The Use of Split Light-Up Aptamer F30 Broccoli

to Track Co-transcriptional of RNA Nanoparticles

By
Melina Ines Richardson

In partial fulfillment of a NanoSURE Program Research Project

under the supervision of Dr. Afonin

Summer 2017
UNC Charlotte
 The Use of Split Light-Up Aptamer F30 Broccoli
to Track Co-transcriptional Assemblies of RNA Nanoparticles
Melina Richardson1, Martin Panigaj2, Weina Ke3, Morgan Chandler3, Sameer Sajja3, Allison
Tran3, Kirill A. Afonin3
1 NanoSURE Research Experiences for Undergraduates Program, University of North Carolina in Charlotte; 2
Institute of Biology and Ecology, Pavol Jozef Safarik University in Kosice; 3 Department of Chemistry, University
of North Carolina in Charlotte, Charlotte, NC 28223;

Abstract:
RNA nanoparticles can be used for specific gene silencing which has implications for future
cancer treatments. However, RNA nanoparticles are not widely used in medicine, partly due to
the time and expense of manufacturing them. Using microorganisms (e.g., yeast or bacteria) to
produce RNA nanoparticles would open avenues for manufacturing large quantities of particles
efficiently. Overall, this project seeks to both transcribevia rolling circle transcription
(RCT)and assemble RNA nanoparticles in vivo. As the mean to trace the assemblies, we chose
to use split light up aptamers (Broccoli F30). Initial steps were taken to establish protocols for
transcribing and assembling a split light up aptamer called Broccoli. Further experiments were
conducted in vitro on the RCT method of transcription using various DNA splints and ligases.
Ligation of the DNA templates for RCT was only sporadically successful and typically had
lower yields than typical RNA transcription. Further experiments will be conducted to adjust
protocols accordingly to promote transcription. In future experiments, plasmids containing DNA
templates of the RNA strand a split aptamerBroc and Coliwould be transformed into
competent E. Coli that produces T7 polymerase. DFHBI fluorescent dye would indicate that the
split Broccoli aptamer transcribed and assembled in the bacteria.

Introduction:

RNA nanotechnology is an emerging field with many potential biomedical applications. Various
RNA nanoparticles such as siRNA, harpinRNA, and microRNA all have potential to be used as
nucleic-acid based drugs that interrupt messenger RNA and stop protein translation4. These types
of treatments have enormous implications for the future of cancer and gene therapy research.
RNA nanoparticles also can be used as personalized, fine-tuned drug delivery systems to treat
diseases more efficiently. However, for RNA based drugs to be effective, there needs to be a
method for mass producing RNA nanoparticles. One way to do this would be to use bacteria to
produce the nanoparticles. The RNA nanoparticles would be transcribed and assembled in vivo.
Then they would be extracted from the cells. However, to make this process work, there must be
a method to verify the assembly of the nanoparticles. Currently, in vitro assembly is verified
using gel electrophoresisa method that would not work for particles inside bacteria cells. To
solve this problem, the Afonin lab utilized a light up aptamer from the S.R. Jaffrey lab to act as a
fluorescent beacon to indicate nanoparticle assembly.
Broccoli a light up aptamer1 a short single stranded RNA that was selected to specifically bind
a target molecule, called DFHNI-1T, and to activate its fluorescence2. Broccoli aptamer was
recently selected by the laboratory of S.R. Jaffrey as an RNA mimic of green fluorescent protein
(GFP)1. It is a RNA strand with 48 base pairs that induces fluorescence (thus the term light-up)
of specific dyes DFHBI and DFHBI-1T when binds to them1. DFHBI-1T is brighter than
DFHBI, thus out of the two dyes, DFHBI-1T was chosen. The Broccoli was chosen for this
study because DFHBI-1T has very low levels of background fluorescence1. This means that the
fluorescent dye DFHBI-1T will only fluoresce in the presence of a complete Broccoli RNA
aptamer. Overall, the F30 Broccoli aptamer in combination of DFHBI-1T was optimal as it
serves as a more accurate indicator than other fluorescent indicators such as green fluorescent
protein.

There were two major goals of this summer project. First to verify and then improve the
assembly of a F30 Broccoli strand that was cut in half into two halves (Broc + Coli). These two
strands were transcribed from two separate DNA sequences, then assembled into a full broccoli
strand. The goal was to create an indicator that would allow visual confirmation of assembly of
RNA nanoparticles.

Figure 1. Predicted secondary structure visualized using NUPACK8. The assembled structure of F30 broccoli and
the point at which it is cut to make single stranded Broc and Coli. Figure was created using Nupack analysis.

The second goal was to begin testing on rolling circle transcription (RCT). RCT is a type of
transcription that has been used to transcribe RNA particles, including mRNA and siRNA. RCT
is a method in which a circular single stranded DNA template is used to quickly translate a large
amount of single stranded RNA with a repeating base sequence4. This project wished to apply
the same method of transcription to the F30 broccoli aptamer.
Figure 2. Schematics of the process of rolling circle transcription. ssDNA template is ligated using a ssDNA circle
ligase. The circularized DNA template would then be combined with at T7 promoter, beginning transcription.

The split Broccoli (Broc and Coli) strands would be transcribed using rolling circle transcription.
In RCT a DNA template is circularized using a ssDNA ligase that bridges the gap between the
two ends of the template 5. Then, the template is ligated with T7 RNA polymerase which begins
the transcription process which continues following the circular template 5. The goal is to use
RCT to transcribe RNA nanoparticles in bacteria and have the strands self-assemble due to the
attraction between the RNAs base pairs. The ability to use bacteria to produce RNA
nanoparticles would open avenues for mass production of RNA based drugs and drug delivery
systems. The F30 aptamer would be used to indicate in vivo transcription and assembly through
fluorescence.

Methodology:
Broc + Coli Assembly
The assembly of Broc and Coli aptamers was successful. The process to create the aptamer was
as follows. First the DNA templates of Broc and Coli were ordered, hydrated, and amplified. The
amplification process is as follows. 1 L of each 0.2 M DNA template (for Broc, Coli, and
Broccoli), 1 L of each 100 M FWD primer (for Broc, Coli, and Broccoli), 1 L of each 100
M REV primer (for Broc, Coli, and Broccoli), 75 L total of 2X MyTaq Mix [from Bioline],
and 66 L total of Ultra-Pure H 2 O (17.5-17.8 M) were combined. The solution was amplified
through polymerase chain reactions in a thermocycler. The amplified DNA was purified using
Ultra-Pure Water (17.5-17.8 M), DNA Binding Buffer [from Zymo Research], and DNA Wash
Buffer [from Zymo Research]. The purified DNA was confirmed using an agarose gel.

Next, the purified DNA was transcribed. Ultra-Pure Water (17.5-17.8 M), 5X Transcription
Buffer, rNTPs (25 mM each of A, C, G, and U), DTT (100 mM), and T7 RNA polymerase were
combined and placed in a 37oC water bath. RQ1 RNase-Free DNase [from PROMEGA] was
added after 3.5 hours. The transcribed RNA was then removed from the water bath 30 minutes
later. The RNA was separated using an 8M urea page gel electrophoresis. Afterwards, the RNA
was precipitated out of the gel using crush and soak buffer. Finally, the two separate RNA
strands Broc and Coli were assembled using the following method.

Broc + coli RNA strands were combined with ultrapure water and melted at 95oC. The assembly
was then ice snapped. While on ice, 5x assembly buffer was added. The strands then were placed
on a heat block at 37oC for 30 minutes. After 30 minutes passed, DFHBI-1T was incubated with
the assembly at the same temperature for 10 minutes. The resulting F30-DFHBI-1T complex
fluorescence and were tested in a 19:1 8% native page gel.

Figure 3. Schematic representation of the assembly of Broc + Coli and then the binding of DFHBI-1T dye. Note
that the exact mechanism of binding between F30 and DFHBI-1T is not known, however it is theorized that DFHBI-
1T binds to a circular shape in the RNA strand.

After confirming that the assembled Broc + Coli aptamer fluoresces, various tests were
performed to optimize the assembly protocol. The assembly was tested without an ice snap, to
see if higher levels of fluorescence could be achieved. The same assembly protocol was followed
except instead of an ice snap, one test sample was incubated at 37oC right after the assembly was
melted at 95oC. The second test sample was incubated at 45oC right after the assembly was
melted at 95oC. After 30 minutes, both samples were incubated with DFHBI-1T for ten minutes
at 37oC. The 45oC and 37oC test samples were run with an ice snapped control sample on a 19:1
8% native page gel. The two test samples both visually exhibited higher levels of fluorescence
when images on a chemidoc imaging system.

Ligation of F30 Broccoli ssDNA Template for Rolling Circle Transcription

The ligation of the F30 template is the first step and critical to beginning tests on rolling circle
transcription. Linearized F30 DNA template was ligated using a circle ligase kit. The DNA
template was combined with the circle ligase, ATP, 10x buffer, MnCl2, and sterile water. The
solution was then placed in a heat block at 60oC for 60 minutes. It was then transferred to a heat
block at 80oC for 10 minutes. The resulting ligated DNA template was run through gel
electrophoresis using a Urea Page gel. The gel was then stained using SYBR Green dye then
imaged using a chemidoc imaging system.

Results and Discussion:

The assembly of Broc and Coli was confirmed by

fluorescence (Figure 4). However equally as
important was the fact that the negative controls of
just Broc and just coli RNA did not fluoresce even
when the entire gel was stained in DFHBI-1T. This
means that Broc + Coli assemblies are an accurate
way confirm RNA particle assembly. This will be
particularly important during in vivo tests where
other indicators typically interact and fluoresce
with other parts of the cell. In the future, the Broc
and Coli RNA strands could potentially be added
to larger RNA nanoparticles to indicate successful
assembly.

Figure 4. Native-PAGE results show the confirmation of the Broc + Coli assembly. DFHBI-1T fluoresced with the
control F30 Broccoli and the Assembled broccoli but not the single stranded Broc or single stranded coli.

The protocol for the Broc + Coli assembly was tested at different temperatures to
find ways to increase the fluorescence of the DFHBI-1T F30 complex (figure 5).
Initially, it was discovered that DFHBI-1T had to be incubated with the
assembly for it to fluoresce. Next, the ice snap portion of the protocol was
removed. This increased fluorescence because an ice snap makes the Bro and
Coli RNA strands fold back on themselves instead of with each other. Thus,
removing it increased the concentration of assemblies.

Figure 5. Native-PAGE stained with DFHBI-1T demonstrate spit Broc and Coli assemblies at
different temperatures. Snap cooling the Broc + Coli assembly showed less fluorescence than
when the Broc + Coli assembly was moved directly from 95 oC to incubate at 37oC and 45oC.
However, there was no visible difference between 37oC and 45oC.
 The successful ligation of the linearized
F30 DNA template opens the door to testing
rolling circle transcription. This in turn
would open the doors to beginning in vivo
studies on transcription and assembly of
RNA nanoparticles in cells. However,
further tests are first needed to optimize the
ligation protocol, as well as test other
methods of ligation. Additionally, in vitro
transcription and self-assembly of a split
Broc and Coli aptamer must also be tested.

Figure 6. Denaturing 8M urea PAGE shows the ligated F30 DNA template. The gel was then stained using SYBR
Green dye then imaged using a chemidoc imaging system. The gel confirms that the ligation occurred. The ligated
DNA template (farthest right) ran a much shorter distance than the F30 control. This means that the ligation
occurred.

Conclusions:
When the light up aptamer F30 Broccoli is split, it still assembles and fluoresces. This is
essential because the aptamer can be used as a fluorescent indicator of the assembly of both F30
but also other nanoparticles. The split F30 broccoli aptamer can be added on to the sequence of a
larger RNA nanoparticle. Once the parts of the nanoparticle assembled, the split Broc + Coli
strands would also assemble. With the addition of DFHBI-1T dye the fluorescence of the
broccoli assembly would also indicate the assembly of the larger nanoparticles. In combination
with the successful ligation of F30, the assembly of the split aptamer also opens ways to test
transcription in vivo. Future experiments will first test and optimize RCT. Afterwards,
experiments will be conducted to transcribevia rolling circle transcriptionand assemble the
split aptamer in cells.
 Bibliography
1. Paige JS, Wu KY, Jaffrey SR. RNA mimics of green fluorescent protein. Science. 2011
Jul 29;333(6042):642-6.
2. Lakhin A.V., Tarantul V.Z., and Gening L.V. 2013 Oct-Dec. Aptamers: Problems,
Solutions and Prospects. Acta Naturae. 5(4): 3443.
3. Oxford dictionaries. 2017. online https://en.oxforddictionaries.com/definition/plasmid
4. Kim H, Park Y, and Lee JB. 2015. Self assembled Messenger RNA Nanoparticles for
efficient gene expression. Nature Scientific Reports.
5. Wang X, Li C, Gao X, Wang J, and Liang X. 2014. Preparation of small RNAs using
rolling circle transcription and site specific RNA disconnection. Nature. 3, e215.
6. Halman JR, Satterwhite E, Roark B, Chandler M, Viard M, Ivanina A, Bindewald E,
Kasprzak WK, Panigaj M, Bui MN, Lu JS, Miller J, Khisamutdinov EF, Shapiro BA,
Dobrovolskaia MA, Afonin KA. Functionally-interdependent shape-switching
nanoparticles with controllable properties. Nucleic Acids Research, 45(4): 2210-2220,
2017.
7. Afonin KA, Kireeva M, Grabow WW, Kashlev M, Jaeger L, Shapiro BA. Co-
transcriptional assembly of chemically modified RNA nanoparticles functionalized with
siRNAs. Nano Letters. 12(10): 5192-5195, 2012. PMCID: PMC3498980
8. J. N. Zadeh, C. D. Steenberg, J. S. Bois, B. R. Wolfe, M. B. Pierce, A. R. Khan, R. M.
Dirks, N. A. Pierce. NUPACK: analysis and design of nucleic acid systems. J Comput
Chem, 32:170173, 2011. (pdf)