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Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA-Bulge Size and Sequence Dependence.

ABSTRACT: In this report, we investigate the efficiency and selectivity of a Zn2+-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that form 4-nt bulges, we identified systems with an improved efficacy (an estimated half-life of ca 7-8 h as compared to 11-12 h for sequences studied earlier) as well as systems with an improved site selectivity (up to over 70% cleavage at a single site as compared to 50-60% with previous targets sequences). For targets forming 3-nt bulges, the enhancement compared to previous systems was even more pronounced. Compared to a starting point of targets forming 3-nt AAA bulges (half-lives of ca 21-24 h), we could identify target sequences that were cleaved with half-lives three times lower (ca 7-8 h), i.e., at rates similar to those found for the fastest 4-nt bulge system. In addition, with the 3-nt bulge RNA target site selectivity was improved even further to reach well over 80% cleavage at a specific site.

SUBMITTER: Murtola M

PROVIDER: S-EPMC6150328 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA-Bulge Size and Sequence Dependence.

Murtola Merita M Ghidini Alice A Virta Pasi P Strömberg Roger R

Molecules (Basel, Switzerland) 20171029 11

In this report, we investigate the efficiency and selectivity of a Zn<sup>2+</sup>-dependent peptide nucleic acid-based artificial ribonuclease (PNAzyme) that cleaves RNA target sequences. The target RNAs are varied to form different sizes (3 and 4 nucleotides, nt) and sequences in the bulge formed upon binding to the PNAzyme. PNAzyme-promoted cleavage of the target RNAs was observed and variation of the substrate showed a clear dependence on the sequence and size of the bulge. For targets that ...[more]

PMID: 29109368

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Project description:Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other molecules, and the energetics of these processes are profoundly influenced by the ion atmosphere that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atmosphere, understanding its properties and effects will require synergy between computational modeling and experiment. Prior computational models and experiments suggest that cation occupancy in the ion atmosphere depends on the size of the cation. However, the computational models have not been independently tested, and the experimentally observed effects were small. Here, we evaluate a computational model of ion size effects by experimentally testing a blind prediction made from that model, and we present additional experimental results that extend our understanding of the ion atmosphere. Giambasu et al. developed and implemented a three-dimensional reference interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helices, and this model predicts that Na(+) would outcompete Cs(+) by 1.8-2.1-fold; i.e., with Cs(+) in 2-fold excess of Na(+) the ion atmosphere would contain an equal number of each cation (Nucleic Acids Res. 2015, 43, 8405). However, our ion counting experiments indicate that there is no significant preference for Na(+) over Cs(+). There is an ∼25% preferential occupancy of Li(+) over larger cations in the ion atmosphere but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, we followed the folding of the P4-P6 RNA and showed that differences in folding with different alkali metal ions observed at high concentration arise from cation-anion interactions and not cation size effects. Overall, our results provide a critical test of a computational prediction, fundamental information about ion atmosphere properties, and parameters that will aid in the development of next-generation nucleic acid computational models.

Project description:Fifty-nine RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters DeltaH degrees, DeltaS degrees, DeltaG 37 degrees, and TM for each sequence were determined. Sequences from this study were combined with sequences from previous studies [Longfellow, C. E., et al. (1990) Biochemistry 29, 278-285; Znosko, B. M., et al. (2002) Biochemistry 41, 10406-10417], thus examining all possible group I single-nucleotide bulge loop and nearest-neighbor sequence combinations. The free energy increments at 37 degrees C for the introduction of a group I single-nucleotide bulge loop range between 1.3 and 5.2 kcal/mol. The combined data were used to develop a model for predicting the free energy of a RNA duplex containing a single-nucleotide bulge. For bulge loops with adjacent Watson-Crick base pairs, neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The proposed model for prediction of the stability of a duplex containing a bulged nucleotide was primarily affected by non-nearest-neighbor interactions. The destabilization of the duplex by the bulge was related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. The stability of a duplex containing a bulged nucleotide adjacent to a wobble base pair also was primarily affected by non-nearest-neighbor interactions. Again, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. However, when one or both of the bulge nearest neighbors was a wobble base pair, the free energy increment for insertion of a bulge loop is dependent upon the position and orientation of the wobble base pair relative the bulged nucleotide. Bulge sequences of the type ((5'UBX)(3'GY)), ((5'GBG)(3'UU)) and ((5'UBU)(3'GG)) are less destabilizing by 0.6 kcal/mol, and bulge sequences of the type ((5'GBX)(3'UY)) and ((5'XBU)(3'YG)) are more destabilizing by 0.4 kcal/mol than bulge loops adjacent to Watson-Crick base pairs.

Dataset Information

Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA-Bulge Size and Sequence Dependence.

Publications

Zinc Ion-Dependent Peptide Nucleic Acid-Based Artificial Enzyme that Cleaves RNA-Bulge Size and Sequence Dependence.

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