DOI: 10.5061/dryad.7d7wm387m
title: Data from: Diverse, distinct, and densely packed DNA nanostar droplets

description: The liquid-liquid phase separation of biomolecules is an important process
 for intracellular organization. Biomolecular sequence combinatorics leads
 to a large variety of proteins and nucleic acids which can interact to
 form a diversity of dense liquid ('condensate') phases. The
 relationship between sequence design and the diversity of the resultant
 phases is therefore of interest. Here, we explore this question using the
 DNA nanostar system, which permits creation of multi-phase condensate
 droplets through sequence engineering of the sticky end bonds that drive
 particle-particle attraction. We explore the theoretical limits of
 nanostar phase diversity, then experimentally demonstrate the ability to
 create 9 distinct, non-adhering nanostar phases that do not share
 components. We further study how thermal processing affects the morphology
 and dynamics of such a highly diverse condensate system. We particularly
 show that a rapid temperature quench leads to the formation of a densely
 packed 2-D layer of droplets that is transiently stabilized by caging
 effects enabled by the phase diversity, leading to glassy dynamics, such
 as slow coarsening and dynamic heterogeneity. Generally, our work provides
 experimental insight into the thermodynamics of phase separation of
 complex mixtures, and demonstrates the rational engineering of complex,
 long-range, multi-phase droplet structures.
description: # Data from: Diverse, distinct, and densely packed DNA nanostar droplets
 Dataset DOI: [10.5061/dryad.7d7wm387m](10.5061/dryad.7d7wm387m) ##
 Description of the data and file structure This document describes the
 data repository associated with the paper Diverse, Distinct, and Densely
 Packed DNA Nanostar Droplets by Aria S. Chaderjian, Sam Wilken, and Omar
 A. Saleh. ### Files and variables This repository consists of a .zip
 archive containing 51 data files, as well as a Mathematica notebook and
 its corresponding PDF. The .zip (
 2025-01-29-DiverseNanostars-DataArchive.zip) contains the following files:
 -This ReadMe file -Three files containing all 4-, 6-, and 8-base DNA
 palindromes with Delta G values calculated from the 2004 SantaLucia
 parameters (see the Mathematica notebook
 GraphStructureOfOrthogonalPalindromes-ForRelease.nb). These files underlie
 Fig. 1D of the main text.      Filenames: *
 AllFourBasePalindromesWithDG.csv * AllSixBasePalindromesWithDG.csv *
 AllEightBasePalindromesWithDG.csv -Three files containing all cliques of
 the maximum size of 3-base orthogonal sticky ends with Delta G within 0.4,
 0.8, and 1.2 kcal/mol of each other. The data is a list of lists of lists,
 namely a list containing all cliques, with each clique containing lists of
 sticky end sequence and corresponding Delta G value. Cliques contain 8, 9,
 and 10 sticky ends for deltaG spread of 0.4, 0.8, and 1.2 kcal/mol
 respectively. These files were generated from the Mathematica notebook
 GraphStructureOfOrthogonalPalindromes-ForRelease.nb.      Filenames: *
 Cliques04 * Cliques08 * Cliques12 -One file
 'DimerConcentrations.csv' containing the concentration of each
 complex that resulted from inputting all sticky end sequences with the
 unpaired T as listed in Fig. 1E of the main text, referred to as strands
 1-9. Each sticky end was input at 12 uM each. This file underlies Fig. 1F
 of the main text. -One file 'All9.ome.tif' which corresponds to
 Fig. 3B-D of the main text. 1 pixel = 0.4887 um. 200 ms exposure time was
 used with 100% LED power. The images are in the order Cy3, Cy5, Atto488.
 No background corrections have been performed (though they were for the
 analysis). -Nine folders 'SingleDark_x.tif' which correspond to
 the single-dark confocal experiments where one NS phase was left unlabeled
 (only visible in brightfield). x in the naming scheme corresponds to the
 NS left unlabeled. Each folder contains 4 files, c0.tif, c1.tif, c2.tif,
 c3.tif, which correspond to Cy3, Atto488, Cy5, and brightfield
 respectively. 1 pixel = 0.22 um. 800 ms exposure time was used for Cy3 and
 Cy5 and 4 s exposure time for Atto488, all with 100% laser power. No
 background corrections have been performed (though they were for the
 analysis). -One video 'PostRamp.ome.tif' which corresponds to
 Fig. 4A of the main text. 1 pixel = 0.733 um. 200 ms exposure time was
 used with 10% LED power. The video begins 40 minutes after the system
 reached 25C with 10 minute intervals. The images are in order (t1, c1),
 (t1, c2), (t1, c3), (t2, c1), (t2, c2), ... where t1 corresponds to the
 first timepoint, c1 corresponds to the Cy3 image, c2 corresponds to the
 Cy5 image, and c3 corresponds to the Atto488 image. No background
 corrections have been performed (though they were for the analysis). -Six
 videos titled 'PostQuench_x.ome.tif' where x corresponds to the
 order in which the video was acquired. All 6 videos are of the same region
 and correspond to Fig. 4B and Fig. 5 of the main text. This was the same
 sample as used in PostRamp.ome.tif, and the same imaging conditions apply
 except that an additional 1.5x magnification setting was turned on, so 1
 pixel = 0.4887 um. No background corrections have been performed. Start
 times (time after reaching 25C) and intervals between frames in the videos
 are as follows, all listed in minutes: ``` x=1    1    5 x=2    260    10
 x=3    1190    10 x=4    2530    10 x=5    3960    20 x=6    7450    10
 ``` ## Code/software A Mathematica notebook titled
 'GraphStructureOfOrthogonalPalindromes-ForRelease.nb' is
 included, saved also as a PDF. This notebook accomplishes the
 graph-theoretic analysis of the set of all 4-, 6-, and 8-base palindromes,
 outputting lists of the largest groups of mutually orthogonal palindromes
 that accomplish a given spread in binding energy, Delta G. More details
 are included in the comments of the notebook.