1. Ethylene Glycol Linkers in Bulge Nucleotides
Abstract
Single stranded RNA folds upon itself to create structural motifs,
such as, hairpin loops, internal loops and bulge loops. Additional hydrogen
bonding and ion interactions occur in the loops regions forming unique
structural features that are recognized in RNA-protein interactions. A helical
structure interrupted by bulges is typically lower in stability than a continuous
helix. In this study we designed RNA and DNA constructs containing varying
length of ethylene glycol linkers to measure the stability of constructs that have
no additional hydrogen bonding interactions in the bulge regions. The role of
metal ions in RNA and DNA is complex as the negatively charged phosphate
backbone of RNA and DNA molecules interacts with positively charged metal
ions. In addition, loop regions often bind to metal ions to stabilize particular
structural features. Thus, examining the stability of linker constructs in various
ionic conditions provides further insights into factors that contribute to RNA and
DNA stability. Thermodynamic denaturation experiments were performed in 10
mM MgCl2 and 1M KCl buffer solutions at a pH of 7. DNA internal loops and
bulge loops showed a decrease in stability with an increase in linker size. Most
constructs showed an increase in stability in the presence of magnesium ions
as compared to 1 M KCl. Further comparative analysis involving RNA linkers
will be pursued.
References
Auffinger, p, Grover, N, Westhof, E, 2011. Metal Ion Binding to RNA. Met Ions
Life Sci.9, 1-35.
Becker, F, W, C, Marsac, Y, Hazarika, P, Mozen, J, Goody, R, Niemeyer, C,
2007. Functional Immobilization of small GTPase Rab6A on DNA-Gold Nano Paricles
by using a Site-specifically attached poly ethylene glycol linker and Thiol place
Exchange Reaction. ChembioChem. 8: 32-36.
Strom, S, Shiskova, E, Ham, Y, Grover, N,. ThermodynamicExamination of One-
to Five-Nucleotide Purine Bugle Loops in RNA and DNA Constructs in Potassium and
Magnesium Ions. RNA 17: 710-717
Xia, D, Mathews, D, Turner, D, Thermodynamics of RNA Secondary Structure
Formation. Univ. Roch. 21-45.
Material and Methods
DNA linker constructs were purchased through Integrated DNA
technologies. Three types of linkers, C3, C9 and C18 equivalent to 1, 3, 6
nucleotides respectively were purchased with the DNA construct 5’ -TAACGT/
linkerX/ATGGCA-3’. RNA was purchased from Dharmacon, the RNA strands
contained the same construct and linkers. Ethylene glycol was used for DNA
and RNA linkers, because of its lack of reactivity (Figure 1). All RNA samples
were TLC purified.
Two different physiological buffers were used, 1M KCl and 10 mM
MgCl2, both at a pH of 7. KCl was used because of the abundance of
potassium found within cells and magnesium was used in order to examine
RNA/DNA metal binding sites and the effect to the overall structure. The DNA
linker constructs were also compared to previous research on DNA bulge
loops and internal loops.
Experiments were performed using nine step serial dilution. All
samples were carried out in a quartz cuvette, with 200 µL of sample each.
Each DNA melt was performed at 260 nm, at 1 °C/min. The temperature
ranged from 0.0 °C to 98.0 °C. Data was collected and analyzed to Meltwin, a
thermodynamic calculating program (Figure 4).
Alyssa Ortega, Neena Grover
Conclusion
An increase in linker size resulted in a lower ∆G°. The purine DNA
bulge loop constructs were generally more stable than the linker constructs but
the internal loop linker constructs were more stable than the 2X2 internal loop. A
magnesium effect was observed for DNA bulge loop linkers but was not observed
for internal loop linkers. Further data involving RNA bulge loop and internal loop
needs to be analyzed. DNA and RNA constructs need to be tested in buffers with
varying pH. Chimeric constructs will also be analyzed in future research.
DNA ∆G° Bar Graph
∆G°
-12
-9
-6
-3
0
DNA bulge loop
C3 C9 C18 A1 A3 A5 G1 G3 G5
KCl MgCl2
Results & Discussion
The C3 bulge loop DNA linker construct had the highest stability in
both buffers. The C3 linker was equivalent to one nucleotide so it was expected
for this linker to have highest stability. The high stability for C3 is consistent with
the adenine and guanine single nucleotide constructs. A larger magnesium effect
was observed in the C3 linker construct compared to the single nucleotide
constructs. The magnesium metal ion has a higher effect on the C3 neighboring
nucleotides than the nucleotide participating in the bulge of A1 and G1 (Strom,
2014). Both C9 and C18 are comparable in stability to each other as well as the
bulge loop. A larger difference in stability between C9 and C18 were expected,
due to bulge size. .
The C3 internal loop is slightly more stable than the C9 internal loop.
The asymmetrical nature of the internal loop linkers may aid overall strand
stability. Surprisingly a magnesium effect was not observed for the C3 and C9
internal loop linker. Only a slight magnesium effect was observed for the 2X2
internal loop. Interestingly, both C3 and C9 linker constructs were more stable
than the 2X2 DNA construct.
DNA ∆G° Bar Graph
∆G°
-14
-10.5
-7
-3.5
0
DNA Interal loop
C3 C9 2X2
KCl MgCl2
Acknowledgments
I would like to thank Neena Grover for allowing me to continue the
linker research that was started in biochemistry II. I would also like to thank
Neena for the constant advising and support in this area of research. I would like
to thank Tanya Cervantes for instruction on how to use the autoclave and
florescence program. I would also like to thank Rachel Wonciar for the all the
help in gathering needed supplies for the lab.
Introduction
RNA molecular structures can provide recognition sites to proteins
and can act as enzymes. Because of the expanding research and interest in
RNA function, structural prediction algorithms are needed. In order to create
such algorithms extensive data on RNA thermodynamics must be collected and
analyzed. Internal loops and bulge loops are two RNA folding motifs that require
further thermodynamic analysis. Currently, preliminary structure prediction
models for bulge loops and internal loops exist. However, little energetic data
has been collected on bulge loop and internal loop nucleotides. Therefore,
structure prediction models are very inaccurate. Thermodynamic data on both
DNA and RNA is needed into order to create a complete story of nucleic acid
structural motifs.
In order to specifically study neighboring nucleotides of these two
structural motifs, the nucleotides that create the bulge and internal loops were
replaced with a non-reactive ethylene glycol linkers. In order to test if the
nucleotide sequence is stabilizing the construct, thermodynamic denaturation
techniques were used in order to compare the ∆G° of each DNA and RNA
construct. The DNA linker constructs were also compared to previous research
on DNA bulge loops and internal loops. Previous research has been done on
purine bulge loop DNA and RNA and 2X2 internal loop DNA (Strom, 2014).
Generally, as the size of the bulge and internal loop increase, stability will
decrease. It is expected that the same will hold true for DNA and RNA linker
Figure 1. Linker structures for RNA and DNA constructs.
Figure 2. DNA Linker Bulge Loop data and DNA Purine Bulge Loop Bar Graph
Data (Strom, 2014).
Figure 3. DNA Linker Internal Loop data and DNA Nucleotide Internal Loop Bar
Graph Data.
C9 C18C3
Figure 4. Van’t Hoff analysis equation for MeltWin