Jimmy Gu

Sequence-function relationships provide new insight into the cleavage site selectivity of the 8–17 RNA-cleaving deoxyribozyme

Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5′ rN18-N1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN18) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8–17 variants, identified from ∼75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ≥0.1 min−1, and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.

In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine–pyrimidine junctions

Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited kobs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ∼1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.

Target-induced and Equipment-free DNA Amplification with a Simple Paper Device.

We report on a paper device capable of carrying out target-induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA-enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.

A DNAzyme Feedback Amplification Strategy for Biosensing

We report a signal amplification strategy termed DNAzyme feedback amplification (DFA) that takes advantage of rolling-circle amplification (RCA) and an RNA-cleaving DNAzyme (RCD). DFA employs two specially programmed DNA complexes, one composed of a primer and a circular template containing the antisense sequence of an RCD, and the other composed of the same circular template and an RNA-containing substrate for the RCD. RCA is initiated at the first complex to produce RCD elements that go on to cleave the substrate in the second complex. This cleavage event triggers the production of more input complexes for RCA. This reaction circuit continues autonomously, resulting in exponential DNA amplification. We demonstrate the versatility of this approach for biosensing through the design of DFA systems capable of detecting a microRNA sequence and a bacterium, with sensitivity improvements of 3-6 orders of magnitude over conventional methods.

Programming a topologically constrained DNA nanostructure into a sensor.

Many rationally engineered DNA nanostructures use mechanically interlocked topologies to connect individual DNA components, and their physical connectivity is achieved through the formation of a strong linking duplex. The existence of such a structural element also poses a significant topological constraint on functions of component rings. Herein, we hypothesize and confirm that DNA catenanes with a strong linking duplex prevent component rings from acting as the template for rolling circle amplification (RCA). However, by using an RNA-containing DNA [2] catenane with a strong linking duplex, we show that a stimuli-responsive RNA-cleaving DNAzyme can linearize one component ring, and thus enable RCA, producing an ultra-sensitive biosensing system. As an example, a DNA catenane biosensor is engineered to detect the model bacterial pathogen Escherichia coli through binding of a secreted protein, with a detection limit of 10 cells ml−1, thus establishing a new platform for further applications of mechanically interlocked DNA nanostructures.

Optimal DNA templates for rolling circle amplification revealed by in vitro selection.

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC-rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.

Sequence Mutation and Structural Alteration Transform a Noncatalytic DNA Sequence into an Efficient RNA-Cleaving DNAzyme

We have previously shown that through test-tube molecular evolution, an arbitrarily chosen noncatalytic DNA sequence can be evolved into a catalytic DNA (DNAzyme) with significant RNA-cleaving activity. In this study, we aim to address the question of whether the catalytic activity of such a DNAzyme can be further optimized using in vitro selection. Several cycles of selective enrichment starting with a partially randomized DNA library have resulted in the isolation of many sequence variations that show notably improved catalytic activity. Bioinformatic analysis and activity examination of several DNAzyme-substrate constructs have led to two interesting findings about sequence mutations and the secondary structure of this DNAzyme: (1) three crucial mutations have transformed the DNAzyme into 8–17, a DNAzyme that has been discovered in multiple previous in vitro selection experiments, and (2) other mutations have allowed this special 8–17 variant to make structural fine-tuning in order to cleave an arbitrarily chosen RNA-containing substrate with a defined sequence. Our study not only showcases the combined power of directed molecular evolution and in vitro selection techniques in turning a noncatalytic nucleic acid sequence into an efficient enzyme, but it also raises the question of whether mother nature has used a similar approach to evolve natural enzymes.

Evolution of an Enzyme from a Noncatalytic Nucleic Acid Sequence.

The mechanism by which enzymes arose from both abiotic and biological worlds remains an unsolved natural mystery. We postulate that an enzyme can emerge from any sequence of any functional polymer under permissive evolutionary conditions. To support this premise, we have arbitrarily chosen a 50-nucleotide DNA fragment encoding for the Bos taurus (cattle) albumin mRNA and subjected it to test-tube evolution to derive a catalytic DNA (DNAzyme) with RNA-cleavage activity. After only a few weeks, a DNAzyme with significant catalytic activity has surfaced. Sequence comparison reveals that seven nucleotides are responsible for the conversion of the noncatalytic sequence into the enzyme. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of the molecular evolution. Our findings demonstrate that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today.

Identification of a toxic peptide through bidirectional expression of small RNAs.

Research in the field of noncoding functional RNA sequences has flourished over the past two decades and has showcased the utility of these nucleic acids, which extends beyond their traditional roles as being the workhorses behind protein translation. A search through the intergenic regions of prokaryotic genomes has unveiled a class of important regulatory elements, known as small RNAs (sRNAs). As their names imply, these RNA sequences are relatively short, typically ranging from 50 to 400 nucleotides (nt) in length. In the E. coli genome alone, over 80 sRNA sequences have been discovered by using a combination of bioinformatics and experimental techniques, though the function of many of these sequences remains unknown. Of the sRNAs that have been characterized to date, the majority of them interact with mRNA targets, thereby modulating their translation initiation or their stability upon binding. Another subset of sRNAs can regulate the activity of protein targets or function as a part of a protein–sRNA complex. Together, these tiny elements govern a number of bacterial stress response pathways and they have been linked to pathogenicity in virulent species. Due to the substantial role of sRNAs in maintaining cellular homeostasis and viability, disruption of their expression is expected to have deleterious effects on the cell. Previously, screening for lethal or growth defective phenotypes upon the overexpression of antisense RNA sequences was used to identify essential genes in Staphylococcus aureus. 9] Here, we implemented a similar approach in an effort to develop a method to isolate sRNA sequences with critical regulatory functions in E. coli. We adopted a tetracycline-inducible system to regulate the expression of the sense and antisense sequences of twelve sRNAs. These sequences were cloned into pNYL-MCS11, a plasmid derived from pZE21MCS1, which is described in an earlier study. The optimized ribosome binding site from the plasmid was removed prior to cloning, such that the sense and antisense sRNAs would be transcribed but not translated when their expression is induced in the presence of anhydrotetracycline (Atc), an analogue of tetracycline that appears to be a more potent yet less toxic inducer. Analyses were subsequently performed in E. coli strain DH5aZ1, which has been engineered to endogenously express the TetR repressor (Scheme 1). Of the sRNAs that were screened, three have been extensively studied and have been shown to regulate different cellular processes (Table 1). These candidates were chosen in order to examine the physiological effects associated with their upand downregulation. The functions of the remaining nine sequences have not yet been characterized. As such, we wish to probe into their biological roles through this screen. Overexpression of all twelve sense sRNA sequences did not affect cell growth