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Polymer Linker DNA Probe Design, Characterization and Comparison with Molecular Beacons

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Title:
Polymer Linker DNA Probe Design, Characterization and Comparison with Molecular Beacons
Creator:
DIAZ, KAREN MARTINEZ ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Base pair mismatch ( jstor )
Beacons ( jstor )
DNA ( jstor )
Dyes ( jstor )
Energy transfer ( jstor )
Fluorescence ( jstor )
Nucleic acids ( jstor )
Polymers ( jstor )
Signals ( jstor )
Space probes ( jstor )

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University of Florida
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University of Florida
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Copyright Karen Martinez Diaz. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
8/31/2006
Resource Identifier:
436098800 ( OCLC )

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POLYMER LINKER DNA PROBE DE SIGN, CHARACTERIZATION AND COMPARISON WITH MOLECULAR BEACONS By KAREN MARTINEZ DIAZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Karen Martinez Diaz

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This document is dedicated to my family; the strength when I am weak, the light when I am in darkness and my support when I am about to fall.

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iv ACKNOWLEDGMENTS I would like to thanks my advisor, Dr . Weihong Tan, for his advice and support, and the Tan research group for all the innumer able memories. I would like to thank my parents Carmen Diaz and Francisco Martinez an d my brothers, Rubier Martinez, Luis F. Cintron and Armando L. Cintr on, for everything that they have done for me, for their love and support throughout the years. I would like to espe cially thanks my sister and best friend, Melissa Martinez, for her unconditional guidance and encouraging words. I would like to express my gratitude to Chaoyong Yang fo r his friendship, patience, support and helpful discussions, Tim Drake fo r the useful science tips and Marie C. Vicens for her guidance and encouragement. Finally, I would like to thanks Orlando M ilians for being on my side throughout the good and hard times.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 Introduction to Molecular Beacon................................................................................2 Introduction to Linear Probe.........................................................................................3 Probe Characteristics....................................................................................................3 2 LINEAR PROBE DESIGN, HYBRIDIZATION AND IN VITRO CHARACTERIZATION..............................................................................................5 Materials and Methods.................................................................................................5 Probe Concentrations....................................................................................................6 Dye Selection................................................................................................................7 Hybridization Experiments and Conditions..................................................................8 Control Experiment....................................................................................................10 Spacer or Polymer Linker Length Effect....................................................................11 Fluorophores Distance................................................................................................12 Selectivity of the Linear Probe...................................................................................14 Salt Concentration......................................................................................................15 Comparison of the Probe with Linker and no Linker.................................................17 Thermal Stability........................................................................................................18 Cell Measurements for the Linear Probe....................................................................19 Conclusions.................................................................................................................20 3 COMPARISON OF MOLECULAR BEACON AND LINEAR PROBE.................22 Fluorescence Probes with Deoxyribonucleases Reaction...........................................22 Selectivity of the Linear Probe and Molecular Beacon..............................................24 Effects of the Presence of Cellular Extract in the Probes Hybridization....................25 Protein Studies............................................................................................................27 Experiment Conditions...............................................................................................27

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vi Hybridization Rates....................................................................................................30 Conclusion..................................................................................................................31 4 SUMMARY AND FUTURE WORK........................................................................32 Summary.....................................................................................................................32 Advantages of the Probe.............................................................................................32 Future Work................................................................................................................33 Probe Optimization..............................................................................................33 Applications.........................................................................................................33 LIST OF REFERENCES...................................................................................................35 BIOGRAPHICAL SKETCH.............................................................................................37

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vii LIST OF FIGURES Figure page 1-1 Hybridization of molecular beacon upon target addition...........................................4 1-2 Hybridization of the lin ear probe upon the addition of the complementary target DNA (X=PEG)...........................................................................................................4 2-2 Absorbance and emission spectra for FAM and Cy5.................................................8 2-3 Emission spectra of linear probe be fore and after target addition. ...........................9 2-4 Hybridization of the probes with target at 300 nM..................................................10 2-5 Linear probe 16 with different targets......................................................................11 2-6 Bar graphs that show the polymer linker effect.......................................................12 2-7 Ratiometric measurements for ta rgets contain A’s and T’s between the fluorophores.............................................................................................................14 2-8 Selectivity of the probe using a single base mismatch.............................................15 2-9 Hybridization of the linear probe in a buffer with different concentrations of magnesium...............................................................................................................16 2-10 Hybridation of the linear probe with the target using di fferent concentrations of NaCl.........................................................................................................................17 2-11 Calibration curve for the pr obe with linker and no linker........................................18 2-12 Melting temperature for the polym er linker probe and no linker probe...................19 2-13 Linear probe hybridized and unhybrid ized states inside of the cell.........................21 3-1 Molecular beacon and linear probe mixed with complementary DNA sequence and DNase I.....................................................................................................................23 3-2 Comparison of selectivity of LP (red) and MB (blue).............................................25 3-3 Linear probe and molecular beacon and additions of the central spinnal ganglion extract solution followed by the complementary sequence.....................................26

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viii 3-4 Molecular beac on hybridization: ( ) target and ( ) protein and target......................28 3-5 Comparison of the linear probe with molecular beacon hybridization in the presence of lactate dehydrogenase (LDH)...............................................................29 3-6 Hybridization rate comparison of molecular beacon and linear probe....................30

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ix Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master in Science POLYMER LINKER DNA PROBE DESIGN, CHARACTERIZATION AND COMPARISON WITH MOLECULAR BEACONS By Karen Martinez Diaz August 2005 Chair: Weihong Tan Major Department: Chemistry Monitoring gene expression in vivo is of great importan ce to biological studies, medical diagnostics and drug discovery. The us e of molecular beacons has enabled gene expression studies with the advantage of a low background and high selectivity. Unfortunately, the use of molecular beacons for intracellular mRNA monitoring has demonstrated a tendency to give false posit ive signals resulting from degradation by nucleases, and nonspecific binding by nucleic acid binding proteins. Here, we are reporting a novel probe for in vivo mRNA monito ring. The probe consists of two single strands of DNA, in which a PEG (polyethylene glycol) linker is used to tether these two sequences together. On the 3' end of one strand, a fluorescence donor has been attached. To the 5' end of the other strand, another fl uorophore acting as an acceptor is labeled. When the target containing the complement ary sequence to the DNA strands is added, each strand binds to its corresponding target sequence bringing the two fluorophores in close proximity and allowing fluorescence reso nance energy transfer to occur. The

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x fluorescence changes of the two fluorophores have been shown to be proportional to the target concentration. Using PEG to tether the two seque nces has shown a higher signal enhancement than the probe without the linker. A series of experiments was performed to optimize this probe, which includes alte ring the PEG linker leng th changing the PEG number of units and the di stance between the two fluorophores. Hybridization kinetic, sensitivity, and selectivity of linear probe were compared to those of molecular beacons. This probe is simple, highly sensitive, and selective and overcomes some of the limitations of molecular beacons. It holds great potential for use in monitoring mRNA expression in living cells.

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1 CHAPTER 1 INTRODUCTION DNA/mRNA detection techniques play an im portant role in disease especially in gene expression studies. Trad itional techniques such as No rthern Hybridization, Reverse Transcriptase Polymerase Chain Reaction (R T-PCR) and Southern blotting are time consuming. Probes such as molecular beac ons (MBs) and the novel linear probe have been developed because they offer seve ral advantages over traditional DNA/mRNA techniques. These fluorescen ce probes have a tremendous impact in drug discovery, disease diagnosis, gene expres sion studies and biomedical fi elds [1-4]. They offer several advantages; first, the ability to perf orm rapid analysis and fully homogeneous that requires no manipulations aside from mixing of the sample and the test solution [9-13]. Second, an important feature of the fluorescence probe analysis is that the recognition of the target and optical reporting occurs simultaneously, which is an advantage for homogeneous high-throughput assays. Third, they are highly selective due to DNA hybridization to complementary sequences and allows for the detection of a single base mismatch [14, 15]. However, the use of MBs for mRNA/DNA hybrid ization studies has several drawbacks. MBs can be difficult to design due to the hairpin structure which requires some expertise. It has also been repo rted that the hairpin structure is not static and can fluctuate between different c onformations [16]. MBs can suffer from degradation by cytoplasmic nucleases and can be destabilized due to protein interactions [7]. They can also simply open momentarily due to thermodynamic fluctuations giving rise to high false positive signal. Nonspecifi c binding is another f actor to take into

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2 consideration for MB use. For these reasons, scientists from different areas (molelcular engineer, chemist, biochemist, biologist etc. ) are still working on the design, optimization and development of new probes that can ach ieve a higher selectivity and sensitivity. Introduction to Molecular Beacon Molecular beacons were fi rst introduced in 1996 [5]. They are single strand DNA probe molecules that consist of a stem and a loop structure [17-19]. Figure 1.1 shows the molecular beacon structure and hybridization upon target addition. The loop sequence is complementary to the single strand target DNA. The stem portion consists of 5 to 7 base pairs complementary to itself so that prior to binding target DNA sequences the structure is in the closed state [20]. Figure 1-1 Hybridization of Mol ecular beacon upon target addition. A fluorophore is covalently linked to the end of one arm (orange color) and a quencher is attached to the end of the othe r arm (blue color). Molecular beacons do not fluoresce when they are free in solution. Ho wever, when they hybridize to a single nucleic acid strand containing a target sequence they undergo a conformational change that restores the fluorescence. Initially MBs were used for nucleic acid detection but they can also be used for mRNA and molecules with higher complexity. The main advantages Complementary Target + Stem Loop

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3 of these probes are: 1) the signal transduc tion mechanism for high sensitivity, 2) target detection without further separation [18], and 3) the ability to distinguish single base mismatches [18-22]. Introduction to Linear Probe In our laboratory we developed a novel oligonucleotide based probe that can be used for hybridization studies and in vivo mRNA/DNA monitoring. The basic principle of the probe is shown in Figure 1-2. This probe consists of two single strands of DNA, in which a polyethylene glycol (PEG ) linker is used to tether these two sequences together [8]. On the 3' end of one strand, a fluorescen ce donor has been attached (green color in Figure 1.2). To the 5' end of the other strand, another fluorophore acting as an acceptor is attached (orange color in Figure 1.2). In the random unhybridized conformation of the probe, the two fluorophores will be away from each other in the absence of a target. When a target (green strand) containing th e complementary sequences to both probes at adjacent positions is added, each strand will bind to its corresponding target sequence bringing the two fluorophores in close proximity and allowing energy transfer to occur. The energy transfer results in the quenchi ng of the donor fluorophore and a fluorescence enhancement of the acceptor fluorophores. Probe Characteristics The linear probe is based on fluorescence wh ich makes this probe very sensitive. It is capable of detecting the target hybridization without the need to separate hybridized and unhybridized probes. This probe also a llows for the detection of a single base mismatch demonstrating the select ivity of the assay. It also ha s most of the advantages of the molecular beacons with the difference that does not cause a high false positive signal in the presence of nucleases and proteins. The linear probe is easier to synthesize and

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4 simpler transduction mechanism (molecular beacon can have more than one conformation upon hybridization) and the hybridi zation rate is faster . These properties allow us to use the linear probe for in vitro and in vivo measurement. This thesis will mainly focus on in vitro characterization of this probe. Figure 1-2 Hybridization of the linear probe upon the addition of the complementary target DNA (X=PEG). +

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5 CHAPTER 2 LINEAR PROBE DESIGN, HYBRIDIZATION AND IN VITRO CHARACTERIZATION In this chapter a complete descri ption of the design, hybridization and characterization of the linear pr obe is reported. The experiment s will show that the linear probe has the potential for not only mRNA/DNA detection capab ilities but also for future applications in biomedical fields given their specificity and sensitivity. Materials and Methods One of the most important steps in the development of the linear probe is the probe design. The probe was designed using part of the -tubulin sequence from Aplysia genes. This gene is highly stable and highly expressed within Aplysia cells. The sequences used synthesized are in the Table 1-1. All DNA synthesis reagents were from Glen Research. The probes and DNA were synthesized with an ABI3400 DNA/RNA synthesizer. FAM core pore glass (CPG) was used for all FA M labeled probe synthesis, while Cy5 was labeled using Cy5 phosphoramidite; 18 Spacer phosphamidite was used for introduction of PEG. For Cy5 labeled probes, ultramild deprotection phosphoramidite were used and then overnight in cubation in 0.05M K2CO3/methanol. The probe and target solutions resulted from deprotection were precipitated in ethanol. The resulting precipitates were then dissolved in 0.5ml of 0.1 M triethylammonium acetate (pH 7.0) for furt her purification with reverse phase highpressure liquid chromatography. RP-HPLC wa s performed on a ProStar HPLC Station

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6 (Varian, CA) equipped with a fluorescent and a photodiode arra y detector. A C-18 reverse phase column (Alltech, C18, 5µM, 250x4.6mm) was used. Table 1-1 Target sequences and the desi gn of linear probe an d molecular beacon Linear Probe: 5 -F1-CTC ATT TTG CTG ATG ACG-(X)n-TGT CTG GGT ACT CCT CC-F2-3 where X represent PEG; F1=Cy5; F2=FAM 5 -Oregon green-AGA GCG CCT CAG GGC-(X)n-GGA AGG AAG GCT GGA-Cy5-3 Molecular Beacon: 5 -FAM-CGC ACC TCC TCC CTC TTT TTG CTG GGT GCG dabcyl-3 Targets: -tubulin 5 -GCT CAT CAG CAA AAT G AG GGA GGA GTA CCC AGA CAG-3 -actin 5 GCC CTG AGG CGC TCT TCC AGC CTT CCT TCC-3 Targets with T’s between the perfect complementary sequences: 5 -GCT CAT CAG CAA AAT GAG T GG AGG AGT ACC CAG ACA G-3 5 -GCT CAT CAG CAA AAT GAG TTT GGA GGA GTA CCC AGA CAG-3 5 -GCT CAT CAG CAA AAT GAG TTT T GG AGG AGT ACC CAG ACA G-3 5 -GCT CAT CAG CAA AAT GAG TTT TTT GGA GGA GTA CCC AGA CAG-3 5 -GCT CAT CAG CAA AAT GAG TTT TTT TT G GAG GAG TAC CCA GAC AG-3 Targets with A’s between the perfect complementary sequences: 5 -GCT CAT CAG CAA AAT GAG A GG AGG AGT ACC CAG ACA G-3 5 -GCT CAT CAG CAA AAT GAG AA G GAG GAG TAC CCA GAC AG-3 5 -GCT CAT CAG CAA AAT GAG AAA GGA GGA GTA CCC AGA CAG-3 5 -GCT CAT CAG CAA AAT GAG AAA A GG AGG AGT ACC CAG ACA G-3 5 -GCT CAT CAG CAA AAT GAG AAA AAA GGA GGA GTA CCC AGA CAG-3 Single base mismatch targets: 5 -GCT CAT CAG CAA AA A GAG GGA GGA GTA CCC AGA CAG-3 (AA) 5 -GCT CAT CAG CAA AA G GAG GGA GGA GTA CCC AGA CAG-3 (AG) 5 -GCT CAT CAG CAA AA C GAG GGA GGA GTA CCC AGA CAG-3 (AC) ‘Random’ sequence target: 5 -CAG TTA CAT TCT CCC AGT TGA TT-3 The first approach to test the new probe was done in a spectrometer (fluorolog-Tau 3). The buffer used for all the hybridization experiments c onsists of 20 mM Tris, 50mM of NaCl and 5 mM of MgCl2 with a pH of 7.5. The so lution consisted of 200 µ L buffer and sufficient amount of probe and target to make a final solution of 300 nM. Probe Concentrations Each probe was purified using RP-HPLC (Variant, Walnut Creek, CA) and the targets using solid phase extraction. Subse quently the absorbances of the linear probes were taken at 260 nm (CAR Y 100 Bio, UV/VIS spectrometer) to calculate the final

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7 concentration of the probes. The concentra tion of the Linear Probe 8 (LP 8), LP 12, LP 16 and LP 20 were 32 µ M, 72 µ M, 80 µ M and 66 µ M respectively and the complementary target concentration was 66 µ M. The name of the probes (LP 8 etc.) is indicative of the amount of PEG units that each probe contains. The following sequence was used: 5’-Cy5-CTC ATT TTG CTG ATG ACG-(X)16-CTG TCT GGG TAC TCC TCC-FAM-3’; where X represents PEG units and where each polymer unit has a length of 23 Å [23]. The reasons for choosing PE G are: 1) provides mobility to the DNA sequences attached and 2) the lack of inte raction with the nucleot ides bases [23]. Dye Selection Dye selection plays an impor tant role in the design of any fluorescence probe. First, the efficiency of the energy transfer de pends greatly on the selected dye, and that is because each dye covers a spectral region. Second, it is necessary to evaluate the experimental conditions because factors such as pH and temperature can also affect the fluorescence characteristics of the fluorophor es. Fluorescein (FAM) and Cy5 were selected as the linear probe dyes. FAM w ill be the donor molecule and Cy5 will be the acceptor. This dye pair was selected for the following reasons; first, the fluorescence emission of the two dyes is completely sepa rated; second, the excitation of Cy5 caused by absorption of the FAM excitation is negligible; and finally because both dyes have a spectrum overlap which allow the FRET or ener gy transfer to occur. Figure 2-2 shows the absorbance and emission spectra for FAM and Cy5. The absorption and excitation of the dyes are: 488 nm and 520nm for FAM and 643 nm and 665 nm for Cy 5. Oregon green and Cy 5 was also used. Oregon green coverts the same spectra region of FAM but it is more stable into the cell than FAM.

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8 Figure 2-2 Absorbance and emission spectra for FAM and Cy5. Excitation of FAM is 488 nm with an emission of 520 nm and for Cy5 the excitation and emission are 643 nm and 665 nm, respectively. Hybridization Experiments and Conditions The goal of the first experiment was to pr ove that the polymer linker connecting the two sequences together does not interfere with the ability of the linea r probe to hybridize. The first measurement was done for linear probe with a spacer length of 8. First, we added 200 µ L of buffer and 1.875 µ L of probe to have a final concentration of 300 nM and followed by the target addition. Figure 2-3 shows the emission spectra wi th 488 nm excitation before and after target addition. The graph clearly shows a significant fl uorescence decrease in 520 nm and an enhancement the fluorescence at 665nm. The decrease and increase in fluorescence from FAM and Cy5 respectively is due to the target ad dition. FAM is the donor dye and Cy5 is the acceptor. The target hybridization results in the quenching of 200300400500600700800 0.0 0.2 0.4 0.6 0.8 1.0 Abs/FLWavelength (nm) FAM-Abs FAM-EM Cy5-Abs C5-Em

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9 the donor fluorophore and enhancement of the acceptor fluorophore. This data showed that the system has undergoes a fluorescen ce resonance energy transfer (FRET) phenomena. 0 5 10 15 20 25 30 35 40 45 50 490540590640690740 Wavelenght (nm)Intensity (A.U.) Figure 2-3 Emission spectra of linear probe be fore and after target addition. Emission spectra of the probe (-), emission spectra of the pr obe after target addition (-). Buffer consisted of 20 mM Tris, 50mM of NaCl and 5 mM of MgCl2 with a pH of 7.5 Similar pattern was found for the rest of the probes. This experiment was very important because it allowed us to know if the probes have proper interaction with the target; it was the proof of con cept. The hybridization result s were analyzed based on the fluorescence ratio of the two dyes, I665nm/I520nm where I665nm and I520nm represent the intensity of the Cy 5 and FAM respectively af ter target addition. Figure 2-4 shows the preliminary results for the hybr idization of the lin ear probes with target. The graph shows that the linear probe can have as high as 22 times signal enhancement which is comparable with molecular beacons. Notice that the linear probe reaches a maximum signal just a few seconds after the target addition, which dem onstrates the rapid hybridization mechan ism of the probe.

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10 0100200300400500600 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 Ratio 665nm/520nmTime (s) LP 8 LP 12 LP 16 LP 20 Figure 2-4 Hybridization of th e probes with target at 300 nM. Control Experiment The next set of experiment was a negativ e control experiment. The goal was to investigate the selectiv ity of the linear probe. The linea r probe should not give any signal with the control target which has a non complementary sequence. The control was synthesized using part of the Manganese S uperoxide Dismutase (MnSOD) sequence as a target: CAG TTA CAT TCT CCC AGT TGA TT (non-complementary sequence, see Table 1.1). We used linear probe 16 w ith the complementary target in 200 µ L of buffer at a final concentration of 300 nM. Figure 2-5 shows the results of the experiments. The signal enhancement occurred when the probe was incubated with the complementary sequence. No signal responses were obser ved for the non complementary sequence. This result not only showed that the linea r probe hybridizes with the complementary target sequence but also is an indi cation of its selectivity as well.

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11 0.0 0.3 0.6 0.9 1.2 1.5 0200400600 Time (s)Ratio (665nm/520nm) Target control Figure 2-5 Linear probe 16 w ith different targets; (-) Control-non complementary sequence, (-) Complementary DNA sequence. Spacer or Polymer Linker Length Effect In order to obtain a better or higher signa l enhancement for the linear probe a series of experiments were conducted. One of the first parameters that we studied was the effect of the polymer linker in the probe hybr idization. The measurements were taken in the spectrometer, with a final probe concen tration of 300 nM. Figure 2-4 and Figure 2-6 show the differences in signal enhancement for the LP 8, LP 12, LP 16 and LP 20 with different spacer lengths in the probes. Linear probe with 16 units resulted in the highest signal enhancement. For this reason we used LP 16 as a probe for the following experiments to observe the results when we va ried other parameters. Figure 2-6 reflects that the spacer allows the two DNA sequences to remain hybridized and when half of the sequence has hybridized to the target, the other half has no other choice than to hybridize as well. As a result, energy transfer can occur between these tw o dyes once hybridization has occurred.

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12 LP8LP12LP16LP200 5 10 15 20 25 Linear Probe with PEG unitsEnhancement of Intensity(665nm/520nm) Figure 2-6 Bar graphs that s how the polymer linker effect. There are several reasons to suspect that destabilizati on of the probes occur when the linker is too large. The lower enhancement of LP 20 could be the result of multiple target binding. The linker is long enough to allow the two DNA strands to hybridize multiple targets and energy transfer is not likely to occur when this happens. Also, a very large linker length makes the si gnal decrease to a point th at the advantage of using linking DNA fragments would disappear [23]. On the other hand, a very short polymer linker is not capable of hybridizing efficiently and destabilizes the equilibrium in such a way that causes a decrease in the fluorescence. Fluorophores Distance To observe how the fluorophores distance a ffects the probes, in terms of energy transfer the following experiment was performe d. The theory behind this experiment is that energy transfer can be po ssible only if the dyes are at certain distances. When the

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13 gap between the donor and acceptor dye is to la rge, little or no signal enhancement will be observe. The probe used was LP 16 and as targets we used thymine and adenine between the complementary sequences. The targets used for these set of experiments were: a) target (no distance between dyes or perfect complementary sequence): GCT CAT CAG CAA AAT GAG GGA GGA GTA CCC AGA C AG, b) target 1T between the dyes: GCT CAT CAG CAA AAT GAG T GG AGG AGT ACC CAG ACA G, c) target that contains 3T between the dyes: GCT CAT CAG CAA AAT GAG TTT GGA GGA GTA CCC AGA CAG and three more targets with 4T, 6T and 8T. The same experiment was performed with using adenine with the ta rget that has the complementary sequence, 1A, 2A, 3A, 4A, 6A and 8A base s between the dyes. Energy tran sfer is directly related to the fluorophores distance; higher the di stance between the dyes, lower signal enhancement. Figure 2-7 shows the results for this experiment where the number of bases added in the target is plotted vers us the ratio (665nm/ 520nm). The highest fluorescence enhancement resulted from the target with the perfect complementary sequence. Energy transfer resulted to be lower when we increased the distance between the fluorophores. The data obtained for A a nd T presented similar pattern, the signal decreases with an increase base insertation. This experiment also tells us that the fluorophores have to be in dire ct contact to obtain highest signal enhancement. FRET efficiency depends on the distance of the donor and acceptor molecules. It is known that FRET is not effective at Förster distances higher than 200 Å for biomolecules and it is recommended that the fluorophores are within 20 and 60 Å for higher energy transfer. Although in this experiment we did not determ ine the Förster distance it was very clear that the two fluorophores are very close in proximity.

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14 -10123456789 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ratio 665nm/520nmNumber of bases inserted data for A data for T Figure 2-7 Ratiometric measurements for targ ets which contain AÂ’s and TÂ’s between the dyes. Selectivity of the Linear Probe It is very important for the linear probe to be able to distinguish between the target molecules and other molecules in order to have biological applications . In an attempt to observe the specificity of the linear probe, the following experiment was conducted. Targets sequences were prepared with a single base mismatch. The perfect complementary sequence is GCT CAT CAG CAA AA T GAG GGA GGA GTA CCC AGA CAG (AT). The next sequences are called AA, AG and AC, because A has a different complementary se quence; GCT CAT CAG CAA AA A GAG GGA GGA GTA CCC AGA CAG (AA), GCT CAT CAG CAA AA G GAG GGA GGA GTA CCC AGA CAG (AG) and GCT CAT CAG CAA AA C GAG GGA GGA GT A CCC AGA CAG (AC). The discrimination or selectivity of the linear pr obe is shown in Figure 2-7. Indeed, the highest signal was produced by the target with the complementary sequence.

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15 The mismatched sequences showed signal enhancements slightly different from each other. Although the signal did not show significant diffe rences, the linear probe has potential for monitoring a single base mismatch in the target, which would be important for single polymorphism detection. The diffe rences in signal could be due to the different affinities that each base has with a non-complementary base. Their distinctions rely in hydrogen bonding between each mismatch base. 0200400600800100012001400 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Ratio of 665nm/520nmTime (s) AA AC AG AT Figure 2-8 Selectivity of the probe using a single base mismatch. Salt Concentration Nucleic acids can interact reversibly w ith different species such as organic molecules, proteins and metal ions [24] and ions such as Mg2+and Na+ can play an important role in the ligands to nucleic acids because they are the major source of electrostatics contributions[25] . It has been well know for y ears that cations can stabilize the DNA duplex. Ions such as Mg2+ and Na+ can have a significantly impact in the hybridization of oligonucleotides base probes with the target . Studies have shown that Mg2+ coordinates with the phosphate bac kbone in the DNA to make a secondary structure. Due to the importance of thes e ions in the hybridization, the following experiment was done: where the concentration of Mg2+ and Na+ was varied in buffer

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16 solution. Also as part of the characterizati on of the probe, the buffer condition that leads to the highest and fastest fluorescence enhancem ent was investigated. The results of the hybridization of the linear pr obe can is shown in Figure 2-8, where a linear probe concentration was 300nM, target 300 nM, buffe rs that contain 20 mM Tris, 50 mM NaCl and different concentrations of Mg2+. Magnesium concentration appeared to play an important role in the linear pr obe hybridization as expected. In a real time experiment the hybridization kinetics were faster when 5 mM magnesium concentration was used. Consequently, 5 mM of magnesium concentr ation was used for all of the following experiments unless otherwise is specified. 0 5 10 15 0100200300400500 Time (s)Ratio (665nm/520nm) 0 mM Mg 0.5 mM Mg 1 mM Mg 2 mM Mg 3mM Mg 5 mM Mg 7.5 mM Mg 10 mM Mg Figure 2-9 Hybridization of the linear probe in a buffer with different concentrations of magnesium. In the following experiment we compared the effect in the hybridization when I varied sodium concentration. The Figure 29 shows the hybridization of the linear probe with the target does not change significantly when NaCl c oncentration was varied. The hybridization of the probes resu lted to have similar values once the target was added

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17 which indicate that th e hybridization rate is not affected by the Na+ concentration. The hybridization was almost constant at 150 s in all buffers. Although, previous studies suggest that for this anal ysis it is important Na+ concentration in hybridization rates. -1 1 3 5 7 9 11 13 15 0100200300400500 Time (s)Ratio (665nm/520nm) 0 mM NaCl 5 mM NaCl 10 mM NaCl 25 mM NaCl 50 mM NaCl Figure 2-10 Hybridation of th e linear probe with the targ et using 0, 5, 10, 25 and 50 mM of NaCl. Comparison of the Probe with Linker and no Linker The following experiment was conducted as part of the characterization of the probe. It involves a direct comparison of the linear probe with the linker and the two DNA strands that do not contain the polymer linker tethering these two sequences together. The main objective for this experiment was to investigate the role or the function of the polymer linker in the hybridi zation and equilibrium of the probe. The -tubulin sequence was used for both probes. A calibration curve (See Figure 2-10) was done for both probes where different concentrati ons of target were used. As you can see in the plot the probes have similar detecti on capabilities. The main difference between these two plots appeared to be the dynamic range. The probe with the linker has a larger

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18 dynamic range than the probe without the linke r, this can be expl ain because the linker allows the two sequences to st ay together and the equilibriu m is most likely to occurs over 1:1 ratios. The DNA strands decreas ed the fluorescence when the ratio of probe:target was not larger than 1:1. If we analyze this data in terms of equilibrium we can conclude that it is harder for the strands without the linker to bind the same target and result in energy transfer than the probe with the linker which allow both strands to bind the same target. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0100200300400500600 Probe Concentration (nM)Ratio (665nm/520nm) Linker No linker Figure 2-11 Calibration curve for the probe with linker and no linker. Thermal Stability In order to compare both pr obes (the probe with the pol ymer linker and the probe without), another experiment was performed where the thermal stab ility was studied. The probes were hybridized using 1:1 ratio a nd at a final concentration of 300 nM. The fluorescence intensity was monitored at 488nm, 520 nm and 665nm from 15 ºC to 95 ºC. Using a water bath (RTE-111 from Neslab) th e melting temperatures were found for the respective hybridized probes. The melting te mperature for both probes is show in the

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19 Figure 2-11. It is plotted as the derivativ e versus the temperature. The derivative provides a mathematical formulation of the rate of change in this case the change in the fluororescence over the range of temperature. The melting temperature of the linear probe and the probe with no linker were 67 and 30°C respectively. The highest melting temperature of the linear probe in dicates that it is more stab le than the probe without the polymer. This demonstrated the advantage of using a PEG linker to tether two DNA strands together, that is, the PEG dramatically increase loca l concentration of one strand to the other. The local concentration effect facilitates probe hybrid ization and stabilizes the hybridized product. This stab ility allows the linear probe to maintain the equilibrium even at high concentrations in the presence of target. This is im portant in hybridization studies because the hybridization of the linear probe will not likely depend on the temperature below 67ºC. 102030405060708090 -0.030 -0.025 -0.020 -0.015 -0.010 -0.005 0.000 0.005 Tm=67oC Tm=30oCdF/dTTemperature oC Probe with PEG Probe no PEG Figure 2-12 Melting temperature for the polymer linker probe and no linker probe. Cell Measurements for the Linear Probe Fluorescence probes are useful for in vitro and in vivo measurements because of the sensitivity in either of these two environments. Until now we have investigated the behavior of the linear probe in vitro , but it is very important to determine the response of

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20 the linear probe into the cell for practical applications. Cellular measurements for the linear probe (Figure 2-12), showing the hybrid ized (top) and unhybridized (bottom) states inside a cell. On the left, detection at 520 nm and the right is the detection at 665 nm; detection at Oregon green dye nm and Cy5 respectively. Fo r this purpose we used Oregon green instead of FAM because Oregon is more stable in the cell; however bothe dyes have the same absorption and excitati on wavelength. The sequence targeting was actin 5 -Oregon green-AGA GCG CCT CAG GGC-(X)n-GGA AGG AAG GCT GGACy5-3 and the complementary target is -actin 5 GCC CTG AGG CGC TCT TCC AGC CTT CCT TCC-3 . The image demonstrated the f easibility of using the linear probe inside a cell. The two pictures at th e top are the linear pr obe hybridized with its complement. The green color represents th e emission of Oregon green dye and the red color represents the Cy5 signal when the syst em is excited at 488 nm and the target is present in the cell. The two pictures on the bottom show the ce ll excited at 488 nm without target, which means that the probe did not show any hybridization signals because the signal in Cy5 was not appreciab le. This experiment was completed as preliminary data to demonstrate how the linear probe works inside of a cell. As seen in the image, there is a significant enhancement which indicates that the LP has potential for molecular recognition in vivo . Conclusions In this part of our work, the linear prob e was developed and characterized. First, the result showed that when the linear probe hybridizes is capable of having as high as 22 fold signal enhancement which is comparab le with the fluorescence enhancements found in molecular beacons. Second, the highest signal enhancement for the linear probe resulted when the fluorophores were in very close proximity for the energy transfer to

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21 occur. Third, the spacer length ideal for this type of probe is 16 units of the linker PEG. We confirmed this with a series of expe riments that proved th e equilibrium may be affected with low or high PEG units. We al so noticed that the li nker not only makes the probe simple but also adds an extra degree of flexibility to the probe without adversely interfering with the hybridization of the DNA strands. The specificity of the probe was also investigated and we found that the linea r probe is capable of differentiating a single base mismatch in the target sequence. Th is finding plays an important role for the detection of a single nucleotide mutation in gene expression studies. The linear probe demonstrates functionality in “ in vivo” or “ in vitro ” environment using relatively low concentrations (as low as a few nM). All th e parameters were studied to determine the best set of conditions where the maxi mum signal enhancement can be reached. Figure 2-13 Linear probe hybridized and unhybr idized states inside of the cell.

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22 CHAPTER 3 COMPARISON OF MOLECULAR BEACON AND LINEAR PROBE Molecular beacons and linear probes have been developed as tools for molecular recognition. These fluorescence probes have ex cellent detection capabilities. In this chapter a comparison of molecular beacons and linear probe will be discussed in terms of hybridization rates, deoxyribonuc lease (DNase) resistance, e ffect of protein binding and performance in cellular extract. Fluorescence Probes with Deoxyribonucleases Reaction There are many types of that DNases cleave the backbone of the DNA;. Some DNases can cleave a single stranded DNA, othe rs just double strand DNA; some are able to cut anywhere along the chain and others ha ve very specific sequence requirements. Deoxyribonuclease I was used fo r this set of experiments. It cleaves at the phosphodiester linkage adjacent to pyrimidine nucleo tides, yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The molecular beacon used for this experiment targeted the same se quence as the linear probe (FAM-CGC ACC TCC TCC CTC TTT TTG CTG GGT GCG -dabcyl and FAM-CTC ATT TTG CTG ATG AGC (X)n CTG TCT GGG TAC TCC-Cy5 for the linear). The buffer (20mM Tris, 50 mM NaCl, 5mM MgCl2 at a pH of 7.5), target; GCT CAT CAG C AA ATT GAG GGA GGA GTA CCC AGA CAG (300nM) a nd probe concentration ( 300 nM) were the same as well. The results for the experiment performed for both molecular beacon and linear probe are shown in Figure 3-1. Molecular beacon hybridization with the complementary sequence resulted in a ~12 fold enhancemen t and ~16 signal enhancement was found for

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23 DNase cleavage. These results demonstrated that molecular beacons in the presence of DNase give false positive signals. This phenomenon can be explained because DNase cuts the beacon sequence and dest roys the integrity of the st ructure. Consequently, the fluorophore is no longer quenched by the quenche r resulting in the fluorescence of the dye. Signal from MBs caused by DNase was actually higher than the complementary sequence, which is because the quencher and the fluorophores separate completely in the case of DNase digestion. While the fluorophore and quencher remain linked to each other by one fragment of the double strande d DNA in the cDNA case [6]. Conversely, the linear probe with the complementary sequence has a 16 fold enhancement while no false positive signal was observed for the DNase . However, a slight signal decrease was noticeable as a result of the linear probe degr adation. The decrease can also be explained because the DNase cut the probe in pieces cau sing a degradation of the linear probe and therefore a decrease in the fluorescence intensity. 0100200300400500600700 0 2 4 6 8 10 12 14 16 18 Intensity (A.U.)Time (s) MB-cDNA MB-DNase LP-cDNA LP-DNase Figure 3-1 Molecular beacon and linear prob e mixed with complementary DNA sequence and DNase I. Buffer: 20mM Tris, 50 mM NaCl, 5mM MgCl2 at a pH of 7.5.

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24 Selectivity of the Linear Probe and Molecular Beacon The selectivity of the probe is very important for all t ypes of applications. The degree of selectivity for any probe is characterized by the ability to recognize minor differences in target sequences. In this expe riment we investigated the selectivity of the probe when a single nucleotide in the targ et sequence was varied. Three hundred nanomolar concentrations were used for the probes and targets. Figure 3-2 shows the response of molecular beacons and linear probe to different targets. The sequences used for targets were: GCT CAT CAG CAA AA A GAG GGA GGA GT A CCC AGA CAG (AA), GCT CAT CAG CAA AA G GAG GGA GGA GTA CCC AGA CAG (AG) and GCT CAT CAG CAA AA C GAG GGA GGA GTA CCC AGA CAG (AC). Under the experimental conditions molecu lar beacons showed about 8 fold signal enhancement with the perfect complementary sequences (AT, data not shown). In the following test we changed the T which is the perfect complementary sequence of the probes to A. We also tested G and C and th e signal enhancement decreased for all of the base mismatches. The same experiment was run with the linear probe (conditions were not changed). It also showed the highe st signal enhancement with the perfect complementary sequence; AT which resulted in a signal increase of 20 (data not shown). A single base mismatch, AA, AC, and AG ta rgets showed a decreased in the signal intensity. The signal enhancem ent of the linear probe was higher than the molecular beacon. However, in terms of selectivity th e linear probe was slightly lower than the molecular beacon. The advantage of the mol ecular beacon over the linear probe leaves us room for future probe optimization. The degree of selectivity can be dramatically change varying the probe parame ters such as fluorophore pair.

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25 AAACAGAT 0.0 0.2 0.4 0.6 0.8 1.0 Relative Signal enhancement Figure 3-2 Comparison of selectivity of Lin ear Probe (red) and Mo lecular Beacon (blue). Both probes were hybridized under the sa me conditions. The selectivity of the probes to each target was normalized to th e selectivity of perfect match target AT. Effects of the Presence of Cellular Extract in the Probes Hybridization Recent studies on molecular beacons have demonstrated that molecular beacons suffer degradation in the presence of cellula r extract. This experiment was done using sequences from Aplysia Californica . The sequences were synthesized to develop experiments into Aplysia neurons in our laboratory. Briefly, Aplysia is a gastropod mollusc suited for neurobiology mainly because of its large neurons (they are among the largest in the animal kingdom) [28]. Their simplicity with only a few hundred neurons, make this animal suitable to performed studi es that conduct us to a better understanding of molecular events. The central spinal gangl ion extract (A ganglion is cluster of nerve cell bodies on the posterior root of a spinal nerve) was used for this experiment and similar results were found for central nervous system extract (data not show). The buffer use for this analysis contained 20mM of Tris, 50 mM of NaCl and 5mM of MgCl2. At a final concentration of 300 nM the probes were tested in the presence of a solution from

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26 central spinal ganglion extract. The Figur e 3-5 shows the fluores cence intensity of both probes. Molecular beacons have a 4 fold signal enhancement in the presence of the central spinal ganglion solution. On the contra ry, the linear probe di d not have any signal enhancement when the same extract amount wa s added. This experiment tells us that molecular beacons suffer of many conformati onal changes in the pr esence of cellular extract which explains the fluorescence enha ncement that we observed. On the other hand, the intensity of the LP remains almost constant in the presence of the extract. A slight increase in the background of the probe can be explained by the addition of extra material into the solution. 0 1 2 3 4 5 6 7 8 02004006008001000 Time (s)Intensity (A.U.)Central Spinal Ganglion Extract solution Target Figure 3-3 Linear probe ( ) and molecular beacon ( ) that contains the sequence of A plysia . Additions of the central spinna l ganglion extract solution followed by the complementary sequence (linear probe).

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27 Protein Studies The interaction of proteins with nucleic ac id has been very important in molecular biology for years. Protein-nucle ic acid interaction can invol ve changes in conformations of both macromolecules and techniques able to identify these interactions are essential. Techniques such as DNA footprinting, filter binding assays, X-ra y crystallography and fluorescence spectroscopy have been used to study protein-nucleic acid interaction [26] and the information obtained can vary for each methodology. Fluorescence spectroscopy can be an excellent method because of the high sensitivity and low sample volume havving, fast and real time detection capabilit ies. In the following experiment we are taking the advantage of the fl uorescence signal to detect the protein binding event. Recent studies have shown that molecular beacos can interact with proteins causing a significant fluorescent enhancement [7]. Molecular beacons in the closed state are destabilized in the presence of protein in the solution allowing the fluorescence energy transfer to occur [7]. Experiment Conditions The protein used in the experiment was lactate dehydrogenase (LDH). LDH is an enzyme in the glycolytic cycle that catalyzes the reversible interconvertion of lactate and pyruvic acid [26]. The five different isoenzymes of LDH bind ssDNA. LDH was purchased from Sigma (St. Louis, MO). Th e sequence of the molecular beacon used for this experiment was: FAM-CGC ACC TCC TCC CTC TTT TTG CTG GGT GCG dabcyl and F2-CTC ATT TTG CTG ATG AGC (X)n CTG TCT GGG TAC TCC-F1 for the linear probe. The fluorophore attached to the molecular beacon was FAM and dabcyl as a quencher. In the experiment the probe, protein and target were fixed at 300 nM and the buffer contained 20 mM of tr is, 50 mM NaCl and 5mM of MgCl2. Figure 3-7 shows

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28 the result for this experiment. First, in a real time experiment the hybridization of the molecular beacon was done with the perfect complementary target; which showed a twelve fold enhancement. Using the same experimental conditions, the protein was added to the buffer with the MB and allowe d to incubate until the signal reached a plateau; then the complementary target wa s added and the intensity was recorded for both. The effect of protein on the molecu lar beacon hybridization can be observed in Figure 3-6. The addition of the protein cause d destabilization in the molecular beacons and therefore separation of the fluorophore/que ncher which resulted in an increase in intensity. Figure 3-4 Molecular b eacon hybridization: ( ) target and ( ) protein and target. The same experiment was performed usi ng the linear probe an d the comparison of the linear probe with molecular beacon was plotted in Figure 3-7. Addition of LDH to 0 2 4 6 8 10 12 0 500 1000 1500 2000 Time (s) Intensity (A.U.) Protein addition Target addition

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29 molecular beacon solution resulted in 5 fold increase in fluorescence intensity. On the other hand the linear probe had just 1.5 fold enhancement; this result supported our theory that the linear probe doe s not bind with the protein. 0 2 4 6 8 10 12 Probe + LDHProbe + LDH+ TargetProbe + TargetEnhancement Molecular Beacon Linear Probe Figure 3-5 Comparison of the linear probe w ith molecular beacon hybridization in the presence of lactate dehydrogenase (LDH). The high signal enhancement of the mo lecular beacon is the result of the conformational change induced by the presence of LDH. In a real time experiment, the target or complementary sequence was added and the fluorescence enhancement recorded. As a result, the hybridization changes of the probes were obtained in the presence of protein. The figure below shows the enhancement for each probe in the presence of target. For the MB and LP, the enhancement resulted in 2.5 and 7, respectively. The low signal for the MB with the complementary target was the result of the opening of the probe caused by LDH bi nding. The MBs did not show high signal enhancement for the target because some of the beacons were already open because of the presence of the protein in the solution. Conversely, LP has a hybridization of 7 fold which is the same signal value in the absence of the protein. One of the most significant

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30 differences of the LP and MBs is the protei n addition. When one is considering the use of MBs for intracellular detection one must be very careful. These probes can interact with proteins to form either a DNA/protein complex or to simply open the MB causing a high false positive signal and the integrity of the assay can be at risk. Hybridization Rates The aim of this experiment was to unde rstand and compare the kinetics of the linear probe and molecular beacons. Three hundred nanomolar concentrations of probe and target were used for this experiment in buffer. The hybridization of the probe was done in a real time experiment and the data was recorded in the Figure 3-8. When we compare the hybridization rate of these two pr obes, LP has a higher rate of binding than the molecular beacon. It was also evident th at the hybridization of the LP reached the maximum intensity or the plateau in less than 400 seconds of target addition. MBs in the same amount of time were still increasing in their intensity. 0 2 4 6 8 10 12 14 16 18 02004006008001000 Time (s)Intensity (A.U.) MB LP Figure 3-6 Hybridization rate comparis on of molecular beacon and linear probe.

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31 This interesting finding let us think that i ndeed the linear probe is having a faster and more efficient hybridization mechanism than that of the molecular beacons. This is probably because the LP has the flexible linker allowing the tw o strands to stay together in a single open state (not open or closed state like MBs) which is something that does not happen with MBs. The structure of MBs (loop and stem) fluctuates between different conformations in the absence of target and that could be a major concern for the hybridization rate. It takes mo re time for the MBs to reach the equilibrium because of the conformational changes that are going through in the presence of target. Conclusion In this chapter we have compared the linear probe and the molecular beacons in terms of hybridization rates, deoxyribonuclease (DNase) resi stance, effect of protein binding and performance in cellu lar extract. These are important approaches because allow us to determine the conditions in whic h each probe work bests and their advantages and disadvantages in different conditions. For example, if one wants to work in an environment where DNase can be an issue, pr obably the use of molecular beacons is not a good idea because of the degradation of MBs. The same behavior is visible in the presence of proteins. Probably one of the mo st important advantages of the LP over the MBs is the fast hybridization rate which re duces significantly the time of the assay.

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32 CHAPTER 4 SUMMARY AND FUTURE WORK Summary In this work we have characterized the linear probe and demonstrated the possibilities of using this probe for in vitro and in vivo analysis. Here we also investigated the effect of the polymer linke r on the assay under different conditions. Our primary goals for this work were to explore the effects of a probe with a flexible linker for DNA/mRNA detection analysis, find the opt imal working conditions for the linear probe and make a comparison of the linear probe with molecular beacons. Despite the differences that exist between a molecula r beacon and a linear probe (in terms of structure, hybridization mechanis m, sensitivity and selectivity) , we have to recognize that these probes have a great potential for real time analysis of DNA/mRNA and biological applications. Advantages of the Probe The developments for nucleic acid detecti on techniques are in demand and continue to increase for innumerable applications in molecular biology. For example, the linear probe, which is one of the newest devel opments for nucleic acid detection, takes advantage of a fluorescent signal transducti on mechanism that enables a very sensitive analysis at low concentrations and sample volumes. The signal can increase more than 20-fold when is used under the best condi tions. Also LPÂ’s have the advantage of detection without separation which is a ma jor problem for some existing methods. Another advantage of LP is their specificity; th ey have the potential to detect as low as a

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33 single nucleotide mismatch. Overall, molecu lar beacons showed higher sensitivity and selectivity than linear probe. However, the linear probe has the advantage of minimizing false positive signals in the presence of DNase and cellular contents which is important for molecular recognition inside the cell. Future Work Probe Optimization The linear probe synthesized contained FAM and Cy5. FAM has been known to have some pH dependency, low stability and hi gh photobleaching. Although, we have proved that the probe works well with FAM and Cy5 as dye pair; it is imperative for us to try other fluorophores to impr ove the energy transfer a nd enable higher fluorescence intensities for in vitro and in vivo analysis. The used of a dye pair with a bigger spectra overlap which allows a more efficient FR ET has been suggested. Another important improvement for the analysis will be working at picomolar concentrations. It will allow us to perform more sensitive assays and detect targets that are at a minimum concentration into the cell. Applications The linear probe was designed for in vivo DNA/RNA detection. However, their use should not be limited to only this; it can also be very useful as a surfaceimmobilizable biosensor. Ex tensive research has been fo cused on the development of biosensors for proteins, nucleic acids and molecular recognition in general. As a biosensor the linear probe will have the potenti al to detect DNA target s in real time with high sensitivity. RNA detection has been a challenge in molecular biology for the last years. Molecular beacons posses have the possibility of degradatio n by nucleases or opening by

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34 binding proteins when placed in cells [6]. Conversely, the linear probe has been demonstrated to not be dependent on the envi ronment and able to hybridize in almost any condition without adversely affecting the det ection of the target but it will be very important to perform in vivo experiments to generate more data that shows the linear probe for mRNA/DNA detection in cells. Monitoring gene expr ession is not an easy task but the LP promises to be a useful strate gy for gene expression studies in molecular biology.

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35 LIST OF REFERENCES 1. S. Hanash; Nature (London); 2003 , 422, 226. 2. P. F. MacGregor and J. A. Squire; Clin. Chem .; 2002 , 48, 1170. 3. O. Noya, M. E. Patarroyo, F. Guzman, and B. A. De Noya; Curr. Protein Peptide Sci. ; 2003 , 4, 299. 4. B. Solomon; George S. Wise; Immunization Against Alza imerÂ’s disease and Other Neudegenerative Disorders ; Publisher: Springer-Verlag, Berlin, Germany; 2003 . 5. S. Tyagi and F. R. Kramer; Nature Biotechnol .; 1996 , 14, 303. 6. JJ. Li, R. Geyer, W. Tan; Nucleic Acids Research ; 2003 , 28, e52. 7. X. Fang, JJ. Li, W. Tan, Anal. Chem .; 2000 , 72, 3280-3285. 8. K. Martinez, C. Yang, W. Tan; 2005 in preparation. 9. X. Fang, JJ. Li, J. Perlette, K. Wang, W. Tan; Anal. Chem .; 2000 , 72, 747A-753A. 10. K. Wang, JJ. Li, X. Fang, S. Schuster, M. Vicens, S. Kelley, S. Lou, JJ Li; B iomedical Photonics Handbook , Edited by Vo-Dinh Y. Washington: CRC Press; 2003 ; 57.1-57.2. 11. A. Tsourkas, G. Bao, Briefings in Funtional Genomics & Proteomics ; 2003 ; 1 (4), 372-384. 12. J. Perlette, J. Li, X. Fang, S. Schuster, J. Lou, W. Tan; Rev. Anal. Chem .; 2002 , 21, 1-14. 13. S. Tyagi, DP. Bratu, FR Kramer; Nat. Biotechnol .; 1998 , 16, 49-53. 14. H. Wang, J. Li, H. Q. Liu, Q. Mei, Y. Wang, J. Zhu, N. He, Z. Lu; Nucleic Acids Research ; 2002 , 30:e61. 15. S.A.E Marras, F.R. Kramer, S. Tyagi; Generic Analysis : Biomolecular Engineering ; 1999 , 14, 151-156. 16. G. Bonnet, O. Krichevsky, A. Libchaber; Proc. Natl. Acad. Sci .; 1998 , 95, 86028606.

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36 17. D.L. Sokol, X.L. Zhang, P.Z. Lu, A.M. Gewitz, Proc. Natl. Acad. Sci. USA 95; 1998 11538–11543. 18. J. Perlette, W. Tan; Anal. Chem .; 2001 , 73, 5544–5550. 19. T. Matsuo, Biochim. Biophys. Acta . 1379 ; 1998, 178–184. 20. X. Fang, J.W.J. Li, J. Perlette, W. Tan, K. Wang; Anal. Chem. 2000 , 11, 2921– 2922; 21. W. Tan, X. Fang, J. Li, X. Liu; Chem. Eur. ; 2000 , J. 6 , 1107–1111. 22. T. Drake, W. Tan; Applied Spectroscopy ; 2004 , 58, 9, 269A-279A. 23. E. Knoll, T. Heyduk; Analytical Chemistry ; 2004 , 76, 1156-1164. 24. M. Blackburn, M.J. Gait; Oxford University Press; Oxford, UK; 1996 , 331. 25. V.K. Misra, K.A. Sharp, R.A. Friedman, B.J. Honing; J. Mol. Biol . 1994 ; 238, 245263. 26. G.C.Kneale., Ed. DNA-Protein Interactions: Principles and Protocols ; Humana Press: Totowa, NJ, 1994 . 27. R.L. Searcy; Diagnostic Biochemistry, McGraw-Hill: New York, 1969 ; p. 336. 28. Björn Brembs; http://www.brembs.n et/learning/aplysia/aplysia.html; Science Magic ; Freie Universität Belin, July, 2005 .

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37 BIOGRAPHICAL SKETCH Karen Martinez is from Las Piedras, Puerto Rico. She was raised in a small town until she was 17 years old when she decided to go to college. She graduated magna cum laude with a B.S. in chemistry from Universi ty of Puerto Rico, Cayey campus, in 2001. She worked in a company when she realized that she could be more than just a technician. In 2003 she joined the graduate sc hool at the University of Florida. She would like to pursue her Ph.D. in chemistry.