Split Deoxyribozyme Probe for Efficient Detection of Highly Structured RNA Targets

University of Central Florida STARS

Honors Undergraduate Theses UCF Theses and Dissertations

2018

Split Deoxyribozyme Probe For Efficient Detection of Highly Structured RNA Targets

Sheila Raquel Solarez University of Central Florida

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Recommended Citation Solarez, Sheila Raquel, "Split Deoxyribozyme Probe For Efficient Detection of Highly Structured RNA Targets" (2018). Honors Undergraduate Theses. 311. https://stars.library.ucf.edu/honorstheses/311 SPLIT DEOXYRIBOZYME PROBE FOR EFFICIENT DETECTION OF HIGHLY STRUCTURED RNA TARGETS

SHEILA SOLAREZ

A thesis submitted in partial fulfillment of the requirements for the Honors in the Major Program in Biological Sciences in the College of Sciences and the Burnett Honors College at the University of Central Florida Orlando, Florida

Spring Term, 2018

Thesis Chair: Yulia Gerasimova, PhD

ABSTRACT

Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis. [1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques. [2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which uses a double-labeled fluorogenic substrate as a reporter – has been introduced. In this project we developed an assay based on sDz probe to detect yeast tRNAPhe as a proof-of-principle highly structured target. An sDz probe was designed specific to tRNAphe that could efficiently unwind stable secondary and tertiary structure of the target RNA thereby providing an efficient tool for tRNA detection. [3] The efficiency of the developed sDz probe was compared with a currently used state-of-the-art hybridization probe – molecular beacon probe.

The results obtained in the project further demonstrate the power of sDz probes for the detection of highly structured RNA analytes. The split probes show signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.

ii DEDICATION

For my family, for keeping me grounded while I reach for the stars.

iii

ACKNOWLEDGMENTS

I would like to express my deepest gratitude to Dr. Yulia Gerasimova for her patience, kindness, and guidance. As well as Dr. Charissa de Bekker and the Gerasimova lab for their continued support.

iv TABLE OF CONTENTS Introduction ...... 1

Methodology ...... 5

Molecular beacon probe assay ...... 5

Split deoxyribozyme probe assay ...... 5

Results and discussion ...... 7

Molecular beacon design ...... 7

Split deoxyribozyme probe design ...... 9

Conclusion ...... 14

References……………………………………………….……………………………………….15

v LIST OF TABLES

Table 1: Oligonucleotides used in the study……………………………………………………6

vi LIST OF FIGURES

Figure 1: Secondary and tertiary structure of yeast tRNAphe……………………………….……..1

Figure 2: MB probe in the absence or presence of the analyte…………….…..…………………2

Figure 3:Secondary structure of tRNA with a fragment complementary to the MB probe……...... 3

Figure 4: Split deoxyribozyme probe design ……….……………………………………………..4

Figure5: Fluorescent response of the MB probe to various analytes ………………..…………...8

Figure 6: Split deoxyribozyme probes designs used.………….………………………………. ....9

Figure 7: Comparative analysis of the two sDz probe designs - sDz-long and sDz-short .…. …11

Figure 8: Dependence of sDz-short signal on yeast tRNAphe concentration……….………… …12

Figure 9: Linear dynamic range of yeast tRNAPhe with sDz-short or MB probe…………… ...... 13

vii INTRODUCTION

phe Figure 1: Secondary and tertiary structure of yeast tRNA : Secondary (A) and tertiary (B) structure of yeast tRNAphe, which was chosen as a model analyte in the study.[10] The analyte fragment targeted by two subunits of the sDz probe is indicated using blue and orange curves. The 3’- terminal nucleotides of the acceptor stem serve as a “toehold” for the probe binding.

Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information, they serve to activate and deliver amino acids to the site of protein synthesis. One of the twenty amino acids is carried by a tRNA and is added to the polypeptide chain according to the messenger RNA (mRNA). In addition to protein synthesis, tRNAs also serve as regulators for gene expression. For example, uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell.[1] Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis. [1] tRNAs are composed of 73-

1 90 nucleotides and have a secondary structure that can be schematically represented as a cloverleaf (Figure 1A). [2] In addition, the loops of tRNA interact to form tertiary structure that adds to the stability of tRNA structure (Figure 1B), which represents a challenge for the detection of tRNA molecules by conventional techniques.

Figure 2: MB probe with and without the presence of the analyte [4]

The methods that detect the presence of RNA in vitro include the northern blot and reverse transcription polymerase chain reaction (RT-PCR). For in vivo detection of RNA, fluorescent and non-fluorescent probes are used. [3] These methods are useful in detecting single stranded RNA, however most functional RNA have secondary and tertiary structures. Previous research has been done that use Molecular Beacon (MB) probes to detect the presence of highly structured RNAs related to various biological functions and diseases. [3] They have important features such as a conformational constraint in form of a stem loop, fluorescent signal immediately after the hybridization event, and reversible binding to the analyte (Figure 2). [4] At the same time, the MB probes are not very efficient in binding to structured nucleic acid targets due to the stem-loop structure of the probe itself. [5] In this project, a MB probe was designed and used to detect yeast tRNAphe, which was our model analyte (Figure 3).

Figure 3: Secondary structure of tRNA with complementary to the MB probe shown.

To increase the efficiency of the probe binding to such targets, a split approach to the probe design has been suggested. [3,5,6]. In this approach, a target-recognition site of the probe is split into two parts, each of which is elongated by a signal reporter-binding fragments. It has been shown that a split probe using an MB probe as a reporter can efficiently unwind and detect even a DNA/RNA target folded in a 13 nt-long stem. [7] More importantly, this probe can differentiate between the two targets differing a single nucleotide. At the same time, the MB- based split probe suffers from relatively high detection limit, since it binds to the correspondent target in 1:1 ratio. In addition, it has limited use in complex biological mixtures, such as cell cultures. For example, the MB probe can be opened up by biocomponents of those mixtures causing high background. [8] To enable signal amplification and lower detection limits, another

3 split probe - split deoxyribozyme (sDz) probe, which uses a double-labeled fluorogenic substrate as a reporter – has been introduced (Figure 4). [8] It was proved that sDz probe can efficiently unwind secondary structure of E.coli rRNA and detect the probe-targeted rRNA fragments with low detection limits. [8] In this project an sDz probe was designed to unwind the secondary and tertiary structure of yeast tRNAPhe, thereby providing an efficient tool for tRNA detection without the need of prior annealing the probe to its target. The probe was tested on its performance for the detection of yeast tRNAPhe as a model analyte. The limit of detection of the assay was determined and the efficiency of tRNA detection using the sDz probe was compared with that of the MB probe.

Figure 4: Split Deoxyribozyme Design Split deoxyribozyme is designed by splitting the catalytic core of a deoxyribozyme phosphodiesterase into two subunits, which re-assemble only in the presence of a specific nucleic acid analyte. This re-forms the catalytic core and the deoxyribozyme cleaves the reporter, which has a fluorophore and a quencher at the opposite side from the cleavage site. Therefore, analyte-dependent cleavage of the reporter triggers fluorescence increase.

4 METHODOLOGY Molecular Beacon Probe Assay

For MB probe assay, the samples (120µl) containing 50 nM of the MB probe and defined amount of yeast tRNAphe (0-1000 nM) in a reaction buffer containing 50 mM Tris-HCl, pH 7.4, and 50 mM MgCl2 were prepared. Each sample was split into two tubes, and one set of samples was incubated for 15 min at room temperature followed by measuring the fluorescence at 517 nm upon excitation at 485 nm was then measured using a fluorometer in a quartz cuvette

(3 mm path). The second set of samples was annealed by heating for 5 min at 95°C and then incubating at room temperature prior to measuring the fluorescence. The data from three independent experiments was averaged, with standard deviations as errors. The data was processed in Excel.

Split Deoxyribozyme Probe Assay

sDz probes were designed and their performance analyzed in silico using Nupack

3 software. For the fluorescent sDz assay, the samples (60µl) containing 5 nM sDza, 5 nM of

phe sDzb, and 200 nM of the reporter (Mz substrate), and a defined amount of yeast tRNA in a reaction buffer containing 50 mM HEPES-NaOH, pH 7.4, 50 mM MgCl2, 20 mM KCl, 120 mM

NaCl, 0.03% Triton X-100 was incubated for one hour at 30°C. Fluorescence of the samples at

517 nm upon excitation at 485 nm was then measured using a fluorometer in a quartz cuvette (3 mm path).

5 Name Sequence

Yeast 5’- GCGGAUUUAGmCUCAGU*U*GGGAGAGCGmCCAGACOUGOAAYAPC5 tRNAPhe UGGAGG7UCC5U GU5PCGA1UC CACAGAAUUC GCACCA-3’

DNA 5’-GACTGAATATCTGGAGGTCCTGTTCGATCCACAGAATTCGCACCA-3' Analyte Dza 5'-CCAGGGAGGCTAGCTCAGGACCTCCAGATTTTCAGTC-3'

Dzb 5'-TGGTGCGAATTCTGTGGATCGATCAACAACGA GAGGAAAC-3'

Mz 5’-AAGGTT(FAM)TCCTCguCCCTGGGCA-BHQ1- 3’ Substrate MB Probe 5’-FAM-CGCGACGAACACAGGACCTCGCG-BHQ-3’

Table 1: Oligonucleotides used in the study* *Yeast tRNAphe contains 11 modified bases: 2-methyl-guanosine (2MG), 5,6-dihydrouridine (DHU), N2- dimethylguanosine (M2G), O29-methyl-cytidine (OMC), O29-methyl-guanosine (OMG), the Y base or wybutosine (YG), pseudouridine (PSU), 5-methyl-cytidine (5MC), 7-methyl-guanosine (7MG), 5-methyl-uridine (5MU), and 1- methyl-adenosine (1MA)10. For the MZ substrate, FAM is the flourophore used in this study and BHQ is the quencher that inhibits it from fluorescing. The lowercase “gu” is where the MZ substrate is cleaved because the catalytic activity of the sDz strands act as a ribonuclease.

6 RESULTS AND DISCUSSION

Molecular Beacon Design

To compare the efficiency of the developed sDz probe with currently used hybridization probes, an MB probe targeting the least stable arm of yeast tRNAphe target (according to the

Mfold software) was designed. The MB probe was used at the optimal Mg2+ concentration and at the same ambient temperature that was used for sDz assay. Results from an optimal probe and using it in optimal conditions to measure their fluorescence in the presence of yeast tRNAphe was compared to the results of the sDz strands.

The MB probe was first used with its complement probe to test fluorescence. Figure 5A shows increase in fluorescence as the concentration of the complement increases until it reaches a saturation point. This shows that the short and secondary structure-free oligonucleotide complementary to the loop portion of the MB probe can open up the probe, which results in the concentration-dependent increase in fluorescence.

Figure 5B shows fluorescence of MB probe in the presence of yeast tRNAphe. Yeast tRNAphe was annealed at 95o C with the MB probe prior to the assay and left at room temperature for 5 minutes. Annealing of the probe with tRNA results in slight fluorescence increase, which is triggered only by tRNA at relatively high concentration (Figure 5B, blue curve). The same concentration of the that would result in the maximum fluorescence in case of the MB complement triggered almost no increase in case of the highly structured tRNA. Without prior annealing of the probe with tRNA, no significant increase in fluorescence was observed even at high analyte concentration (Figure 5B, orange curve). Therefore, the MB probe targeting

7 the least stable arm of the tRNA (based on the in silico analysis), failed to unwind the secondary

and tertiary structure of the analyte and detect it.

A A

30 25

20 15

Flouresence 10 5

0 0 20 40 60 80 100 120 Complement Concentration (nM) B 8 Annealed tRNAphe tRNAphe

Flouresence 3

0 0 100 200 300 400 500 600 700 800 900 1000 tRNA Concentration (nM)

Figure 5: Fluorescent response of the MB probe to various analytes.

A. Different concentration of the linear analyte complementary to the loop fragment of the MB probe. B. Different concentrations of yeast tRNAPhe . Fluorescent response of the samples of tRNA annealed with the MB probe prior to the analysis is shown by blue curve. Orange curve shows the data obtained without annealing.

8 Split Deoxyribozyme Probe Design

The sDz probe consists of the two strands Dza and Dzb, each of which has a partial sequence of an RNA-cleaving deoxyribozyme catalytic core (Figure 4). In addition, the terminal fragments of the strands are complementary to the tRNA target and a signal reporter (Mz

Substrate, Table 1), which is a double-labeled oligonucleotide with a fluorophore and a quencher at the opposite sides of a cleavage site. The cleavage occurs at the phosphodiester bond between the two ribonucleotides embedded in the deoxyribonucleotide reporter. In the absence of a tRNA target, the fluorescence of the Mz substrate is low, since the fluorescence of the fluorophore is quenched due to the close proximity of the quencher. When the tRNA target is present, Dza and

Dzb strands bind to the abutting positions of the target, thus re-forming the deoxyribozyme catalytic core. This results in the cleavage of the Mz substrate, which separates the fluorophore from the quencher resulting in the fluorescence increase. The fluorescent signal is amplified due to high turnover of the deoxyribozyme catalytic action.[8]

Figure 6: sDz Probes designs used in the study. The two probes differed by the length of the reporter-binding arms: sDz-long and sDz-short had 11/10-nt and 8/7-nt duplex between the reporter and the reporter-binding arms.

9 To enable efficient binding of the sDz probe to the tRNA target, the target-binding arms of the Dza and Dzb strands were designed long enough to unwind the stable secondary/tertiary structure of tRNA. The 3’-terminal nucleotide of the acceptor stem serve as a “toehold” for one of the strands, which starts unwinding the secondary structure making it easier for the second strand to bind (Figure 4). The sequences of the Dza and Dzb strands, Mz reporter and the target are shown in Table 1. To initially test and optimize the sDz probe, two probes were designed that differed in length of the reporter binding arms (Figure 6). Their performance was studied using a

DNA analyte corresponding to the targeted fragment of the yeast tRNAPhe (shown in Table 1 as

“DNA analyte”). The results shown in Figure 7A show that sDz-long has higher fluorescence in the presence of 4 nM and 40 nM analyte than sDz-Short. However, as shown in Figure 7B, sDz- short has a higher signal-to-background ratio, which was calculated as the ratio of fluorescent signal of sDz probes triggered in the presence of the analyte to the signal without the analyte present. sDz-long had longer reporter binding arms, which could have caused formation of the catalytic core, then binding and cleavage of the reporter which contributed to the higher background.

10 A 40

25 Blank 20 4 nM

Flouresence 15 40 nM

0 sDz-long sDz-short

B 4

3.5

2.5

2 4 nM 40 nM S/B Ratio 1.5

0.5

0 sDz-long sDz-short

Figure 7: Comparative analysis of the two sDz probe designs - sDz-long and sDz-short. A. Fluorescent signal of the probes in the absence or presence of 4 nM or 40 nM DNA analyte. B. Signal-to- background ratio of the two sDz probes signal triggered by 4 nM or 40 nM DNA analyte.

The sDz-short design was then used to study dependence of the signal on yeast tRNAphe concentration. The results are shown in Figure 8. There is increase in fluorescence as the concentration of yeast tRNAphe increases, which means that the signal indeed resulted from unwinding the yeast tRNAphe by the analyte binding arms of sDz-short followed by cleavage of the fluorogenic substrate by the action of the formed sDz catalytic core. Figure 9 shows the linear dynamic range and the limit of yeast tRNAphe detection by sDz-short or MB probe. It can be seen that the signal for the MB probe does not reach the limit of detection (LOD) threshold at tRNA concentration below 100 nM. At the same time, in case of the sDz probe, the LOD was calculated to be 4 nM (Figure 9, blue dots and the best fit line).

30 Fluorescence 20

0 0 10 20 30 40 50 60 70 80 90 Concentration nM

Figure 8: Dependence of sDz-short signal on yeast tRNAPhe concentration.

12 y = 0.115x + 0.9713 R² = 0.96715 10

sDz-Short 6 LOD MB 4

0 0 10 20 30 40 50 60 70 80

Figure 9: Linear dynamic range of yeast tRNAPhe with sDz-short or MB probe. The fluorescence that is correspondent to the limit of detection (LOD) threshold is shown by the dashed line.

13 CONCLUSION

In this project an assay was developed based on an sDz probe to detect yeast tRNAPhe as a proof-of-principle highly structured target. The developed probe was more efficient in detecting tRNA than MB probe, which is one of the state-of-the-art hybridization probes. The

MB probe failed to unwind the secondary and tertiary structure of the tRNA. The results obtained in the project will further demonstrate the advantage of the split probes with signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.

14 REFERENCES

1. Raina, Medha, and Michael Ibba. “tRNAs as Regulators of Biological Processes.” Frontiers in Genetics 5 (2014): 171. PMC. Web. 17 Apr. 2018. 2. Voet, Donald, et al. Fundamentals of Biochemistry: Life at the Molecular Level. 4th ed., Wiley, 2008. 3. Li, J., et al. “Detection of Small, Highly Structured RNAs Using Molecular Beacons.” Analytical Methods, vol. 9, no. 20, 2017, pp. 2971–2976., doi:10.1039/c7ay00341b. 4. Kolpashchikov, Dmitry M. “An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions.” Scientifica, vol. 2012, 2012, pp. 1–17., doi:10.6064/2012/928783. 5. Grimes J., Gerasimova Y.V., Kolpashchikov D.M.(2010) Real-time SNP Analysis in secondary structure-folded nucleic acids. Angewandte Chemie International Edition Engl.,49, 8950-8953. 6. Kolpashchikov, Dmitry M. “A Binary Deoxyribozyme for Nucleic Acid Analysis.” ChemBioChem, vol. 8, no. 17, 2007, pp. 2039–2042., doi:10.1002/cbic.200700384. 7. Gerasimova, Yulia V., et al. “Deoxyribozyme Cascade for Visual Detection of Bacterial RNA.” ChemBioChem, vol. 14, no. 16, 2013, pp. 2087–2090., doi:10.1002/cbic.201300471. 8. Gerasimova, Yulia V., et al. “Expedited Quantification of Mutant Ribosomal RNA by Binary Deoxyribozyme (BiDz) Sensors.” Rna, vol. 21, no. 10, 2015, pp. 1834–1843., doi:10.1261/rna.052613.115. 9. Gerasimova, Yulia V., et al. “Cover Picture: RNA-Cleaving Deoxyribozyme Sensor for Nucleic Acid Analysis: The Limit of Detection (ChemBioChem 6/2010).” ChemBioChem, vol. 11, no. 6, Jan. 2010, pp. 729–729., doi:10.1002/cbic.201090020. 10. Shi, H., and P.b. Moore. “The Crystal Structure of Yeast Phenylalanine tRNA at 1.93 A Resolution.” RNA, Feb. 2000, doi:10.2210/pdb1ehz/pdb.