extend dna duplex at both terminals?

[Randy Bin Lin]

I have a DNA duplex with coordinates. But there are two base pairs missing at both terminals. I hope to use 3DNA to add the missing base pairs while they still maintain the alpha helix of B-DNA. Is it possible to do it via 3DNA?

Randy Bin Lin

[xiangjun]

3DNA should be applicable here, at least in principle. As always, please use a concrete example to illustrate exactly what you want to achieve, then I may offer more specific help.

Xiang-Jun
 

[Randy Bin Lin]

Xiang-Jun, sorry for my post being too general. for instance, I have a sequence of DNA duplex, with coordinates available:

CTCGATGCCATC

I want to extend the sequence at both ends by 2, turning it into

CTCTCGATGCCATCAC

hope to preserve the backbone of the common sequence and add the two base pairs following alpha helix (say canonical B conformation).

thank you for your advice.

[xiangjun]

Okay, "analyze" your DNA duplex the normal way. You will get a file named 'ref_frames.dat'. Then run 'frame_mol' to reorient your duplex to the reference frame of one terminal pair (say the right-side C). Build a fiber B-DNA model of your preferred sequence (e.g., CAC), and then reset it to its 1st base-pair reference frame. Since two structures have a common reference frame, you can extract the "extended" part of the fiber model to the original structure. Repeat the same procedure for the other end, you should get what you want.

The above text description may sound a bit abstract, but the basic idea is very simple and generally applicable. If you work through a concrete example step-by-step, and meet any technical problem, please post back.

Xiang-Jun

[Randy Bin Lin]

xiang-jun, here's what I did following your suggestions:

find_pair 1ax6.pdb stdout
frame_mol -1 ref_frames.dat 1ax6.pdb 1ax6_ref_frames.pdb
fiber -b -seq=ctc ctc.pdb
frame_mol -1 ref_frames.dat ctc.pdb ctc_ref_frames.pdb

I used the first base pair at 5' end before extension as reference frame.

after visualizing both output molecules, they don't seem to share the common 5' CG base pair. What did I do wrong here?

Randy Bin Lin

[xiangjun]

For the fiber model (CGC), re-orient it using its 3'-terminal C, i.e., use -3 instead of -1 for 'frame_mol'.

Does this help?

Xiang-Jun

[Randy Bin Lin]

Xiang-jun, yeah, that's my bad. -3 should be the correct one to use.

now it looks close, but the sugar and base don't superimpose very well. since we use the same reference frame, they are supposed to match well for the base pair, right?

[xiangjun]

That's understandable and case-specific. Please attach data files and images to make your point unambiguous.

Xiang-Jun
 

[Randy Bin Lin]

1ax6 is available from this link: http://www.rcsb.org/pdb/files/1AX6.pdb.gz

with that, everything is reproducible with 3DNA commands:

find_pair 1ax6.pdb stdout
frame_mol -1 ref_frames.dat 1ax6.pdb 1ax6_ref_frames.pdb
fiber -b -seq=ctc ctc.pdb
frame_mol -3 ref_frames.dat ctc.pdb ctc_ref_frames.pdb

load the output pdbs file into visualization package of choice, it's in the attachment. green stick is 1ax6, cyan is ctc. I think the superimposition could be closer.

[xiangjun]

I think the superimposition could be closer.

You are absolutely right.

To get what you'd expect, run "find_pair" immediately after your fiber model, as below:

fiber -b -seq=ctc ctc.pdb
find_pair ctc.pdb ctc.bps
frame_mol -3 ref_frames.dat ctc.pdb ctc-frame-3.pdb

The point is: one should use the 'ref_frames.dat' file corresponding to the structure to be reoriented.

Try the process again and report back how it goes.

Xiang-Jun

[Randy Bin Lin]

The point is: one should use the 'ref_frames.dat' file corresponding to the structure to be reoriented.

xiang-jun, it worked like a charm! thanks a lot.

Can you elaborate a little more what it really means in ref_frames.dat for each base pair? My guess is for different ref_frames.dat somehow they share a common coordinate system or something like that. that is why when one 'frame_mol' a duplex based on its own ref_frames.dat, different molecules align very well against each other.

Randy Bin Lin

[xiangjun]

it worked like a charm! thanks a lot.

Glad to hear. Could you please summarize the procedure in detail from a user's perspective and post it at the section "Users' contributions"? That'd benefit the whole 3DNA community, including yourself.

what it really means in ref_frames.dat for each base pair?

As you quoted, I said "The point is: one should use the 'ref_frames.dat' file corresponding to the structure to be reoriented." The fix-named file 'ref_frames.dat' is derived from a specific structure, thus it makes no sense to use it to reorient a bp in another structure.

Xiang-Jun

Indeed, Randy Bin Lin posted the recipe "build dna bulges and extend dna duplex at both terminals via 3dna", as requested -- thanks! [Noted added on Monday, 2012-06-11]

[Randy Bin Lin]

thanks, Xiang-Jun. I will do that.