This document describes the engineering of an aptamer nanomachine called ART (ATP Regulated T1C) that can selectively detect T cells based on cancer metabolite levels. The design hybridizes an ATP-sensing aptamer with a T cell-binding aptamer so that ATP levels regulate cell binding. Testing showed ART1.2 destabilizes its primary helix and binds cells only in the presence of ATP, validating the hypothesis. Future work may increase stability, add protection, and conjugate it to drugs for targeted chemotherapy delivery based on cancer metabolite levels. The student acknowledged help from mentors in designing and testing the ART aptamer system.

1. ENGINEERING A DNA APTAMER NANOMACHINE
PLATFORM FOR T CELL DETECTION BASED ON
CANCER METABOLITES
Jared C. Lenn, Bronx High School of Science
Under mentorship of Dr. Mallikaratchy, CUNY Lehman College

2. INTRODUCTION
• Selective chemotherapy: a drug that only targets cancer cells
• Typically use a cell-surface approach
• Increasing selectivity: metabolites in the microenvironment
• Aptamers are short single stranded DNA or RNA sequences that
have been selected to bind specifically to a target molecule
• Their binding properties arise from folding into secondary structures
A hairpin loop

3. INTRODUCTION
Question: Can I make a two stage aptamer that targets cancer based on both cell-
surface markers and metabolites?

4. DESIGN
• Hypothesis: By hybridizing an ATP
sensing aptamer and a T Cell binding
aptamer, I can use ATP levels to regulate
cell binding.
• T Cell binding is controlled by the secondary
helices
• Closed hairpin → Binds to cell
• Helix destabilization: When ATP binds, the
primary helix breaks and the secondary helix
forms.
Primary helix ATP Binding
Secondary helices

5. DESIGN
• ART: ATP Regulated T1C
• Two key design considerations
• Length of primary helix
• Too long → wouldn’t bind
• Too short → would bind without ATP
• Connection between ATP and T1C
aptamers
ART1.2
(updated
design)
ART1.1
(initial
design)

6. SEQUENCES
Aptamer Sequence
Anti-ATP 5’-CCTGGGGGAGTATTGCGGAGGAAGG-3’
T1C 5’ - GCCGC GGGGTGGGTCTAGTGTGGATG TTTAGGGG
GCGGC - 3’
ART1.1 5’ - 6FAM - AC GCCGC CACCT GGGGGAGT ATTGCGGAGGA
AGGTT - PEG36 - GCCGC GGGGTGGGTCTAGTGTG
ATGTTTAGGGG GCGGC GT - IBFQ - 3’
ART1.2 5’ - 6FAM - CA CCT GGGGGAGTATTGCGG AGGA AGG - PEG36 -
AATGG GC GGGGTG GGTCTAGTGTGGATGTTTAGGGG GC
CCAGG TG - IBFQ - 3’

7. FRET PAIRING

8. RESULTS: FLUORESCENCE SPECTROPHOTOMETRY
ART1.2 Releases Cell Binding Domain After
ATP Addition • Measured fluorescence of ART in a
spectrophotometer
• Fluorescence increase is evidence of FRET
unpairing
• Only ART1.2 has an increase in fluorescence when
ATP is added
• ATP causes helix destabilization, releasing T1C

9. RESULTS: FLOW CYTOMETRY
ART1.2 binds to
cells when ATP
is added

10. DISCUSSION
• We have developed a DNA aptamer based nanomachine that binds to T Leukemia Cells when activated by the
metabolite ATP.
• We designed ART1.2 after ART1.1 failed to respond to ATP binding, and it validated the hypothesis by:
• Destabilizing its primary helix on addition of ATP
• Binding to cells when ATP was added (only after stabilization)
• ART is modular
• Future steps:
• Increase stability at high temperatures
• Exonuclease protection for in vivo use
• Conjugation to a chemotherapeutic moiety such as gemcitabine
• Validation that ART internalizes upon receptor binding

11. ACKNOWLEDGEMENTS
• Thank you to
• Dr. Mallikaratchy, for accepting me into her lab, giving me the resources and encouragement to
complete my project, and advising me on experimental design.
• Dr. Lina Freage, for her indispensable supervision, mentorship in aptamer design, and for teaching
me fluorescence techniques.
• Dr. LaGrassa, for her amazing guidance as I conducted my project and wrote my paper.
• The rest of Mallikaratchy Lab, for all of their help and support!

12. SELECTED REFERENCES
• Huizenga DE, Szostak JW. (1995) A DNA Aptamer that binds adenosine and ATP. Biochemistry, 34(2):656-65.
• Markham N.R. and Zuker M. (2008) UNAFold: Software for Nucleic Acid Folding and Hybridization. Data,
Sequence Analysis, and Evolution, J. Keith, ed., Bioinformatics: Volume 2, Chapter 1, pp 3-31, Humana Press Inc.
• Pellegatti, P., Raffaghello, L., Bianchi, G., Piccardi, F., Pistoia, V., & Di Virgilio, F. (2008). Increased level of
extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase. PloS one, 3(7), e2599.
doi:10.1371/journal.pone.0002599.
• Tang Z, Mallikaratchy P, Yang R, Kim Y, Zhu Z, Wang H, Tan W. (2008) Aptamer Switch Probe Based on
Intramolecular Displacement. Journal of the American Chemical Society, 130(34): 11268-11269.
• You M, Litke JL, Jaffrey SR. (2015) Imaging metabolite dynamics in living cells using a Spinach-based riboswitch.
Proceedings of the National Academy of Sciences of the United States of America. doi:
10.1073/pnas.1504354112
• Zumrut HE, Ara MN, Maio GE, Van NA, Batool S, Mallikaratchy, PR. (2016). Ligand-guided selection of aptamers
against T-cell Receptor-cluster of differentiation 3 (TCR-CD3) expressed on Jurkat.E6 cells. Analytical
biochemistry, 512, 1–7.