An unexpected identification of an anti-Annexin A2 aptamer. A paper presentation that I did last month. Enjoy! :) This must have been the most easiest to understand paper that I've ever read :) Very well written piece!

1. Impact factor: 3.73
5-year IF: 4.24

2. Introduction
What is Cell-SELEX?
• SELEX against live pathogenic organisms
• Target? Cell surface biomarkers/ membrane proteins that
are difficult to purify in their native conformation
• Theoretically, many surface proteins = many aptamers
specific to each surface protein can be co-evolved
• Uses mock cells from a similar cell line for negative
selection
• Advantage:
 Decreased speed of aptamer selection
• Disadvantage:
 Increased difficulty in aptamer identification
 Negative selection step is imperfect (aptamers that bind
to both mock cell line as well as target cell)

3. Source: (Sefah et al., 2010) From Nature Protocols

4. Introduction
What is Annexin A2?
• ANXA2 gene
• Annexin family - calcium-dependent
phospholipid-binding protein family
• 2 forms: monomeric (intracellular) and heterotetrametric
(extracellular)
• Function:
 As an autocrine factor which heightens osteoclast formation and
bone resorption
 Sorts endosomes inside the cell
 Involved in anticoagulant reactions outside the cell
 Regulating cell functions (angiogenesis, proliferation, cell migration
and adhesion)
Degradation Cells that resorp
boney tissues
Being a substance secreted by a cell and acting on
surface receptors of the same cell

5. What they have done?
• Target surface protein & cell: ETBR , modified hamster CHO-K1
cell line
• Negative selection: normal/wild-type hamster CHO-K1 cell line
• 15 rounds of Cell-SELEX -> 26 sequences that are predominantly
amplified
• Assessed affinity of these
sequences
• Pretreated cells with Endothelin-1
• Competitive binding assays – to
check where the 3 aptamers bind
to on the cell surface (and is it a
common target?)
• 7 high-affinity BUT bind to
both cell lines!
• Only 3 aptamers unable to
bind.
• Binding of aptamer ACE13
not affected by ACE26 or
ACE4. ACE4 and 26 are
affected by each other =
common target.

6. What is ETBR?
• Not EtBr
• G-protein coupled receptor
• Is overexpressed in low-stage tumors and in cancer
cells (melanoma, breast cancer etc)
• When activated by Endothelin-1, will initiates a
phosphatidylinositol-calcium secondary messenger
system

7. What they have done?
• Evaluation on Human breast cancer cell line (MCF-7)
 ACE13 aptamer – no binding
 ACE4 and ACE26
– 10x higher binding affinity
– Cell pretreatment with Endothelin-
1 does not affect affinity
 Doesn’t recognise human
form of its target
 OR target not present on
MCF-7 cells
 Target is NOT ETBR
 BUT target is highly expressed
on the surface of MCF-7
Result: Failed to select for an aptamer against ETBR. ACE4 was chosen
for further study.
ACE4 VS ACE26 -> ACE4 demonstrated a better affinity on cells

8. ACE4 aptamer
Target identification (Trial and error method)
1. Cells treated with trypsin/ proteinase K
Completely abolished binding. Target possibly a
membrane protein
2. Protein purification via biotinylated ACE4 aptamer
Cells that bind to ACE4 aptamer was purified first.
Then mild lysis produces proteins that are bound to
ACE4

9. Results
• B – Beads only (To eliminate non-
specific binding)
• C – beads with scrambled ACE4 seq
that is already confirmed to be unable
to bind to target.
• ACE4 – Beads with ACE4 aptamers
• Not very clean way of cell protein
purification
• Excise band -> trypsin digestion ->
nano-LC MS/MS
• A protein match was obtained:
Annexin A2

10. Results
Characterization of the aptamer
• Binding affinity: 10.5±4.6nM
• Control = scrambled seq of ACE4 aptamer
• Studies have reported an increased in expression of Annexin A2 in
cancer tissues as compared to normal tissues. - they decided to test it
on healthy cells vs MCF-7 using ACE4 aptamer.
(nM)

11. Results

12. Results
Characterization of the aptamer
• Studies shown that aptamers can usually be internalized. Fluorescent
microscopy -> ACE4 aptamer can be efficiently internalized.
• In vivo targeting of MCF-7 tumors in murine models using ACE4
aptamers
 Fast distribution – signal detected throughout the body within a few minutes of
injection.
 Rapid elimination – within 3 hours, fluorescent signal was detected in bladder.
 Quantify fluorescent signal in tumor (via fluorescence diffuse optical tomography
[fDOT] imaging) after 3 hours of injection = 14 times higher signal for ACE4
aptamers as compared to control and scrambled sequence of ACE4 in tumor.
 But is the difference due to different resistant to nucleases? (scrambled seq less
resistant?) – Tests show that both aptamer and scrambled seq experienced 40%
degradation after 3 hours.

13. Limitations
• Cell-SELEX is difficult to balance and somewhat bias.
 Annexin A2 is not particularly abundant on CHO cells (~30,000
targets per cell) but inside the cells, it functions as an RNA-binding
protein and hence, may be favorable enough to generate aptamers.
 Despite having negative selection step, the final aptamer pool still
binds to both wild-type CHO and recombinant CHO cells
Further work
• Check whether ACE4 aptamer has a neutralizing effect on
Annexin A2.

14. Conclusion
• Possible applications of this ACE4 aptamer:
 In vivo imaging of tumors overexpressing Annexin A2
 As a target-specific drug delivery tool since it can be
internalized into cells that have Annexin A2 and
worked fine as demonstrated in in vivo conditions.
• Undesirable aptamers (Recycled aptamers) isolated
during cell-SELEX deserve to be studied despite being
selected for target that is other than the intended
one.

15. Thank you!