Hello Farzad.

This interesting question will give me the opportunity to explain something that is not very well shared, in my opinion.

After analyzing quite some "aptamer-target" structures by X-ray crystallography, a few groups [e.g. Somalogics/L. Gold in Boulder Colorado or Tan's group at the University of Florida] have told that they have NEVER observed any similarity between the "possible" shape of an aptamer based on MFold analysis or any other software supposed to analyse DNA/RNA structures and the observations made using structure analysis.  

You can analyze the structure of many oligos and found that in some particular conditions a given oligo will form a duplex [that can be B, Z, A] or a quadruplex but it does not preclude that once bound to the target protein/target the oligo will form a completely different structure.

Nobody knows either what the effect of PEG could be on the structure of aptamer and though most are PEG labeled, when the modification is not present during the selection of the aptamer sequence, that may be a problem, or not...   

What is pretty clear though is that the sequence of 1411 [GGT GGT GGT GGT TGT GGT GGT GGT GG] or of GRO29A [TTT GGT GGT GGT GGT TGT GGT GGT GGT GG] cannot form a canonical duplex.

Also I do not know of any paper showing a structural analysis of 1411 and demonstrating that it forms a G-quartet.

And for what I know none of the Somalogic apatmers that have been studied as co-crystal with target contain G-quartet.

I am not convince that we have ANY evidence allowing to say that G-rich oligos are more likely to be good aptamers BECAUSE of the propensity to for G-tetrads/quartets.

Hope that helps.

Hello Marc

First of all thank you very much for your kind and thorough response and I cant' help admitting that I completely agree with all you discussed above. Actually besides my concern about the possible adverse effects of PEG conjugation, that the minor of it would be the reduction of molecular dynamics and hence binding affinity, my foremost concern is the likely participation of two separate Apt. strands in the formation of the final 3-D conformation. As you can see through the attached image if there are two strands involved, the final macromolecular conformation would present two 5' phosphate termini, namely a homobifunctionalaptamer upon amination of 5' endings. If this really happens, drug or polymer conjugation to AS1411 would occur at both 5' termini, for some purposes perhaps a privilege, for others a catastrophe! Therefore, I'm really confused and don't know how to obtain conclusive visions making sure that I wont proceed blindly!... 

Have a look at these "prestigous" articles below

One of them (see the figure in it) has hypothesized that AS1411 presents two 5' Phosphate end groups amenable for further functionalization such as amination to get conjugated with drugs or PEG chains, the other assumes that the molecule is just a single strand quartet able to be labled from one side only! Interesting contradiction, isn't it ?!

Dear Farzad,

All I can say is that all the versions of AS1411 used in clinical trials that I am aware of where devoid of 3'- or 5'-phosphate group.  I do not know why one P is mentioned on the figure.  And while a 5'-phosphate can be modified as you described, this is not the method of choice those days.  I'd rather add the amino-group during the synthesis of the oligonucleotide.  That is more efficient.  

About the other question, I need to think a little bit more, and read the papers you provided.

Dear Marc,

I'm exactly talking about the 5' amino modifications made directly during the synthesis process, which enables the resulting oligonucleotide to be conjugated with another moiety (PEG, dyes, other polymers, etc.) via a specific coupling reaction (e.g. EDC/NHS). Actually my dilemma is whether the final conformation of AS1411 has two strands involved or just one? two strands means two 5'-Amines at both termini, and this could be a limitation for some conjugation purposes...

I've read all the papers that you have shared and researched that case a bit.  

It seems clear that there is a consensus that AS1411 works as a dimer binding nucleolin.  Of great interest concerning the structure of those GRO is the NAR paper of the Bates - Miller group in 2003 [attached].

They conclude - and this is not the entire conclusion - A previous study of backbone-modified GRO29A analogs (22) suggested that while G-quartet formation is necessary for activity, it is not sufficient, and binding to the GRO-binding protein is the primary determinant of activity. [...] Active structures included quadruplexes with a variety of conformations, suggesting that biological activity depends on subtle features of the quadruplex, rather than simply recognition of the G-quartet motif or a four-stranded structure. Biological activity requires both a reversible UV melting profile at 295 nm and the ability to compete for the GRO-binding protein (nucleolin) in the

EMSA. The results presented in this report show that there is no simple recognition element associated with activity. This supports our hypothesis that protein binding (and thus biological activity) is mediated by recognition of the specific shape of the quadruplex groove, which will depend on many factors and could theoretically be similar in quadruplexes with very different sequences.

I would add that based on work at Somalogic on quite many different aptamers [most if not all NOT G-rich and NOT tetraplex forming] a HYDROPHOBIC POCKET or GROOVE is critical for the activity of the aptamer.

As for your dilemma, yes is a homo-dimer is formed between two identical strand, you will have 2 aminos in the dimer.  Depending on the orientation of the 2 strands in the dimer, those 2 aminos will be on the "same side" and that could create some steric hindrance if conjugates are large, OR they could be on opposite sides of the dimer, that would not create problems, in my opinion.

I did not find any data, result, discussion that would allow me to be more specific.

I hope this helps.

Best regards

Dear Marc 

Thank you very very much for the precious time devotion and this excellent response.

I totally agree with you and will read the paper you shared 

Best wishes and regards

it forms a  left-handed quadruplex structure. Please see the the following paper. 

Proc Natl Acad Sci U S A. 2015 Mar 3;112(9):2729-33. doi: 10.1073/pnas.1418718112. Epub 2015 Feb 18.

Structure of a left-handed DNA G-quadruplex.

Chung WJ1, Heddi B1, Schmitt E2, Lim KW1, Mechulam Y2, Phan AT3.