Biomod/2012/UCSD/tRiton Nano Architects/Experiment: Difference between revisions

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==Oligo Sequences==
==Oligo Sequences==


The following oligo sequence is the aptamer of choice for lysozyme as it has a binding affinity of kd = 2.8 ± 0.3 nM as determined by fluorescence anisotropy [1]. According to previous research [2], a binding affinity in the order of nanomolar is sufficient for protein targets.  
The following oligo sequence is the aptamer of choice for lysozyme as it has a binding affinity of Kd = 2.8 ± 0.3 nM as determined by fluorescence anisotropy [1]. According to previous research [2], a binding affinity in the order of nanomolar is sufficient for protein targets.  


Aptamer Sequence: 5' – GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGC ATG CGG C – 3'
Aptamer Sequence: 5' – GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGC ATG CGG C – 3'


Secondary Structure of the ssDNA Aptamer with flanking primers
Secondary Structure of the ssDNA Aptamer with flanking primers --


[[Image:ssDNAsecondary.JPG|center|thumb|350px]]
[[Image:ssDNAsecondary.JPG|center|thumb|350px]]
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ΔG Aptamer – Lysozyme system
ΔG Aptamer – Lysozyme system
ΔG: -11.7 kcal/mole
ΔG: -11.7 kcal/mole
The above value is calculated using the following equation, while Kd for chosen aptamer is known:
[[Image:equation1.JPG|center|200px]]


ΔG Aptamer - Complement system
ΔG Aptamer - Complement system
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Variant 2 (''see Figure 3''): -8.76 kcal/mole
Variant 2 (''see Figure 3''): -8.76 kcal/mole


[[Image:VARIANT2.JPG|center|800px]]
[[Image:VARIANT2.JPG|center|thumb|800px]]
<center>''Figure 3: This image is obtained from OligoAnalyzer, www.idtdna.com''</center>
<center>''Figure 3: This image is obtained from OligoAnalyzer, www.idtdna.com''</center>


The difference between variant 1 & variant 2:
The difference between variant 1 & variant 2:
Variant 1 binds strongly in its center, whereas Variant 2 binds strongly on the 5' end where the fluorophore is bound. We will test both variants for both stability and sensitivity.


==Fitting Parts Together==
==Fitting Parts Together==
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a. Add aptamer-complement duplex (1 nM) to phosphate buffered saline solution (pH 7.4).
a. Add aptamer-complement duplex (1 nM) to phosphate buffered saline solution (pH 7.4).


b. Measure fluorescence in the absence of target. Use excitation wavelength of 648 nm and except emission wavelength at 668 nm for Cy5 fluorophore.  
b. Measure fluorescence in the absence of target. Use excitation wavelength of 648 nm and expect emission wavelength at 668 nm for Cy5 fluorophore.  


c. Measure fluorescence in the presence of target. Excitation and emission wavelengths, same as above.
c. Measure fluorescence in the presence of target. Excitation and emission wavelengths, same as above.
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[[Image:DRAWING1.JPG|center|thumb|800px]]
[[Image:DRAWING1.JPG|center|thumb|800px]]


''Figure 4. Expected fluorescence spectra of detector system before and after the addition of target - Lysozyme''
<center>''Figure 4. Expected fluorescence spectra of Lysozyme Detector before and after addition of lysozyme.''</center>
 
'''Figure X. Fluorescence spectra of Lysozyme Detector before and after addition of lysozyme.'''


Similar measurements can be performed for the same detector for the following cases
Similar measurements can be performed for the same detector for the following cases

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        <li><a href='http://openwetware.org/wiki/Biomod/2012/UCSD/tRiton_Nano_Architects#Abstract:_Fluorescent_DNA_Aptamer_for_lysozyme_detection'><span>Abstract</span></a></li>
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Methods

Oligo Sequences

The following oligo sequence is the aptamer of choice for lysozyme as it has a binding affinity of Kd = 2.8 ± 0.3 nM as determined by fluorescence anisotropy [1]. According to previous research [2], a binding affinity in the order of nanomolar is sufficient for protein targets.

Aptamer Sequence: 5' – GCA GCT AAG CAG GCG GCT CAC AAA ACC ATT CGC ATG CGG C – 3'

Secondary Structure of the ssDNA Aptamer with flanking primers --

Figure 1: This figure is adapted from Lammertyn’s “Selection and Characterization of DNA Aptamers for Egg White Lysozyme.” The figure shows the start site and end site of the aptamer from 5’ to 3’ respectively.

Aptamer 3’-end is alkyl-thiol modified and 5’-end of complement is modified with Cy5. Both are purchased from Integrated DNA Technologies, Inc.

Sequences

Aptamer flanked by primers with added Poly-A (n=10) tail:

5’-AGC AGC ACA GAG GTC AGA TG GCAGCTAAGCAGGCGGCTCACAAAACCATTCGCATGCGGC CCT ATG CGT GCT ACC GTG AAA AAA AAA AAA – thiol -3'

Aptamer Complement Variant 1:

5' –(Cy5)— TTTCACAATAGCACAATATAGGC - 3'

Aptamer Complement Variant 2:

5' –(Cy5)— TTTCACAATAGCAAAATATAGGC - 3'

Design Principles

The lower the free energy, the more stable a system is. We design our aptamer-complement systems to be higher in free energy relative to aptamer-lysozyme system. Hence, the lower free energy in addition to high affinity of aptamer to lysozyme should enable the detector to be highly sensitive.

ΔG Aptamer – Lysozyme system

ΔG: -11.7 kcal/mole

The above value is calculated using the following equation, while Kd for chosen aptamer is known:

ΔG Aptamer - Complement system

Variant 1 (see Figure 2): -9 kcal/mole

Figure 2: This image is obtained from OligoAnalyzer, www.idtdna.com

Variant 2 (see Figure 3): -8.76 kcal/mole

Figure 3: This image is obtained from OligoAnalyzer, www.idtdna.com

The difference between variant 1 & variant 2: Variant 1 binds strongly in its center, whereas Variant 2 binds strongly on the 5' end where the fluorophore is bound. We will test both variants for both stability and sensitivity.

Fitting Parts Together

We follow adapted protocols from Mirkin’s “Nano-flares for mRNA Regulation and Detection.”

Gold nanoparticle + Aptamer

From literature [3] and [4], a suitable method is adopted to maximize DNA loading onto gold nanoparticles using salting techniques, sonication and temperature. Essentially, it involves adding thiol-modified aptamers to a solution of gold nanoparticles (13 ± 1 nm), waiting for 2 hours, followed by addition of sodium dodecylsulphate (SDS), phosphate buffer (pH=7.4), and sodium chloride. This is further followed by slow incremental addition of sodium chloride until a final concentration of 0.3M.


Gold nanoparticle + Aptamer + Complement

The aptamers now bound on gold nanoparticles are hybridized to their short complements by adding 106 times more complementary strands than aptamers by concentration. For hybridization to occur, the strands are heated to 70˚C and slowly cooled down to room temperature over four hours and stored at 4 ˚C.

Fluorescence Spectroscopy: To verify function and measure selectivity of aptamer

When the lysozyme detector is in a fully hybridized state, the fluorophore is close to the quencher and will result in a low fluorescence signal. In the presence of the target, the lysozyme protein, the short complementary strand with the fluorophore will be released into solution. This will result in increased fluorescence and indicate the binding of aptamer to target.

Protocol:

a. Add aptamer-complement duplex (1 nM) to phosphate buffered saline solution (pH 7.4).

b. Measure fluorescence in the absence of target. Use excitation wavelength of 648 nm and expect emission wavelength at 668 nm for Cy5 fluorophore.

c. Measure fluorescence in the presence of target. Excitation and emission wavelengths, same as above.

d. Compute fold-change in fluorescence.

Figure 4. Expected fluorescence spectra of Lysozyme Detector before and after addition of lysozyme.

Similar measurements can be performed for the same detector for the following cases

a. Detector + lysozyme

b. Detector + lysozyme + proteins of similar structure

A high fluorescence for lysozyme only, will demonstrate the aptamer’s high specificity.

References

1. Tran, Dinh T., et al. "Selection and characterization of DNA aptamers for egg white lysozyme." Molecules 15.3 (2010): 1127-1140.

2. Nutiu, Razvan, and Yingfu Li. "Structure-switching signaling aptamers." Journal of the American Chemical Society 125.16 (2003): 4771-4778.

3. Prigodich, Andrew E., et al. "Nano-flares for mRNA regulation and detection." ACS nano 3.8 (2009): 2147-2152.

4. Mirkin, Chad A. (Wilmette, IL, US), Lytton-jean, Abigail K. R. (Chicago, IL, US), Hurst, Sarah J. (Evanston, IL, US) 2010 "Maximizing Oligonucleotide Loading on Gold Nanoparticle". United States 20100099858 http://www.freepatentsonline.com/y2010/0099858.html


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