In vitro selection of DNAzymes selective for monovalent metal ions and their development as sensors in living cells and organisms
Beyond the function of carrying genetic information, the recognition function (DNA aptamers), the structural function (DNA origami and DNA nanodevices), and most interestingly, the catalytic function (deoxyribozymes or DNAzymes) of DNAs have been explored since 1990s, which attract widespread interest. DNAzymes, fully biocompatible but not naturally identified, commonly use metal ions as cofactors for catalysis, and some of them show exceptional selectivity for target metal ions over other metal ions, granting DNAzymes ideal advantages in metal ion sensing. To obtain such ion selective DNAzymes, in vitro selection methodology is exclusively used, and has resulted in a spectrum of RNA-cleaving DNAzymes selective for different types of metal ions, which have been leveraged for the development of environmental or live cell metal ion sensors. Potassium ion (K+) is the most abundant metal ion in all living cells and plays important biological roles. Hence, methods to detect K+ in living systems with high selectivity are invaluable to understand the precise mechanisms of K+ in physiological events. As the first step of live detection of K+, DNAzymes that are selective for K+ were obtained through in vitro selection and are being validated and truncated, which is recorded in the Chapter 2. The first K+-selective RNA-cleaving DNAzymes has been obtained from an in vitro selection, showing more than 16% cleavage yield by 20 hours under selection conditions with over 30-fold selectivity over Na+ and over 100-fold selectivity over other metal ions, and without restrictions by anion types. Since the proposal of “in vitro evolution” in 1989 and three independent successes in 1990 by the Joyce, Szostak, and Gold labs, in vitro selection (or SELEX) has been broadly used in obtaining and improving the performance of functional nucleic acids. To improve the effectiveness of selection, evolutionary stringency or pressure are usually applied during the selection process. However, how to introduce these selection pressures and how certain selection pressure works is still largely unclear. To answer some of these basic questions, branched selections under different selection pressures were compared by the next-generation sequencing (NGS) in the Chapter 3. Analyzing the evolution process during in vitro selections under different selection pressures revealed that introducing negative selection at the early rounds but not late rounds of selection favors the enrichment of target-selective sequences. Also, enhancing selection pressure by decreasing positive selection time may not always lead to high yield DNAzyme sequences but can enrich highly selective and high initial rate sequences. DNA handling methodologies (PCR amplification, precipitation, extraction, etc.) make studying DNA functions possible in molecular biology. Almost all current DNA handling protocols use Na+ and K+ containing buffers. However, Na+ and K+ are suggested to help with DNA folding into higher order structures, like the G-quadruplex (G4) structure, and block DNA polymerase elongation and PCR efficiency. Li+ ion and some organic cations do not favor the G4 structure formation, and thus could potentially be used as substitutions for Na+ and K+ in DNA handling protocols. In the Chapter 4, Li+ based PCR, ethanol precipitation, and PAGE gel extraction protocols have been developed to achieve a better efficiency compared with standard or commercialized protocols. Organic cation tris(hydroxymethyl)aminomethane (Tris) based DNA PCR method was also developed and achieved improved amplification yield for G4 containing sequences and high GC DNAs compared with commercial protocols. Li+ has been used for bipolar disorder (BD) therapy for more than 70 years, yet its cellular enrichment has not been fully understood, mainly caused by a lack of live cell selective Li+ imaging approach. Based on recent success of obtaining Li+-selective DNAzyme 20-4 by Dr. Claire McGhee in the Lu lab, the kinetics (kobs ∼0.14 h−1) and selectivity (> 100-fold) of this DNAzyme has been studied, and a sensor working under physiological conditions had been constructed from the DNAzyme in the Chapter 5. The sensor displays a remarkable detection range down to submilimolar for Li+ in cell imaging, overlapping with the biomedical relevant concentration range of Li+ in BD therapy. The first highly selective Li+ imaging at low millimolar concentration range was achieved in living HeLa cells. Metal ion profiling with spatial and temporal precision is limited by a shortage of methods to probe metal ions with activity control at specific space and time, especially in vivo. To overcome this limitation, the work in the Chapter 6 developed a Zn2+-specific near-infrared (NIR) DNAzyme nanoprobe for in vivo Zn2+ profiling with spatiotemporal activity control in live zebrafish together with Mr. Kang Yong Loh. By conjugating photocaged DNAzyme sensors onto lanthanide-doped upconversion nanoparticles (UCNPs), a deep tissue penetrating NIR light was converted into 365 nm emission for DNAzyme sensor activation and metal ion detection. Genetically encoded fluorescent proteins (FPs) have been applied for metal ion detection but restricted to a limited number of metal ions. On the other hand, DNAzyme-based metal ions sensors for cellular imaging largely relies on costly synthesis and a tricky protocol for sensor annealing and delivery. By conjugating the advantages of various FPs in convenient readout and diverse DNAzymes in metal ion selectivity, a new class of FP and DNAzyme-based genetically encoded ion sensor was developed together Dr. Mengyi Xiong and recorded in the Chapter 7. Using Mg2+-specific 10-23 or Zn2+-specific 8-17 RNA-cleaving DNAzymes, the expression of FPs was regulated to show ratiometric fluorescent readout for different levels of metal ions.
Deoxyribozyme, DNAzyme, in vitro selection, metal ion, lithium, potassium, zinc, magnesium, DNA handling, PCR bias, GC rich sequence PCR, sensor, live imaging, in vivo imaging, metal ion profiling, fluorescence protein sensor, genetically encoded sensor
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