,382

3. Robert Penchovsky's Short Biography - ACS Synth Biol - 2012, 21615063, Q1 (Biochemistry, Genetics and Molecular Biology), IF – 5,382