Labeled Oligonucleotides for Twisted Structure

According to standard procedures
labeled staple 110 (helix 4) with ddUTP Atto 550
labeled staple 164 (helix 29) with ddUTP Atto 647N
finally, labeled oligonucleotides were dissolved in 50µl TE buffer.

For concentration measurements, absorption coefficients of unlabeled staples 110 and 164 were determined:
ε₂₆₀(staple 110) = 3.5·10⁵ M^-1cm^-1
ε₂₆₀(staple 164) = 3.3·10⁵ M^-1cm^-1

Fluorescence microscope slides of BM12_4/29

• In order to measure the spatial separation of the two fluorophores, we decided to take pictures with red and green illumination (each picture is separately illuminated with its color). From this distance we want to evaluate the twist of the arms
• therefore we took the BM 12_4/29 structure, in which ATTO 550 is incorporated in helix 4 and ATTO 647N in helix 29
• pipet plan for the concentration of DNA binder solution: Image:20111018 BM12 4 29 pipettplan.xlsx

BM12_Spermine_1to7

• In this sample every 7th base pair a DNA binder should have bound
• Prepared microscope slides as indicated in the wiki: Immobilizing structures with biotin-oligos for TIRF
• BM12_4/29 was diluted 1 to 5000 with FOB20

BM12_Spermine_1to21

• In this sample every 21st base pair a intercalator should have bound
• Prepared microscope slides as indicated in the wiki: Immobilizing structures with biotin-oligos for TIRF
• BM12_4/29 was diluted 1 to 5000 with FOB20

Measurement

From each sample, 100 picture pairs (green and red) were made.