RNA Nanostructures Design Characterization and Applications Methods in Molecular Biology 2709 Kirill A. Afonin (Editor)

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Volume 2709
Methods in Molecular Biology
Series Editor
John M. Walker
School of Life and Medical Sciences, University of Hertfordshire, Hatfield,
Hertfordshire, UK
For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the
research protocols and methodologies in the critically acclaimed
Methods in Molecular Biology series. The series was the first to
introduce the step-by-step protocols approach that has become the
standard in all biomedical protocol publishing. Each protocol is
provided in readily-reproducible step-by-step fashion, opening with an
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complete the experiment, and followed by a detailed procedure that is
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introduced by series editor Dr. John Walker and constitute the key
ingredient in each and every volume of the Methods in Molecular
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protocols from the series are indexed in PubMed.
Editor
Kirill A. Afonin
RNA Nanostructures
Design, Characterization, and Applications
Editor
Kirill A. Afonin
Department of Chemistry, UNC Charlotte, Charlotte, NC, USA
ISSN 1064-3745 e-ISSN 1940-6029

Methods in Molecular Biology
ISBN 978-1-0716-3416-5 e-ISBN 978-1-0716-3417-2
https://doi.org/10.1007/978-1-0716-3417-2
© The Editor(s) (if applicable) and The Author(s), under exclusive

license to Springer Science+Business Media, LLC, part of Springer
Nature 2023
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Preface
The study of RNA improves our understanding of cellular processes and
the origin of various diseases. Rationally designed functional RNA
nanostructures benefit from the inherent biological properties of RNA
and its capacity to assemble from a diverse set of structural and
interacting motifs. RNA nanostructures are attractive for a broad range
of biomedical applications and clinical use because of their controllable
architectures and proficiency in responding readily to biological
environment changes.
This book is an extensive resource of detailed protocols that
renowned experts in computer-assisted design and characterization of
RNA nanostructures, assessment of immunology of nanomaterials,
biosensing, RNA nanotechnology, and drug delivery have organized.
This collection of in-depth chapters addresses this field’s dimensions,
covering RNA nanostructures’ design and characterization, which
outlines their production, storage, and immunorecognition assessment
protocols. This book also highlights a diverse set of biomedical
applications and delivery approaches for therapeutic RNA
nanoparticles.
This collection will interest a broad audience due to its
interdisciplinary nature aiming to address essential topics and
concerns in the growing field of RNA nanotechnology.
Kirill A. Afonin
Charlotte, NC, USA
Contents
Part I Computational Design and In Silico Studies of RNA
Nanostructures
1 Molecular Dynamics Simulations of RNA Motifs to Guide the
Architectural Parameters and Design Principles of RNA
Nanostructures
Valentina Abondano Perdomo and Taejin Kim
2 Computer-Assisted Design and Characterizationof RNA
Nanostructures
Christina J. Bayard and Yaroslava G. Yingling
3 Combining Experimental Restraints and RNA 3D Structure
Prediction in RNA Nanotechnology
Jian Wang, Congzhou M. Sha and Nikolay V. Dokholyan
4 Structural Characterizationof Nucleic Acid Nanoparticles Using
SAXS and SAXS-Driven MD
James Byrnes, Kriti Chopra, Lewis A. Rolband, Leyla Danai,
Shirish Chodankar, Lin Yang and Kirill A. Afonin
Part II Production and Storage of Functional RNA Nanostructures
5 Metalated Nucleic Acid Nanostructures
Douglas Zhang and Thomas Hermann
6 Bioconjugation of Functionalized Oligodeoxynucleotides with
Fluorescence Reporters for Nanoparticle Assembly
Erwin Doe, Hannah L. Hayth and Emil F. Khisamutdinov
7 Light-Assisted Drying for the Thermal Stabilization of Nucleic
Acid Nanoparticles and Other Biologics
Susan R. Trammell
8 Preparation of Nucleic Acid Aptamer Functionalized Silver/Gold
Nanoparticle Conjugates Using Thiol-Substituted Oligonucleotides
Joshua D. Quarles, Allen T. Livingston, Ashley E. Wood and
Timea Gerczei Fernandez
Part III Characterization of RNA Nanostructures
9 Thermodynamic Characterizationof Nucleic Acid Nanoparticles
Hybridization by UV Melting
Megan Teter, Ross Brumett, Abigail Coffman and
Emil F. Khisamutdinov
10 Structural Characterizationof DNA-Templated Silver
Nanoclusters by Energy Dispersive Spectroscopy
Damian Beasock and Kirill A. Afonin
11 Small Volume Microrheology to Evaluate Viscoelastic
Properties of Nucleic Acid-Based Supra-Assemblies
Akhilesh Kumar Gupta, Joel Petersen, Elizabeth Skelly,
Kirill A. Afonin and Alexey V. Krasnoslobodtsev
12 Characterizationof RNA Nanoparticles and Their Dynamic
Properties Using Atomic Force Microscopy
Alexander J. Lushnikov, Yelixza I. Avila, Kirill A. Afonin and
Alexey V. Krasnoslobodtsev
Part IV Intracellular Delivery and Immunorecognition of RNA
Nanostructures
13 Synthesis of Mesoporous Silica Nanoparticles for the Delivery
of Nucleic Acid Nanostructures
Tamanna Binte Huq and Juan L. Vivero-Escoto
14 Assessment of Intracellular Compartmentalization of RNA
Nanostructures
Yasmine Radwan, Kirill A. Afonin and M. Brittany Johnson
15 Discriminating Immunorecognition Pathways Activated by RNA
Nanostructures
Leyla Danai, M. Brittany Johnson and Kirill A. Afonin
16 Detection of Nanoparticles’ Ability to Stimulate Toll-Like
Receptors Using HEK-Blue Reporter Cell Lines
Edward Cedrone and Marina A. Dobrovolskaia
17 Characterizationof PAMAM Dendrimers for the Delivery of
Nucleic Acid Nanoparticles
Yelixza I. Avila, Laura Rebolledo, Melanie Andrade-Muñ oz and
Kirill A. Afonin
Part V RNA and DNA Nanostructures Designed for Biomedical
Applications
18 Reverse Transfection of Functional RNA Rings into Cancer Cells
Followed by in Vitro Irradiation
Renata de Freitas Saito, Isabella Nevoni Ferreira,
Maria Cristina Rangel and Roger Chammas
19 Aptamer Conjugated RNA/DNA Hybrid Nanostructures
Designed for Efficient Regulation of Blood Coagulation
Lewis A. Rolband, Weina Ke and Kirill A. Afonin
20 Detection of Multiplex NASBA RNA Products Using Colorimetric
Split G Quadruplex Probes
Maria S. Rubel, Liubov A. Shkodenko, Daria A. Gorbenko,
Valeria V. Solyanikova, Yulia I. Maltzeva, Aleksandr A. Rubel,
Elena I. Koshel and Dmitry M. Kolpashchikov
21 Synthesis of DNA-Templated Silver Nanoclusters and the
Characterizationof Their Optical Properties and Biological
Activity
Elizabeth Skelly, Lewis A. Rolband, Damian Beasock and
Kirill A. Afonin
22 Dynamic Nanostructures for Conditional Activation and
Deactivation of Biological Pathways
Yasmine Radwan, Laura P. Rebolledo, Martin Panigaj and
Kirill A. Afonin
23 Anticoagulant Activity of Nucleic Acid Nanoparticles (NANPs)
Assessed by Thrombin Generation Dynamics on a Fully Automated
System
Renata de Freitas Saito, Bá rbara Gomes Barion,
Tania Rubia Flores da Rocha, Alex Rolband, Kirill A. Afonin and
Roger Chammas
Index
Contributors
Kirill A. Afonin
Nanoscale Science Program, Department of Chemistry, University of
North Carolina at Charlotte, Charlotte, NC, USA
Melanie Andrade-Muñoz
Yelixza I. Avila
Bárbara Gomes Barion

Laborató rio de Hemostasia do Hospital das Clínicas da Faculdade de
Medicina da Universidade de Sã o Paulo, Sã o Paulo, SP, Brazil
Christina J. Bayard
Department of Materials Science and Engineering, North Carolina State
University, Raleigh, NC, USA
Damian Beasock
University of North Carolina at Charlotte, Charlotte, NC, USA
Ross Brumett
Chemistry Department, Ball State University, Muncie, IN, USA
James Byrnes
Brookhaven National Laboratory, Upton, NY, USA
Edward Cedrone
Nanotechnology Characterization Laboratory, Cancer Research
Technology Program, Frederick National Laboratory for Cancer
Research, Frederick, MD, USA
Roger Chammas
Comprehensive Center for Precision Oncology, Centro de Investigaçã o
Translacional em Oncologia (LIM24), Departamento de Radiologia e
Oncologia, Faculdade de Medicina da Universidade de Sã o Paulo and
Instituto do Câ ncer do Estado de Sã o Paulo, Sã o Paulo, SP, Brazil
Shirish Chodankar
Kriti Chopra
Abigail Coffman
Leyla Danai
Marina A. Dobrovolskaia
Nanotechnology Characterization Laboratory, Cancer Research
Technology Program, Frederick National Laboratory for Cancer
Research, Frederick, MD, USA
Erwin Doe
Department of Chemistry, Ball State University, Muncie, IN, USA
Nikolay V. Dokholyan
Department of Pharmacology, Penn State College of Medicine, Hershey,
PA, USA
Department of Engineering Science and Mechanics, Penn State
University, State College, PA, USA
Department of Biochemistry and Molecular Biology, Penn State College
of Medicine, Hershey, PA, USA
Department of Chemistry, Penn State University, State College, PA, USA
Department of Biomedical Engineering, Penn State University, State
College, PA, USA
Timea Gerczei Fernandez

Department of Chemistry, Physics, Geology and the Environment, Sims
Building, Winthrop University, Rock Hill, SC, USA
Isabella Nevoni Ferreira

Renata de Freitas Saito

Daria A. Gorbenko
Laboratory of DNA-Nanosensor Diagnostics, ITMO University, Saint
Petersburg, Russia
Akhilesh Kumar Gupta

Department of Physics, University of Nebraska Omaha, Omaha, NE, USA
Hannah L. Hayth
Thomas Hermann
Department of Chemistry and Biochemistry, University of California,
San Diego, CA, USA
Center for Drug Discovery Innovation, University of California, San
Diego, CA, USA
Program in Materials Science and Engineering, University of California,
San Diego, CA, USA
Tamanna Binte Huq

Department of Chemistry, Nanoscale Science Program, University of
North Carolina, Charlotte, NC, USA
M. Brittany Johnson
Department of Biological Sciences, University of North Carolina at
Charlotte, Charlotte, NC, USA
Weina Ke
Emil F. Khisamutdinov
Taejin Kim
Physical Sciences Department, West Virginia University Institute of
Technology, Beckley, WV, USA
Dmitry M. Kolpashchikov
Department of Chemistry, University of Central Florida, Orlando, FL,
USA
Burnett School of Biomedical Sciences, University of Central Florida,
Orlando, FL, USA
Center for Forensic Science, University of Central Florida, Orlando, FL,
USA
Elena I. Koshel
Petersburg, Russia
Alexey V. Krasnoslobodtsev
Allen T. Livingston
Alexander J. Lushnikov
Nanoimaging Core Facility at the University of Nebraska Medical Center,
Omaha, NE, USA
Yulia I. Maltzeva
Petersburg, Russia
Martin Panigaj
Department of Chemistry, University of North Carolina, Charlotte, NC,
USA
Valentina Abondano Perdomo

Physical Sciences Department, West Virginia University Institute of
Joel Petersen
Joshua D. Quarles
Yasmine Radwan
Department of Chemistry, University of North Carolina at Charlotte,
Charlotte, NC, USA
Maria Cristina Rangel

Laura Rebolledo
Laura P. Rebolledo
Department of Chemistry, University of North Carolina, Charlotte, NC,
USA
Department of Biological Sciences, University of North Carolina,
Charlotte, NC, USA
Tania Rubia Flores da Rocha
Laborató rio de Hemostasia do Hospital das Clínicas da Faculdade de
Medicina da Universidade de Sã o Paulo, Sã o Paulo, SP, Brazil
Alex Rolband
University of North Carolina, Charlotte, NC, USA
Lewis A. Rolband
Aleksandr A. Rubel
Laboratory of Amyloid Biology, Saint-Petersburg State University, Saint
Petersburg, Russia
Maria S. Rubel
Petersburg, Russia
Congzhou M. Sha
PA, USA
Department of Engineering Science and Mechanics, Penn State
University, State College, PA, USA
Liubov A. Shkodenko
Petersburg, Russia
Elizabeth Skelly
University of North Carolina, Charlotte, NC, USA
Valeria V. Solyanikova
Petersburg, Russia
Megan Teter
Susan R. Trammell
Department of Physics and Optical Science, University of North Carolina
at Charlotte, Charlotte, NC, USA
Juan L. Vivero-Escoto
Department of Chemistry, Nanoscale Science Program, University of
North Carolina, Charlotte, NC, USA
Jian Wang
PA, USA
Ashley E. Wood
Lin Yang
Yaroslava G. Yingling
Department of Materials Science and Engineering, North Carolina State
University, Raleigh, NC, USA
Douglas Zhang
Department of Chemistry and Biochemistry, University of California,
San Diego, CA, USA
Part I
Computational Design and In Silico
Studies of RNA Nanostructures
© The Author(s), under exclusive license to Springer Science+Business Media, LLC,
part of Springer Nature 2023
K. A. Afonin (ed.), RNA Nanostructures, Methods in Molecular Biology 2709
https://doi.org/10.1007/978-1-0716-3417-2_1
1. Molecular Dynamics Simulations of

RNA Motifs to Guide the Architectural
Parameters and Design Principles of
RNA Nanostructures
Valentina Abondano Perdomo1 and Taejin Kim1
(1) Physical Sciences Department, West Virginia University Institute of
Taejin Kim
Email: taejin.kim@mail.wvu.edu
Abstract
Molecular dynamics (MD) simulations can be used to investigate the
stability and conformational characteristics of RNA nanostructures.
However, MD simulations of an RNA nanostructure is computationally
expensive due to the size of nanostructure and the number of atoms.
Alternatively, MD simulations of RNA motifs can be used to estimate the
conformational stability of constructed RNA nanostructure due to their
small sizes. In this chapter, we introduce the preparation and MD
simulations of two RNA kissing loop (KL) motifs, a linear KL complex
and a bent KL complex, and an RNA nanoring. The initial solvated
system and topology files of each system will be prepared by two major
force fields, AMBER and CHARMM force fields. MD simulations will be
performed by NAMD simulation package, which can accept both force
fields. In addition, we will introduce the use of the AMBER cpptraj
program and visual molecular dynamics (VMD) for data analysis. We
will also discuss how MD simulations of two KL motifs can be used to
estimate the conformation and stability of RNA nanoring as well as to
explain the vibrational characteristics of RNA nanoring.
Key words Molecular dynamics simulations – RNA motif – RNA

nanostructure – AMBER – CHARMM – NAMD
1 Introduction
Since RNA tectoRNA [1–5] was built in the early 2000s, RNA
nanotechnology has rapidly developed computationally and
experimentally. The various shapes of RNA nanostructures have been
built using numerous RNA motifs. The examples of RNA motifs are kink-
turn motif, junction motif, pseudoknot, kissing loop hairpins, GNRA
loop-receptor, triple helical scaffold, and G- quadruplex. Examples of
RNA nanostructure shapes, which are constructed by RNA motifs, are
triangle [6–8], square [9], hexagonal ring [10–12], cubes [13–16], and
polyhedron [17, 18]. The various shapes of RNA nanostructures also
have been investigated to develop diverse biomedical applications, such
as drug delivery [19–21], gene therapy [14, 22], and molecular beacon
[23–31].
Several computational methods have been developed to design RNA
nanostructure. Examples of computational design tools are RNA2D3D
[32], NanoTiler [33], Assemble2 [34], INFO-RNA [35], and NUPACK
[36]. Once an initial RNA nanostructure is computationally generated,
molecular dynamics (MD) simulations can be used to fix steric crashes
in the nanostructure as well as to investigate the stability and
conformational changes of nanostructure. Most commonly used MD
simulation packages are Assisted Model Building with Energy
Refinement (AMBER, https://ambermd.org/) [37], Nanoscale
Molecular Dynamics (NAMD, https://www.ks.uiuc.edu/) [38],
Chemistry at Harvard Macromolecular Mechanics (CHARMM, https://
www.charmm.org/) [39], and GROningen MAchine for Chemical
Simulations (GROMACS, https://www.gromacs.org/) [40]. Each MD
simulation package uses specific atomic interaction parameters and
topological information, which are called force fields (FF). AMBER,
CHARMM, and GROMACS have their own FF, and the NAMD platform
can accept these FF to run MD simulations. In this chapter, we will
introduce the use of AMBER and CHARMM FF to run MD simulations
using NAMD. The atomic interactions in AMBER MD simulation are
described by the below equation.
The first three terms describe short-range interactions in two

(bonded), three (angle), and four (dihedral) atoms. The last two terms
calculate long-range van der Waals and electrostatic interactions,
respectively. Atomic potentials in CHARMM are described by additional
potential components. Besides short- and long-range interactions,
additional terms such as improper interactions, two-body Urey-Bradley
term, and CMAP term are included as below.
The above atomic potentials are applied to describe the molecular

behavior of all types of biomolecules, such as DNA, RNA, proteins, and
lipids. However, the unique behavior of each biomolecule is
characterized by specific FF. Thus, when users prepare MD simulations,
it is necessary to choose proper FF for biomolecules as well as the most
recent FF.
In this chapter, we introduce how to prepare initial structure and
topology files of DNA and RNA motifs as well as an RNA nanostructure.
To provide a wide range of selecting FF for user’s research, we
introduce how to prepare initial systems and run MD simulations using
both AMBER and CHARMM FFs. In Subheading 3.1, it will be explained
how to apply AMBER FF to prepare the initial solvated system and
topology file for a linear RNA motif. In Subheading 3.2, it will be
explained how CHARMM FF can be applied to a bent RNA motif and an
RNA nanostructure. The MD simulations of these systems will be
performed by NAMD package. For post MD simulation data analysis, we
will introduce the use of AMBER cpptraj program [41] and visual
molecular dynamics (VMD) [42]. The cpptraj is a very convenient and
powerful text command for data analysis, while VMD provides basic
data analysis tools based on a graphic interface. The detailed
explanations of cpptraj and VMD are described in Subheading 3.3.
In Note 1, we will introduce how MD simulation can be failed due to
the small periodic boundary box, especially when the biomolecule
experiences a large conformational change during MD simulations. In
Notes 2 and 3, we will also briefly describe how MD simulations of RNA
motifs can be used to estimate the conformational stability of RNA
nanostructure as well as to understand its vibrational mode.
2 Materials
In this section, we briefly introduce AMBER, CHARMM, and NAMD. We
will also briefly introduce VMD and Discovery Studio Visualizer (DSV),
which can be used for the visualization of molecular structure and MD
trajectory, molecular editing, and basic data analysis.
2.1 AMBER
AMBER (https://ambermd.org/) is a comprehensive biomolecular MD
simulation package. The most recent AMBER FFs can be found in the
AMBER website (https://ambermd.org/AmberModels.php). However,
it is strongly advised that users check the details of FF in the most
recent AMBER reference manual (https://ambermd.org/Manuals.php)
before preparing MD simulation. AMBER MD simulation can be
performed by one of two commands, sander (simulated annealing with
NMR-derived energy restraints) or pmemd (particle mesh Ewald
molecular dynamics). The pmemd command shows better performance
in terms of computation speed. In addition, the performance of MD
simulation can be further improved when the graphics processing unit
(GPU) is used by pmemd.CUDA command. More detailed information
about sander, pmemd, and pmemd.CUDA can be found in the most
recent AMBER reference manual. Besides MD simulations, AMBER
package also provides a comprehensive data analysis program, which is
called cpptraj. The cpptraj can perform various data analysis. Examples
of fundamental data analysis by cpptraj are molecular geometry
measurements, such as distance, angle, dihedral angle, root-mean-
square deviation (rmsd) calculations, and hydrogen bond (HB)
interactions. The functionality and data file treatments by cpptraj
command can be found in the most recent AMBER reference manual.
2.2 CHARMM and NAMD

Most recent CHARMM FF and detailed information can be found from
the MacKerell lab website (http://mackerell.umaryland.edu/charmm_
ff.shtml). NAMD is an MD simulation platform, which can run MD
simulation of a biomolecular system prepared by AMBER, CHARMM, or
GROMACS FF. In the Method section, it will be explained how to run MD
simulations using NAMD when the initial structure and topology file are
prepared by AMBER FF (Subheading 3.1) or CHARMM FF (Subheading
3.2). Most recent NAMD package can be downloaded from NAMD
website (https://www.ks.uiuc.edu/Development/Download/
download.cgi?PackageName=NAMD). This website provides two
different versions of NAMD. One version is for using the central
processing unit (CPU) system and the other version is for NVIDIA
CUDA-based GPU system. The basic data analysis can be performed by
either AMBER cpptraj or VMD.
2.3 VMD (Visual Molecular Dynamics)

VMD provides 3D visualization of biomolecules. It can visualize static
structure or MD trajectory of AMBER, CHARMM, and GROMACS. VMD
also provides basic data analysis tools, such as atomic distance, angle
along three atoms, dihedral angle along four atoms, RMSD, HB
interactions, and salt bridge. VMD also can be used to generate initial
solvated structure and topology files using CHARMM FF. More details of
generating initial structure and topology are explained in Subheading
3.2. The most recent VMD version can be downloaded from VMD
website (https://www.ks.uiuc.edu/Development/Download/
download.cgi?PackageName=VMD).
2.4 Discovery Studio Visualizer (DSV)

One of the unique functionalities of DSV is that users can build or edit
nucleic acids, proteins, or small molecules. DSV also provides cleaning
atomic clashes, which is a similar process of energy minimization of MD
simulation. Besides these functionalities, DSV provides atomic
geometry measurements, HB interactions, and visualization of
CHARMM MD trajectory. Recent DSV can be downloaded from https://
discover.3ds.com/discovery-studio-visualizer-download.
3 Methods
MD simulation of a large RNA nanostructure, which is built by self-
assembly of multiple RNA motifs, is computationally very expensive
because of the size of the system and number of atoms. Alternatively,
MD simulation of an RNA motif can be used to predict the
conformational stability of the RNA nanostructure with a small cost of
computational power. In this section, we introduce how to prepare the
initial solvated system and topology files of RNA motifs and an RNA
nanostructure using AMBER (Subheading 3.1) and CHARMM
(Subheading 3.2) FFs. We also introduce NAMD protocols for MD
simulations. The basic data analysis using AMBER cpptraj commands
and VMD will be explained at Subheading 3.3.
3.1 MD Simulation of a Linear RNA Motif

To demonstrate how to determine the stability of a linear RNA motif by
MD simulations, we use the dimerization initiation site (DIS) of HIV-1
RNA (PDB ID: 2FCX.pdb), which forms a kissing loop (KL) interaction.
We modify the sequence of HIV-1 DIS to prepare two mutated kissing
loop complexes. The bases of the KL-1 complex form full Watson-Crick
base pairings, while the KL-2 complex forms non-Watson-Crick base
pairings. The sequence of two DIS and initial crystal structure are
plotted in Fig. 1. The bases of the DIS are replaced by DSV. In this
section, the preparation of the solvated KL complexes and topology files
using AMBER FF as well as NAMD protocols for MD simulations will be
discussed.
Fig. 1 (a) The initial structure of the linear RNA motif (PDB ID: 2FCX.pdb). (b) The
DIS sequence and the final MD structure of the KL-1 complex. (c) The DIS sequence
and the final MD structure of the KL-2 complex
3.1.1 Preparation of Initial Structure and Topology

Using AMBER FF
Two RNA KL complexes may have steric crashes due to base
modifications by DSV. The steric crashes can be repaired by energy
minimization. To minimize the structure, it is necessary to generate the
initial structure and topology files which MD simulation can accept. The
initial structure and corresponding topology files using AMBER FF can
be generated by the AMBER tleap command as below.
tleap
source leaprc.RNA.OL3
mol = loadpdb KL.pdb
saveamberparm mol KL.prmtop KL.inpcrd
Besides tleap, xleap, which is a window interface, can be also used.
The command source loads RNA.OL3 force field for RNA. The command
loadpdb loads the input pdb file, KL.pdb. The saveamberparm command
saves the result coordinates to inpcrd format and topological
information to prmtop format.
The energy minimization can be performed by the below AMBER
command:
sander -i min.in -o min.out -p KL.prmtop -c KL.inpcrd -r KL-EMIN.rst -x
KL-EMIN.x
Sander is one of main MD simulation programs in AMBER. Min.in is
an input file which details are listed below.
&cntrl
imin=1, ntx=1, drms=0.01,
irest=0, ntxo=1, cut=15.0,
ntpr=100, ntwx=100, ntwe=100,
nsnb=20,
maxcyc=30000, ncyc=1000,
igb=1,saltcon=1.0,
ntt=0,offset=0.13,
gbsa=1, ntb=0,
&end
The detailed explanations of each command in the input file can be
found in the most recent AMBER manual. KL-EMIN.rst is the minimized
structure, and KL-EMIN.x is the trajectory file. Trajectory file can be
visualized by VMD. In VMD, select File → New Molecule → Browse →
select KL.prmtop → select AMBER 7 Parm → Load → select KL-EMIN.x →
select AMBER Coordinates → Load.
The minimized structure, KL-EMIN.rst, can be converted to pdb
format by the AMBER command, ambpdb, as below.
ambpdb -p KL.prmtop -c KL-EMIN.rst > KL-EMIN.pdb
The converted pdb file (KL-EMIN.pdb) is used for solvation and
ionization for explicit MD simulations. The solvated system can be
generated by tleap as below.
tleap,
source leaprc.RNA.OL3
source leaprc.water.tip3p
loadAmberParams frcmod.ions234lm_1264_tip3p
molEMIN = loadpdb KL-EMIN.pdb
solvateBox molEMIN TIP3PBOX 20.0 0.8
addions molEMIN K 0
addionsRAND molEMIN K 22 CL 22
addionsRAND molEMIN MG 1 CL 2
saveamberparm molEMIN KL-Solvated.prmtop KL-Solvated.inpcrd
savepdb molEMIN KL-Solvated.pdb
quit
The command source loads RNA.OL3 force field and water.tip3p
force field for KL complex and water, respectively. The command
loadAmberParams loads ion force field, frcmod.ions234lm_1264_tip3p.
The command solvateBox places water molecules around the KL
complex with 0.8 Å gap and the thickness of 20 Å water layer from the
KL complex. The significance of water box size is discussed in Note
1. The command addions adds K+ ions to neutralize the KL complex.
Extra ions can be added to increase salt concentrations. In this example,
extra K+, Cl−, and Mg2+ are added to set 50 mM of KCl and 2 mM of
MgCl2. The number of extra ions can be calculated using formula below
based on the volume of the water box.
AMBER tleap command generates a log file to record detailed

information of tleap. The volume of the water box can be also found in
the log file after the solvateBox command is executed. The solvated
structure and topology files are saved by saveamberparm command.
The resultant structure is also saved by pdb format using savepdb
command.
3.1.2 Initial Minimization and Equilibration

The first step of MD simulation is the energy minimization of the entire
system (KL complex, water, and ions). After minimization, equilibration
is applied to water and ions for a given temperature, while the KL
complex is fixed. VMD can be used to generate a pdb file, which
identifies fixed atoms. To generate the pdb file for fixed atoms, in VMD,
select File → New Molecule → Browse → select KL-Solvated.prmtop →
select AMBER7 Parm → Browse → select KL-Solvated.inpcrd → select
AMBER7 Restart → Load. Then, type below commands to VMD console
window.
vmd > set all [atomselect top all]
vmd > set Fixatom [atomselect top "resname A C G U G5 C3"]
vmd > $all set beta 0
vmd > $Fixatom set beta 1
vmd > $all writepdb KL-Fixed.pdb
vmd > set center [measure center $all]
The above commands assign index 1 to the beta column of atoms
that belong to the residue names, A, C, G, U, G5, and C3. Atoms with
index 1 in the beta column are recognized as fixed atoms during NAMD
simulations. The result is saved to pdb format (KL-Fixed.pdb). The
command set center detects the center of the water box in (x, y, z)
coordinates. This coordinate will be used for the periodic boundary
conditions in the NAMD config file (line 48 in the EminEQ-I.conf in Table
1).
Table 1 The list of input files for minimization, equilibration, and MD simulations.
These files can be used to run NAMD simulations with AMBER FF. If the system is
prepared by CHARMM FF, use the config files listed in Table 2
Initial minimization and equilibration are performed using NAMD
by the below command with the config file, EminEQ-I.conf.
namd2 +p number of cores EminEQ-I.conf > EminEQ-I.ene
The above command is for using a CPU system to run MD
simulation. Depending on the available computational resource, users
can specify the number of CPU cores to run MD simulation after the flag
+p. EminEQ-I.conf is an input config file (see Table 1 for the details).
Note that the commands for AMBER in EminEQ-I.conf are specified in
bold text. If a system is prepared by CHARMM FF, # AMBER Input
section (line 6–9) must be removed. In addition, switching (line 17)
must be on and switchdist (line 18), and pairlistdist (line 19) in the #
Force-Field Parameters must be activated by removing # symbol. There
are a few more sections that should be noted. The fixed atoms in the KL
complex are activated (line 32). The initial periodic box is defined (line
49), while the pressure control is deactivated (line 65). Under this
condition, NVT simulation will be performed. To control temperature,
Langevin dynamics (line 83–87) is employed. EminEQ-I.ene is an output
file, which contains the molecular mechanics information, such as
pressure, temperature, energy terms in bonding, angular, dihedral, van
der Waals, and electrostatic interactions. The initial minimization and
equilibration generate the following output files.
KL-EminEQ-I.coor, KL-EminEQ-I.vel, KL-EminEQ-I.xst, KL-EminEQ-
I.restart.xsc.
KL-EminEQ-I.restart.coor, KL-EminEQ-I.restart.vel, KL-EminEQ-
I.restart.xsc, and KL-EminEQ-I.dcd.
The first group of files are the final coordinates, velocities, and
periodic boundary information (xst and xsc files), respectively. The
second group of files are restart files of coordinates, velocities, periodic
boundary information, and NAMD trajectory files (KL-EminEQ-I.dcd.),
respectively. Restart files are periodically updated by NAMD command,
restartfreq. More detailed information about config file and output file
can be found in the recent NAMD manual (http://www.ks.uiuc.edu/
Research/namd/).
3.1.3 Constrained MD Simulation

Once water and ion molecules are equilibrated at the target
temperature, it is necessary to apply another minimization and then
equilibrate the entire system while holding the KL complex with a weak
constraint. To generate a constraint file, load initial solvated system and
topology files (KL-Solvated.prmtop and KL-Solvated.inpcrd) to VMD as
described in Subheading 3.1.2. To assign constraints to the KL complex,
type the below commands to the VMD console.
vmd > set all [atomselect top all]
vmd > set ConstraintAtom [atomselect top "resname A C G U G5 C3"]
vmd > $all set beta 0
vmd > $ConstraintAtom set beta 0.5
vmd > $all writepdb KL-Constraint.pdb
Here, the beta column is set to zero, while atoms which belong to
the residue names, A, C, G, U, G5, and C3, are set to 0.5 kcal/(mol∙Å ). If a
biomolecular behavior is sensitive to equilibration, it may need to apply
strong initial constraint to the molecule and gradually reduce
constraints during multiple constrained MD simulations. The
constrained MD simulation can be performed by the below command.
namd2 +p number of cores EminEQ-II.conf > EminEQ-II.ene
The details of EminEQ-II.conf are listed in Table 1. Note that
coordinate, velocity, and periodic boundary files from the previous
simulation are specified as input files (line 2–6) in the EminEQ-II.conf.
In addition, the fixed KL complex is turned off (line 37), while the
constrained KL complex is activated (line 45). For the constrained MD
simulation, NVP is changed to NPT by turning off the initial boundary
box in line 54 and activating Langevin pressure control in line 71–81.
3.1.4 Final Equilibration and Product MD

Simulations
Once constrained MD simulation is completed, release constraints by
turning it off (line 45) in the EQ-III.conf file (see Table 1) to run the final
equilibration MD. This time, all components in the system, KL complex,
ions, and water, will be equilibrated at the target temperature. The
NAMD command for the final equilibration is below.
namd2 +p number of cores EQ-III.conf > EQ-III.ene
The details of the EQ-III.conf file are listed in Table 1. After the final
equilibration MD simulation is completed, the product MD simulation
can be performed using the below NAMD command.
namd2 +p number of cores MD.conf > MD.ene
The details of the MD.conf file are listed in Table 1. The MD.conf will
run to produce a 6 ns-long MD trajectory (3,000,000 step ×
2 fs = 6,000,000 fs = 6 ns). Longer MD trajectory can be produced by
running consecutive MD simulations using coordinate, velocity, and
periodic boundary files of the previous MD run. The results of MD
simulations are discussed in Note 2.
3.2 MD Simulation of a Bent RNA Motif and RNA

Nanoring
In this section, we introduce MD simulations of a bent RNA and an RNA
nanoring (Fig. 2). The bent RNA is composed by kissing loop
interactions between two dumbbell-shaped RNA motifs [11]. The RNA
nanostructure has a ring shape, which is constructed by six dumbbell-
shaped RNA motifs. Each dumbbell-shaped RNA has slightly different
sequences in the stem. The initial solvated structure and topology will
be prepared with CHARMM FF using VMD. Load the initial pdb
structure (bentRNA.pdb) to VMD by the below procedure.
File → New Molecule → Browse → select bentRNA.pdb → select file
type as pdb → Load.
Fig. 2 (a) The initial structure of the bent RNA, which is built by two dumbbell-
shaped RNA motifs. The red dotted arrow indicates the length of the dumbbell-
shaped RNA motif, and the blue dotted lines indicate the bending angle of the bent
RNA. (b) The final MD structure of the bent RNA. (c) Top and side views of the initial
structure of RNA nanoring. (d) Top and side views of the final structure of RNA
nanoring
Generate structure and topology files with CHARMM FF as below.
Extensions → Modeling → Automatic PSF Builder → define Output
basename as bentRNA. Then, follow below steps in the Automatic PSF
Builder window.
Step 1: Load default CHARMM FF and structure files. If newer
CHARMM FF and structure files are available, delete default FF and
structure files, and click the Add button to load newer versions of
FF. Click the Load input files button to complete Step 1.
Step 2: Select the type of molecules. In this case, select Nucleic Acid.
Then, click the Guess and split chains using current selections
button.
Step 3: Detailed information of the loaded structure will show up
in the window box in Step 3. Select the strand, and click the Edit
chain button to confirm the First Atom and the Last Atom index are
correct as well as the 5′ (5TER) and the 3′ (3TER) ends being
properly defined. If everything is okay, click the Create Chain
button. Now, Automatic PSF Builder will generate bentRNA.pdb
(structure) and bentRNA.psf (topology) files.
To run explicit MD simulation, it is necessary to solvate the system
with ions. To solvate the KL complex, in the VMD menu, select
Extensions → Add Solvation Box → make sure that the previously
generated structure and topology files (bentRNA.pdb and bentRNA.psf)
are loaded under Input → Define the name of solvated system in the
Output (e.g., bentRNA-sol) → Check Use Molecule Dimensions option.
Enter the water box padding size in the Box Padding → Click the Solvate
button. As discussed in Note 1, the size of water box padding should be
carefully defined to avoid the violation of periodic boundary
conditions. Now, the solvated structure and topology will be generated
by pdb (bentRNA-sol.pdb) and psf (bentRNA-sol.psf) file formats,
respectively.
To ionize the solvated system, load the solvated system (bentRNA-
sol.pdb and bentRNA-sol.psf) to VMD. In the VMD menu, select
Extensions → Modeling → Add Ions → Define the name of the ionized
system in the Output prefix (e.g., bentRNA-solion). VMD provides six
default salt types, NaCl, KCl, CsCl, MgCl2, CaCl2, and ZnCl2. Choose the
proper salt type for your simulation. In the section of Ion placement
mode, users can select Only neutralize system with select salt type,
Neutralize and set salt concentration to used defined salt concentration
in mol/L, or User-defined number of ions. Once the preferred salt
conditions are determined, click the Autoionize button. The ionized
structure and topology will be generated as pdb and psf file formats,
respectively.
When the solvated system with ionization is prepared, the explicit
MD simulations can be performed with the same MD protocols
described in Subheadings 3.1.2, 3.1.3 and 3.1.4. However, the AMBER
part in NAMD config files must be removed. In Table 2, NAMD input
files, EminEQ-I.conf, EminEQ-II.conf, EQ-III.conf, and MD.conf, are listed.
Notice that # AMBER Input is removed. In addition, commands for
CHARMM FF are included in bold text. For example, in EminEQ-I.conf
file, paraTypeCharmm is on (line 10); CHARMM parameter,
par_all36_na.prm, is defined (line 11), switching is on (line 18); and
switchdist (line 19) and pairlistdist (line 20) are defined. The results of
MD simulation of the bent RNA will be discussed in Note 3.
Table 2 The list of input files for minimization, equilibration, and MD simulations.
These files for NAMD run with CHARMM FF. If the system is prepared by AMBER FF,
use the config files listed in Table 1
The same procedure can be applied to generate the initial solvated
system and corresponding topology files of RNA nanoring. The MD
simulation of RNA nanoring can be performed by the same protocols in
Subheadings 3.1.2, 3.1.3 and 3.1.4. with NAMD config files in Table 2. A
brief discussion about MD simulation of RNA nanostructure will be
discussed in Note 3.
3.3 Data Analysis Using cpptraj and VMD

3.3.1 cpptraj
Cpptraj is one of the AMBER sub-programs that provides a wide range
of data analysis tools. The basic command to run cpptraj is
cpptraj -p topology file <cpptraj-input file> cpptraj-output file
Cpptraj-input file can contain multiple data analysis action
commands. Below is an example of a cpptraj-input file.
trajin MD-trajectory.dcd
center : 9-16,34-41
strip :TIP3
strip :POT
strip :CLA
distance dist1 :2@N3 :20@N3 out DataOutPut.dat
angle ang1 :4@N1 :5@N1 :6@N1 out DataOutPut.dat
dihedral dihe1 :4@N1 :5@N1 :6@N1 :7@N1 out DataOutPut.dat
hbond :9-16,34-41 avgout DataOutPut-hbond.dat
rmsd first out DataOutPut.dat :1-88
trajout trajectory-NoWAT.dcd
Trajin command loads MD trajectory. Trajin can load MD
trajectories of major MD simulation packages, such as AMBER, NAMD,
and GROMACS. The command center brings the RNA residues 9–16 and
34–41 to the center of the water box. The command strip removes the
specified residues from the system. In this example, ions (POT and CLA)
and TIP3 water molecules are removed from the MD trajectory, which
can significantly reduce the size of MD trajectory file. The command
distance measures the distance between N3 atoms at the residue 2 and
20. The command angle measures the angle of N1 atoms at the residue
4, 5, and 6. The command dihedral measures the dihedral angle along
N1 atoms at the residue 4, 5, 6, and 7. Instead of specific atoms, the
group of atoms, residues, or the group of residues can be used to
measure their distance, angle, or dihedral angles. In this case, the center
of mass of the selected regions will be used to measure the geometrical
data. The command hbond detects HB interactions between acceptors
and donors in residues 9–16 and 34–41, where the kissing loop
interactions are established. The command avgout calculates the
occupancy, average hydrogen bond distance, and average hydrogen
bond angle between a donor and an acceptor. Note that the output file
name of hbond is different from that of other commands. The command
rmsd measures root-mean-square-deviation of the MD trajectory with
respect to reference structure. Here, the rmsd of residue 1–88 is
calculated with respect to the structure at the first frame. More detailed
information of the cpptraj program can be found from the most recent
AMBER manual (https://ambermd.org/Manuals.php).
3.3.2 VMD
VMD can visualize the final structure of MD simulation or MD
trajectory. In addition, VMD provides basic data analysis tools to
measure atomic distance, angle, dihedral angle, rmsd, and HB
interactions. For data analysis, load the structure in a pdb or coor file
with topology file or an MD trajectory with topology file. For example,
in VMD, select File → New Molecule → Browse → select bentRNA-
solion.psf → select CHARMM, NAMD, XPLOR PSF → Load → Browse →
select coor (structure) or dcd (trajectory) file → select NAMD Binary
Coordinate for coor file or CHARMM, NAMD, XPLOR DCD Trajectory for
dcd file → Load. To measure atomic distance, angle, and dihedral angle,
follow the below procedures.
Atomic distance: Press 1 on the keyboard and select two atoms using
the mouse point.
Atomic angle: Press 2 on the keyboard and select three atoms using
the mouse point.
Dihedral angle: Press 3 on the keyboard and select four atoms using
the mouse point.
To save measured distance, angle, or dihedral data, select Graphics
in the VMD menu → Labels → select Bonds for atomic distance, Angles
for angle, or Dihedrals for the dihedral angle → select the data in the
window box to save → click the Save button → define the name of data
file → click the OK button. The output data file contains frame numbers
which start from 0 in the first column and list measured values in the
second column.
To measure RMSD using VMD, select Extensions → RMSD Visualizer
Tool → Define Atom Selection. In this case, type nucleic → In the
Reference, select self for Molecule ID and 0 for the Frame as Reference
structure → click the RMSD button → click Plot Result button → In the
MultiPlot window, select File → Export to ASCII vectors → Define the
RMSD file name → Click the Save button.
HB interactions can be analyzed by VMD. In the VMD menu, select
Extensions → Analysis → Hydrogen Bonds → Type nucleic in the Selection
1 (Required) → Define Donor-Acceptor distance (Å) and Angle cutoff
(degrees) for HB interaction scanning → In the section, Calculate
detailed info for:, select All bonds → To save data, in the Output options,
check Write output to files? → Define Output directory → define the
name for output file → click Find hydrogen bonds! button. The hydrogen
bond analysis tool will generate two data files. One file lists the number
of hydrogen bonds in each frame, and the other file lists the donor
residues, acceptor residues, and occupancies of HB interactions.
4 Notes
1. The Importance of Water Box Size
It has been discovered that silver (Ag) atoms bound to cytosine-rich
DNA hairpin and form DNA-Ag nanocluster (NC) [43–46]. The DNA-
Ag NC motif can be used to build nucleic acid nanostructure to
develop an antibacterial therapy [47–49]. Computationally, the
structural stability and conformations of DNA-Ag NC can be
investigated by MD simulations. Previous MD simulation study
reported that the hairpin structure of nucleic acids can be
significantly deformed due to ion bindings or salt concentrations
[50]. In this section, we introduce how MD simulations can be failed
if a nucleic acid hairpin is elongated in a small water box.
The NMR structure of a GAA-hairpin loop (PDB ID: 1JVE) [51] is
used as a template to build a larger DNA hairpin, whose bases are
replaced with cytosines using DSV. The initial solvated system and
topology files of the DNA hairpin are prepared by CHARMM FF, as
explained in Subheading 3.2. The MD simulation of DNA hairpin is
performed by the same protocols described in Subheadings 3.1.2,
3.1.3 and 3.1.4 with NAMD config files in Table 2.
In general, a solvated system for MD simulation is prepared
with the minimum size of the water box to reduce computing time.
However, if the DNA hairpin elongates during MD simulations, the
DNA can break the periodic boundary conditions (PBC). During
50 ns MD simulation, the length of DNA hairpin increases from 25
to 32 Å . The initial and the final structures of DNA hairpin are
plotted in Fig. 3a, b, respectively. To demonstrate the significance of
water box size, two different sizes of water boxes are prepared. The
dimensions of small and big water boxes are (58 Å × 59 Å × 85 Å )
and (78 Å × 79 Å × 95 Å ), respectively. Figure 3c is the result of a
30 ns-long MD simulation, where the DNA hairpin is initially placed
in the small water box. The DNA hairpin is rotated during MD
simulation, and the part of the DNA hairpin is placed beyond the
primary PBC box as it elongates. The elongated hairpin collides
with the mirror image of the DNA in the neighbor PBC box. Under
this condition, the DNA hairpin in the primary box and the 5′ and
the 3′ ends of the DNA in the neighboring box interact with each
other. This interaction remains for the rest of the simulations.
However, when the DNA is placed in the bigger water box in Fig. 3d,
there is enough space between the DNA and boundaries of the
primary box so that the mirror images in the adjacent PBC boxes
maintain enough distance from that of the primary box. Therefore,
when a water box is prepared for a biomolecule, which can
experience a large conformational change during MD simulations, it
is necessary to prepare a large enough water box to avoid the
interactions between the biomolecules in the primary and adjacent
PBC boxes.
2. MD Simulations of Linear RNA Motifs
To design RNA nanostructure, depending on the aim of
nanostructure, it is necessary to implement specific sequences into
the RNA nanostructure. However, modifying sequences can also
alter the stability and conformation of RNA nanostructure. To
demonstrate how RNA sequences affect the stability of RNA motifs,
we prepared two mutated sequences in the DIS of HIV-1 RNA
kissing loop complexes, which are named as KL-1 and KL-2 as
shown in Fig. 1.
The solvated system and topology files for each KL are prepared
using AMBER FF, as described in Subheading 3.1.1. The MD
simulations follow the protocols described in Subheadings 3.1.2,
3.1.3 and 3.1.4. Figure 1 shows the final structures of each KL
complex at 200 ns. The stability of the KL complex is measured by
RMSD using the cpptraj command (Subheading 3.3.1). The RMSD
values of the overall KL-1 complex and its KL region are 4.5 ± 0.5 Å
and 3.7 ± 0.1 Å , respectively. However, the RMSD values of the
overall KL-2 complex and its KL region are 7.8 ± 0.8 Å and
5.2 ± 0.2 Å , respectively. These results indicate that the KL sequence
of the KL-2 complex causes significant deformations. The capability
of self-assembly between two RNA hairpins via KL interactions can
be estimated by monitoring HB interactions. The formation and
breaking down of HB interactions during MD simulations are
monitored using the hbond command in the cpptraj program (see
Subheading 3.3.1). The HB interaction analysis shows that KL bases
in KL-1 form stable HB interactions in all six base pairs during the
entire MD simulations, while KL-2 shows very weak HB
interactions between G11:G38 and A16:G36. Therefore, these MD
simulation results indicate that KL-1 can be used as a motif to build
an RNA nanostructure, such as tectoRNA based on its structural
stability and secure HB interactions, while the KL-2 motif may not
form an RNA nanostructure due to severe structural deformation
and very weak KL interactions.
3. MD Simulations of the Bent RNA Motif and RNA Nanoring
The dumbbell-shaped RNA motif, which is introduced in
Subheading 3.2, forms a KL complex, which is bent by 98° (Fig. 3a).
In addition, a ring shape of RNA nanostructure can be constructed
by six dumbbell-shaped RNA motifs (Fig. 3c). In this section, we
briefly introduce the MD simulation results of the bent RNA and
RNA nanoring.
The initial solvated system and topology files of the bent RNA
are prepared by CHARMM FF using the same protocol described in
Subheading 3.2. The MD simulations are performed by the
protocols described in Subheadings 3.1.2, 3.1.3 and 3.1.4 with
NAMD config files in Table 2. The total number of atoms in the
solvated system is 257,243. The final structure of the bent RNA at
100 ns is plotted in Fig. 2b. To measure the stability and
geometrical characteristics of the bent RNA, the length of the
dumbbell-shaped motif, the bending angle between two dumbbell-
shaped motifs, the overall RMSD, and the RMSD of the KL region are
measured by the cpptraj program. The length of the dumbbell-
shaped RNA motif is measured by the distance between two
phosphate atoms at the middle of each hairpin loop (red dotted
arrows in Fig. 2a. The bending angle between two dumbbell-shaped
motifs is measured at three locations, the center nucleotide in one
hairpin loop, two center nucleotides at the KL region, and the
center nucleotide in the other hairpin loop (blue dotted line in Fig.
2a). The initial length of a dumbbell-shaped motif is 54.1 Å , while
the average lengths of each dumbbell-shaped motif in the bent RNA
are 59.7 ± 2.5 Å and 61.7 ± 2.3 Å , respectively. The initial bending
angle is 98° and the average bending angle is 126.7 ± 4.5°. The
average length and bending angle are calculated from the last 50 ns
trajectory. RMSD values are measured with respect to the initial
structure. The overall RMSD of the bent RNA with respect to the
first frame is 7.6 ± 0.7 Å . The most interesting results are the RMSD
of the KL region and the stability of HB interactions. The average
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door.
I climbed out on my hands and knees as she instructed me to do.
She guided me along a dark hall about two yards wide through a
door to the opposite side and into a room. Sitting at a table in front of
me were the two men who had previously visited my dugout. A
candle on the table was the only light in the room. The windows were
tightly covered with heavy cloth. These things my eyes took in as the
woman led me to a chair facing the men. My mind was calm but my
body shook uncontrollably.
The man, my first visitor, was the spokesman. “Don’t be afraid,” he
began. “You know we are trying to help you.”
His voice was reassuring and my body calmed a little.
“We are in great danger,” he continued. “Spies have been
everywhere, searching for missing bodies. If anyone comes near
you, and tries to speak to you, pretend to be deaf and dumb. Make
signs with your hands but never speak to anyone, not even to us,
unless we first speak to you. We cannot be careful enough.” In a
softer tone he added, “I grieve to inform you—the others are no
more. I can tell you nothing more.”
He paused deferentially. I understood. That subject was closed
between us.
In a moment he went on, “It is becoming too dangerous to remain
here. We must go away, but first you must accustom yourself to the
outdoors. Ahead of us is a long, strenuous journey. We dare not risk
the daylight, so the trip must be made after dark. Tonight will be a
starter. Tomorrow we will see you again.”
XXX
RECOVERY
I was dismissed. It was over. I was not to be killed. My body
throbbed with gladness.
The woman was standing beside me. She led me to the door and out
into the open night. The air was sweet and fresh, so noticeable after
being accustomed for so long to musty air. I breathed deeply to
refresh my whole being. It was so long since the last time I had seen
the night all lighted up with golden stars so near and yet so far. This
was the most beautiful night and the saddest one I could remember.
The horizon seemed to be far away, sad and quiet as if the world
were in a deep slumber. But probably this very minute some one was
facing a death sentence.
A heavy stone lay on my soul. I could find no consolation. Never will
I see them again. My sisters fresh as rosebuds, in the very morning
of their lives, and now withered away before they even blossomed,
their youthful faces now covered with the cold earth, vanished
completely and forever before my very eyes. My heart was aflame
with a grief that was tearing my soul.
Were these the same stars which looked down on us at Tsarskoe
Selo? At Livadia? The same as on our cruises, when Father’s stories
about the heavens seemed so real? Father could see them no more;
nor Mother, nor sisters, nor my brother Alexei. Knowing that their
eyes were closed forever, filled me with a loneliness I could not bear.
How could the stars go on shining as if nothing had happened? How
could the air be so sweet and fresh when such foulness had taken
place? If my family could not breathe this fresh air, how could I? And
how could I gaze on this heavenly grandeur?
All about me reminded me of my family. I had no more tears but I
cried inwardly. I lost my balance. I collapsed on the ground. The man
and the woman were watching me. They rushed to help me to my
feet, and back into the house and down the ladder.
Again I sank into the damp earthiness of my tiny room, thankful for
its darkness. Here I felt closer to my family. Outside the stars could
sparkle, the air could be fresh, but inside the contrast could not flaunt
itself before me. I need not look at a world untouched by our tragedy.
In the middle of the night I awoke to find myself walking about my
dugout. I was completely confused, with no idea of where I was. My
bare feet sank into the soft dirt. At last my foot touched the straw rug
which led me to my bed, and in bed was still the welcoming warmth
of the stone wrapped in a cloth.
Walking around with bare feet had given me a slight cold. The
woman was upset; she attributed the setback to the night air, to my
first trip outdoors. The men called early in the morning. They were
annoyed that I had a cold but agreed with the woman it was best to
defer another trip upstairs until I felt better. I heard them say, “Speed
up her recovery.”
I lay still another night wrapped in my thoughts. Though the men had
said that my family was no more, still I would not believe it How
could they be so sure? I would never cease to hope, since I had
learned how difficult it was to die. To me their nobility, their trust in
God, their character were more impressive than the grandeur of the
night. God would not fail them.
But we were separated. That was certainty. Somewhere I would be
deposited to face an indifferent world, a world that would not bend its
knee to me. I must suppress my identity and make a new life, all
alone. God knows how I missed them all. I lay there, one minute
hoping, then despairing. I felt close to them in a world which was not
theirs nor mine. The woman continued her care of me; I was
suspended between two worlds belonging neither to this one nor the
one to come. The idea crossed my mind several times that suicide
was an easy escape from my misery, but my strong faith based on
years of prayer would not permit this lack of will power.
Soon my cold faded away and once more the woman had me
climbing the ladder. This time she stood on a chair, put both hands
under my arms and lifted me up the few rungs through the trap door.
In a few moments I was again in the presence of the same two men.
As before, the only light came from the candle on the table. The
windows had the same heavy coverings. I almost said, “Good
evening,” but remembered not to, just in time. With no preliminaries
the spokesman started immediately.
“We are very much interested in straightening out a few things,” he
said. “We want to ask you some questions.”
I was all fear. At Tobolsk, at Ekaterinburg, questions meant traps.
Surging through me, I remembered my Mother’s advice: “Answer
courteously but give no information.”
“Were they unkind to you in Tobolsk?”
I wanted to tell the truth. In spite of my impaired speech, they wanted
me to answer their questions.
“Very unkind, the last days,” I answered.
“What sort of things did they do to you?”
I hesitated. There had been so much torture and suffering, it was
difficult to begin with any specific detail. In contrast to the final
outcome, the treatment we endured at Tobolsk suddenly seemed
trivial. I could not mention anything specific, not yet. The tragedy
towered over all events, making all others seem unimportant.
“Everything to contribute to our unhappiness and humiliation,” I said.
“Who were with you in Tobolsk?” he began.
Something I could answer easily. I told him, “Our friends, and
household help.” Their faithfulness excited my memories.
“Did they all go with you to Ekaterinburg?”
While in thought, I raised my eyes and saw a door on my right side
which apparently led into another room. Through a crack I saw a
bright light and a shadow flitting across the crack in a sort of
rhythmical motion, as if someone were swinging back and forth. My
thoughts were distracted for a moment. My training came to my aid. I
could see without betraying what I saw. I hid my surprise at
discovering that there were other people in the house besides the
two men and the woman. I dismissed my curiosity since I was
beginning to have confidence in these men. Their questions must be
answered.
I began, “Some went first with Father, Mother, and Marie, Dr. Botkin,
Prince Dolgorukov, a maid and Father’s valet. When our parents left
Tobolsk the others stayed to take care of us children who were left
behind to go later when Alexei should be better.”
What kind of quarters did we have in Ekaterinburg? Did we have
enough to eat? Did we ever go outdoors? Did we see our friends?
How did we pass the time? These questions were meant to pave the
way for more questions that were to come later and to encourage me
to answer them. In a way I was glad to confide my sorrow.
When they were through, one of the men signalled the woman, who
led me outdoors. My second walk in the air. I had decided to take the
outing without once lifting my eyes to the sky. I took short breaths
and leaned heavily on the woman’s arm. We walked around the
house. Guarding us, I sensed rather than saw, was one of the two
men, walking a few feet behind us. At last we were back at the
entrance. The house was dark as we entered the first room and
passed through the open trap door and down the ladder. The trap
door was lowered and I noticed a cloth was nailed on the inside. No
sounds must penetrate through that floor. The exertion and
gratification at my own courage put me into a sound sleep that night.
I awoke stronger and aware of a new milestone in my march to
recovery. Each day the terrifying world I knew nothing of drew closer.
I had almost ceased to struggle to keep from entering this world of
reality. I was being carried like a leaf floating on the surface of a fast-
flowing stream. I could not stop or sink. The prospect of living my life
in good health would have been frightening enough, but now I must
face ill health and loneliness as well. If only crying could oust these
seething tortures.
The woman was busy folding a blanket. When she finished she piled
it on her arm, tossed the pillow on top and lowered her chin on it for
a firm grip on her burden. Now she was climbing the ladder, load and
all. There was something final in the way she climbed. It suddenly
dawned on me that she had spent the many nights close by my side.
Satisfied that I was out of danger, she was henceforth going to sleep
upstairs. I had not been conscious of her presence; misery had
made me deaf and blind. Now that her watchful custody was
removed, I became frightened and longed for the nearness I had not
been aware of.
Without a clock, I had no idea at what time our day started. When
the woman appeared, it was morning. The smoky window became a
panel of gray after she removed its covering. I always listened for a
“Good morning.” But that was all. She never did tell me what I should
call her. If I needed her attention, I whispered, “Lady, please.” I did
overhear the man call her Iliana or Irina. When she combed my hair,
she did it gently. All her services were performed tenderly. I thought I
detected a resemblance between her and the man who was the first
spokesman. I wished I knew something about these men and the
woman.
After an early supper she helped me to get dressed again in the
same grotesque clothes and took me to the room above. The same
men awaited me; the same rhythmical shadow flitted through the
crack. Previously the questions had been of a general nature. Now
they began to ask me more personal ones. They asked about
Madame Vyrubova. Did she influence the Tsarina? Was she intimate
with Rasputin? Did she live in the palace? I did not want to answer
any of these questions, yet I did not dare to refuse.
All the time the shadow continued to pass over the lighted crack of
the door. Who could it be? Was there someone writing down my
answers? I was terrified. Suddenly I became so exhausted that I
thought I would fall from the chair. My interrogator saw the situation
and excused me. The woman accompanied me outdoors, and we
walked around the house several times. Then I returned to my little
hole. Once more I sank into my congenial darkness. The woman
climbed the ladder, her touch was as soft as a feather. The trap door
opened, then lowered back into place, quietly but firmly.
One day she brought me a piece of meat and vegetables. I refused
to eat meat. The woman had no longer to bandage my wounds; they
had healed sufficiently. Now I felt an itching sensation on my head
and the woman was pleased with my progress. “Your hair will cover it
nicely,” she said. There in the darkness my hair was growing fast. I
was able to braid it.
Ahead of me lay the nightly inquisition. Yet each trip into the
outdoors made me more independent physically. Once more and
many times later I sat in the question room trying to control myself in
the face of the curiosity of these two men and the woman. Now my
Mother was the subject of their questioning.
“Did your uncle come from Germany to see the Tsarina?” The
shadow in the next room seemed poised for my answer. Suddenly I
was glad of the recording. Here was the opportunity to show Mother
as the eager helpmate that she really was, trying to report to Father
the facts as she saw them in his absence. “Did the Tsarina listen to
Rasputin, because she believed in his honesty, foreseeing, and
experience?”
I answered, “Whatever Mother did, she did it only for the good of
Russia. When Father did not agree with Mother, she accepted his
decision as final, knowing she could have no further influence.” I
expressed these thoughts as carefully as I could, hoping whoever it
was would take them down accurately.
Every evening for some two weeks I was questioned. Each period of
questioning lasted about an hour. When each bout was ended I was
burning as with a fever. The questions were personal and plentiful.
The subjects were continually changed, covering the entire Imperial
family and the palace staff, as well as the household employees.
Many of these questions seemed so impertinent to me that one night
I burst out, “Why are you asking me all this?” They replied, “We only
want to know.”
Gradually I began to take in my outdoor surroundings. Not far away I
saw a haystack. Beyond it, a dark vastness, perhaps a forest or low
hills. During my walks around the house, I noticed that it was square
and squat like most peasant houses. I gathered it was made of
wood. The roof was shingled. I tried to find my tiny window and
finally located it behind the camouflage of hay. I could hardly see it.
Now I understood why my room was so dark.
The house stood some distance from the road and a long dirt
driveway extended from the road to the rear of the house. To one
side was a large shed, perhaps a barn, which apparently did not
house any animals since I heard no sounds from it Occasionally I did
hear lowing herds in the distance. Perhaps one of these cows was
responsible for the milk I received each day. There were no signs of
horses or chickens. But several times I heard a faint train-whistle in
the distance. I never saw any arrivals or departures. Did these men
come from the neighboring houses and walk across the fields? What
went on in this house was a complete mystery to me. Did these two
men and the woman live in this house? There was another young
woman. Was she behind the ill-fitting door? I had only seen the hall
and the question room, but I felt sure there was another room in the
house. The furnishings I saw were meagre. Perhaps this was an
abandoned farmhouse or discarded servants’ quarters attached to
someone’s estate. The outside, too, had a deserted appearance. I
noticed some hay scattered near the house. My observations were
all based on what little I could see in the darkness. I felt sure these
people were in someone’s service, or they were using the place as a
temporary hideout.
The men became quite friendly. The woman continued her silent
care of me. The men talked freely, even joked about my recovery.
Without warning, the question I had always suppressed, suddenly
broke out. “Why did you rescue me?” I inquired.
The faces of both men turned red. The man I first met was shocked.
The second man’s eyes blazed with fury. “What is it that you want to
know?” he said.
My feelings were deeply hurt at this coarse reply. My fears returned
in full force. The worst must lie ahead.
It was obvious that I must soon go away, otherwise these people
would pay the penalty. Now I wanted to stay in my dugout forever. It
was mine and I was part of it. I would inscribe my name here, but
where exactly? I had nothing with which to write, no crayon, pen or
pencil. There was a hairpin on the table. Quickly I was out of bed
and standing on the rug. I pulled out the small drawer and set it
upside down on my bed and began scratching my name with the
hairpin. I bore down with all my strength on the bottom of the drawer
and formed the letters and numbers: “A.N.R. 1918.” It could not have
been a legible signature but, such as it was, there it would stay, a
witness to my habitation, these hairpin hieroglyphics.
A few days after this incident the woman seemed to take unusual
pains with my grooming. She braided my hair smoothly. She gave
me a fresh pillow which had a lace edging, sat me up and dressed
me in one of her jackets. She arranged my covers neatly. By the time
she had finished the men were already descending the ladder.
The first man took a small kodak from his pocket and after lighting
several candles snapped my picture. A few days later they returned
with several prints. I had one of these until it disappeared with my
manuscript. The clearest object in the picture was the lace on the
pillow case. How different I looked! My nose was still swollen, my jaw
caved in, my eyes had many light dots.
“Isn’t it a splendid likeness?” the spokesman was saying.
As they turned to climb the ladder, they reminded me of the danger
of their position. “Things are terrible,” they said in unison. “People
are lined up and shot at the slightest excuse.”
I gathered that they were more and more frightened at having me
around. The questioning continued, followed by a walk each time.
How could they think of so many things to ask?
My family would have been surprised at my diplomatic skill. The
mystery continued. I learned no more about the house. After each
outing I was returned to my dugout. Then I began to feel an
undercurrent of excitement which told me that the time of my
departure was near.
One day when I was again in the question room, one of the men
said, “We can not stay here any longer. We are taking you to another
little place. It is very risky. Never forget to be deaf and dumb. Speak
only with your hands. We start early in the morning. You will take a
short walk now, then sleep until we awaken you.”
Our outing was brief. Once more I was hustled down the ladder. The
woman helped me into bed and went away. Some time after
midnight, the woman returned. This hour reminded me of the night at
Ekaterinburg. I began to cry. Her worn, white face told me only too
well that she had worked many hours preparing for our departure.
She brought me a cup of milk. Her hand was shaking. I felt a warm
feeling of appreciation. I too was trembling. I spilled the milk. She
dried the splash on my dress and turned away but our souls were
knit together.
She rushed me to dress in the same clothes I had worn before,
except that the old dress now had a wide hem. It almost reached to
my ankles. My black stockings were well hidden by my high laced
shoes and the long dress. She tied a babushka around my head.
Silently she handed me a label which had apparently been removed
from someone’s coat or dress. Stamped on brown taffeta in gold
were a double-headed eagle and the words, “Mikhailov Moskva.”
Mikhailov was the name of a well-known Moscow firm. I could not
recall having ever seen such a label and could only guess at her
motive in giving it to me. Possibly she thought I would recognize it
and surmise something as to what had happened to its owner. I still
have it.
I asked once more what had become of my old clothes that I had
had on that fatal night. Puzzlingly she replied, “They were so badly
bloodsoaked that I had them burned.” I wished to ask her again what
became of the items I had had hidden in my clothes. I was afraid. I
was at their mercy. I had no choice. There were some fifty large
diamonds and pearls and about twenty-five rubies, emeralds and
sapphires.
I took a final glance at the room and thought of the uncertain future
ahead. Now that I was leaving this tiny spot, I knew that I owed it a
great deal. The only thing I had to give it I had already given—my
initials.
In front of the ladder I knelt down to pray for a moment for my dear
ones. I dreaded to leave the dugout for fear that I might never again
visit this part of the world where the remains of my beloved ones no
doubt were buried—somewhere nearby.
My mind went back to a question I was once asked: Why did I
always stand at some distance from my sisters? I now realized that I
must have unconsciously had the premonition that we would some
day be separated.
I blew out the candle and groped my way through the room to climb
the ladder, and out through the trap door. A clam was being tom from
its shell.
In the hall I was greeted with a rush of fresh air. Our departure was
to be immediate, with no light of any kind. In a few seconds the
woman led me outdoors.
XXXI
WESTWARD TREK
I was conscious of figures mysteriously scurrying back and forth to
the house. My eyes began to focus better and I could see standing a
man who led me to the back of a hay wagon, as if to introduce me to
the scheme devised for my escape. He took my hand that I might
feel him unlatch a little door, then he pushed my arm through the
opening, indicating to me the empty space inside. He guided my
hand to the bottom, to the bedding of hay and the blanket, then the
arched roof to feel the hoops laid closely above. He directed my
fingers to the cloth between the hoops, trying to let me know a
covering had been thrown over to keep the hay from falling through.
He made me touch the sides of the wagon and the arched top
covered with hay. My hiding place was to be this coop, camouflaged
as a load of hay. The ingenious plan revealed once more the risk
these people were taking. I stood hesitating between fear and
appreciation, when suddenly I found myself being lifted feet first
through the tiny door. He handed me a bottle of water and without a
word hooked the little door. Locked up in my little cage, I listened.
Footsteps. It seemed there were many. I was anxious to catch every
sound. I could hear nothing. The wagon bent forward as under a
heavy weight. A stronger lurch and we were in motion.
Now I felt like a little calf being taken to the market. Yet I had reason
to trust these people. Their careful preparation, their discreet silence
could be only for my safety. I could not understand them, but I could
not mistake their kindness. Perhaps someone would meet me at our
destination. The serious, elaborate precautions indicated we were in
great danger. This first part of the journey was no doubt the most
critical. Through the rear of the wagon I could see a light. It was the
sun coming up.
I presume we traveled for hours, the horse moving at a steady and
brisk pace. I could tell when we were going up or down. The road
was rough and I was badly shaken. These country roads were in
poor shape and full of ruts. The dust sifted through the partitioned
door and filled my eyes and nostrils. I felt I was suffocating. I could
hardly breathe. My dry throat stiffened. I reached for the bottle, found
and removed the cork, swallowed some water to wash the dust
down, and sponged my face. My cramped coop was long enough to
permit me to lie full length, and high enough so I could easily turn
over. I could even draw up my knees, though I could not sit up. I
rolled on my stomach and pressed my face against the little door to
catch any current of air. I hoped they would soon stop, but dared not
call out for fear that my voice would betray us. The driver seemed to
know the road well, as the cart did not hesitate in making any of the
turns. It had no springs and it jolted brutally and ceaselessly. My
head was splitting. Would my wounds break open? My face was stiff
and plastered with a mixture of dust and sweat. I was miserable. We
travelled for a long time. Then light began to penetrate through the
door and the wagon took a sudden turn. A plunge down and up
again, then slowly along a level road with many bends. It stopped. I
listened. Had we been halted for searching? Did someone jump
down from the driver’s seat? Some one was coming around the
wagon. It was one of the men, pushing the hay from the little door.
Suddenly the door flew open. In came a rush of fresh air. Feeling
unsteady I tottered into the arms of the woman. I was glad she had
come with us. She smiled, reflecting my pleasure at seeing her. She
held me for a moment before I could walk around to loosen my
tensed body.
It was most exciting to find myself free in the woods in the daylight. I
learned there was no trouble here. We had stopped for a rest and to
care for the horse. My eyes squinted and watered painfully, and I
could distinguish very little. I bathed my eyes and the woman tied a
cloth over them and left me sitting on a log. I lifted the cloth a tiny bit
to accustom my eyes to the light. First I saw some green, then the
forest, then a winding stream and myriads of bushes screening us
from the road—a perfectly secluded spot. Later in the evening we
resumed our journey, stopping several times for a rest. At daybreak
the light started again to seep into my coop. The way the horse
turned indicated we were not here by accident, but had actually
stopped according to plan.
It was a beautiful morning; the dew still on the grass made the air
superb. I could see some deep orange-colored flowers and some
wild asters. I plucked some of those dry seeds from the plants and
tossed them into the air, hoping they would fly through the woods in
the direction where my family was resting. I knew so little of the cruel
facts surrounding my dear ones. If I could only kneel beside their
graves for a silent prayer. But even this was denied me. Water
rushed down a little stream and humming insects flitted about No
prison walls here.
Presently the woman spread the army blanket on the ground,
motioning me to sit on it, and opened a lunch basket. She had some
hard boiled eggs, one for each of us, fried fish, bread. It was a
sumptuous feast, and a glorious feeling there under the shaggy
trees, the profusion of pines and birches, under the deep blue sky—
a typical Siberian scene. The horse stood still munching. The leaves
hung motionless, the birds were quiet; all nature seemed to breath in
suspended and sympathetic silence as we ate our lunch hungrily.
The great sacrifice these people were making impressed me once
more and I felt appreciative. In no time all three were asleep. In order
to make it easy for everybody I had an urge to run away, but
because of the effort these people were making, my conscience
would not permit it. Also probably at the “little place” they knew of,
someone might be waiting for me.
The little brook rumbled on in the quiet of the countryside. It alone
defied silence. The trees stood in a colored hush of yellow, red and
green. The white birch and the trembling aspen beat their wings. All
this indicated it was early fall and nature was ready for its long sleep.
After taking these details of the setting, I realized I, too, was
exhausted.
The men were harnessing the horse and the woman was packing
when I awoke. She was waiting to fold my blanket. I was chagrined
to have been such a poor sentinel, but no harm had resulted. Now
the men were ready. The woman started for the wagon, so I
followed, though I could have enjoyed nature in that spot indefinitely.
In a few minutes I was back in my coop; the men and the woman
were in their places. The wagon bent forward several times and we
were on our way again.
We moved steadily and confidently. The way must have been
perfectly familiar to our driver. I did not hear any other passing
wagons, nor was I conscious of passing through any village. I kept
thinking about the beautiful spot in which we had lunched. That day
God and nature were fused together. There in that beautiful resting
place the family had been with me in spirit guiding me in the very air
I breathed, caressing and encouraging me. I imagined I could hear
Father’s quieting words. Even now I thought those words might be
directing us. The driver seemed so sure of the way I became less
alone, less suffocated, less desperate. I could lie quietly.
Transcended, I simmered in wishful dreams. This was the second
night on the road. The wagon rumbled. Another abrupt turn
awakened me. I listened. The horse halted. One of the men was
opening the door of the coop. He pulled me out and set me on my
feet. We were in the midst of a thicket. The third stop on our journey.
It was twilight, the sky still holding its sunset colors. The colors faded
into the gathering darkness.
The men worked hurriedly, nervously and anxiously to get through
feeding and watering the horse. It was a short stretching period, only
long enough to eat from our basket and let the horse finish his feed.
Not one word broke the evening silence, not one moment was
wasted. Once more I lay in the coop and the horse started out again.
Even in the dark I could feel a certainty of direction. The horse’s feet
met the ground as if the way was not strange to him. We went on
about the same pace as during daylight. Each moment I was
prepared for any difficulty which might arise. So far there had been
no trouble. Would we travel all night? I could not sleep in the chilling
darkness and I did not suppose any of the others did. Suddenly a
lurch to the left, a short run and a halt. No sounds, only poignant
stillness. One of the men helped me out of the cage. I found myself
standing beside a house. The woman opened the door and walked
in. They all seemed to know this house. Though it was dark, they
made their way around, and lighted a candle. The woman led me to
a bed. After she finished with me, she went out of the room.
It seemed but a moment before the woman was awakening me from
my sleep. I was getting used to the inevitable. It was still dark when
she came in with a candle in her hand. She guided me to a table
where milk and bread was laid out for me. Then she led me outdoors
to the wagon which stood ready. I could see clearly by the light of the
stars.
To my surprise, we did not stop at the rear of the wagon. We passed
to the front where one of the men already sat in the driver’s seat.
The second man turned as if to help me up. I looked at the woman,
seeking an explanation. She put her lips to my forehead, pressed my
hand, then gently pushed me toward the man who helped me to the
seat beside the driver. The other man climbed after me. The wagon
started, it had two horses this time. We had left the woman behind! I
suddenly realized the woman had said good-bye. I was never to see
her again. We turned out of the yard, onto the country road. A man
on each side of me and the woman gone. I had misgivings. The men
sat rigid, gazing at the pre-dawn blackness. They were on the alert.
To them, leaving the woman behind was a planned milestone in their
hazardous task. Her part was finished, now it was up to them to
carry on.
The woman was protection to me, the last remnant of a feminine
world. We were still in danger, judging by their alertness. I wondered
why I had not returned to the coop. These men were stoical and
brave to attempt this journey at all. Their continual watchfulness
inspired confidence. One of these men had annoyed me with his
questions several days ago. Now he sat beside me.
Each moment took me farther away from the woman. I had taken her
protection for granted. My feeling for her had been a mixture of
wonder and resentment. I always hoped that some day she would
talk to me and tell me what I wanted to know. Now that day was
gone forever. But she had done her part faithfully and I must be
grateful. That last night at the dugout, when we looked eye to eye,
she understood what I wanted most. I would have liked to put it in
words. That night I had felt close to her. Now she was far from me,
free to return to her accustomed way of life. What was her life? On
our way, had we dropped her at her home? This dugout, where I had
been so long, was it far from Ekaterinburg?
The men spoke of Uktus and Mramorskaya. Was one of these the
location of my dugout? It was an irritating mystery. No part of the
mystery was solved and now the woman was gone without telling me
a thing.
The men kept peering through the darkness, taking advantage of its
unlimited screen to make all possible progress. My inner darkness
exceeded the darkness of the night. At length the outer darkness
lifted to herald the coming dawn. Soon the majestic sun appeared
unchallenged. I was cold and numb until a quick turn of the wagon
made me forget my numbness.
The horses halted and the men stood up to stretch. They jumped
down and helped me to alight. Then handing me the basket and the
army blanket, they began to unharness the horses. This was the
beginning of our fourth day on the road. I attended to the food while
the men cared for the horses. The basket contained a fresh supply of
food—black bread, eggs and a bottle of water. The woman must
have baked the bread for us, while we were asleep. I found a sunny
spot on which to spread the blanket while we ate.
When we finished they jumped to their feet, folded the blanket and
we were off again. I climbed to the driver’s seat, where we sat three
abreast. We were a family of peasants driving between work fields.
My faded clothes fitted perfectly into the scheme. That day the
horses seemed to know the route as well as the driver. They knew all
the byways. They must have driven these roads frequently. From a
hill I could see a village in the distance. I did not ask their names and
the men did not volunteer the information. We did see some people
walking on these roads. We also passed a few wagons.
When I noticed that the men were less tense, I assumed we were
beyond the danger zone. We were all in a more relaxed mood. We
stopped in another spot and laid out the food, but there was very
little left. They were so tired, they hardly could eat The meager meal
was soon finished and they harnessed the horses. One of them took
the blanket and climbed on top of the hay and spread it out. The
other man helped me up; here I was to spend my night. How
imperturbable these men were, so reassuring when I was most
afraid. Their thoughtfulness touched me. Now that the men could not
see me, I could shed the tears I had turned back yesterday. I fell
asleep until the wagon halted again. I sat up in fright. It was a dark
night. The stars were quivering. I heard the straps fall. The whiffle-
tree hit the ground. The horses were stepping out of their traces. We
were to spend the night here. The men stretched outside beside the
wagon.
We had been sleeping for a while when I thought I heard a scream of
some kind. I heard it again. Now it was a shriek, the horses neighed
with a shrill sound and jerked the wagon to which they were tied. The
men sprang to their feet and began to pull some hay off the wagon.
They threw it to the ground and lighted a match to make a fire. Soon
the horses quieted down. I overheard the men saying there were two
wolves.
After this scare it was easy to keep awake. I felt sorry for the men
who were up and down several times during the night. At last dawn
appeared and I heard them stirring in preparation for a new
departure. I combed my hair, using my fingers for a comb and my
palm for a brush, trying to make myself as presentable as possible. I
knew I did not look well groomed but I did not much care. One of the
men helped me down from the wagon, the other brought some water
and poured it into my hands. I washed my face without soap. We
had spent the night not far from a farmhouse known to my
companions.
We emerged from the forests into open fields, then another forest.
Judging by the sun I figured we were moving in a southwesterly
direction. These inscrutable men stopped at another byway for rest
and food. We ate bread and washed it down with water. Not a word
was spoken during lunch. Soon we rattled again along the roads and
country lanes without any special incident. We met a number of frail
men and women, and barefoot children wearing tattered clothes. We

RNA Nanostructures Design Characterization and Applications Methods in Molecular Biology 2709 Kirill A. Afonin (Editor)

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RNA Nanostructures Design Characterization and Applications Methods in Molecular Biology 2709 Kirill A. Afonin (Editor)

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RNA Nanostructures Design

Characterization and Applications

DNA and RNA Origami Methods and Protocols Methods in

RNA Protein Complexes and Interactions Methods and

Clinical Applications of PCR Methods in Molecular

Computational Drug Discovery and Design Methods in

Vaccine Design Methods and Protocols Vaccines for Human

Antifungal Immunity Methods and Protocols Methods in

Proteomics in Systems Biology Methods and Protocols

Cancer Cell Biology Methods and Protocols Methods in

Methods in Molecular Biology

For further volumes: http://​www.​springer.​com/​series/​7651

ISSN 1064-3745 e-ISSN 1940-6029

© The Editor(s) (if applicable) and The Author(s), under exclusive

The use of general descriptive names, registered names, trademarks,

This Humana imprint is published by the registered company Springer

Bárbara Gomes Barion

Timea Gerczei Fernandez

Isabella Nevoni Ferreira

Renata de Freitas Saito

Akhilesh Kumar Gupta

Tamanna Binte Huq

Valentina Abondano Perdomo

Maria Cristina Rangel

1. Molecular Dynamics Simulations of

Key words Molecular dynamics simulations – RNA motif – RNA

The first three terms describe short-range interactions in two

The above atomic potentials are applied to describe the molecular

2.2 CHARMM and NAMD

2.3 VMD (Visual Molecular Dynamics)

2.4 Discovery Studio Visualizer (DSV)

3.1 MD Simulation of a Linear RNA Motif

3.1.1 Preparation of Initial Structure and Topology

AMBER tleap command generates a log file to record detailed

3.1.2 Initial Minimization and Equilibration

3.1.3 Constrained MD Simulation

3.1.4 Final Equilibration and Product MD

3.2 MD Simulation of a Bent RNA Motif and RNA

3.3 Data Analysis Using cpptraj and VMD

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For further volumes: http://www.springer.com/series/7651