Structural representation of DNA enables

characterization of regulatory regions and

properties involved in antimicrobial
resistance transfer
Jan Zrimec
Faculty of Health Sciences, University of Primorska

Ale Lapanje Lab, Institute Joef tefan

Background: Antimicrobial resistance and its transfer

Horizontal
gene
transfer
Background: Possible solutions to AMR problem
1. Antimicrobial compounds with new or improved modes of action
a. Improvement of resolving coding and noncoding regions and analysis of regulatory regions

2. Controlling the transfer of AMR genes

a. If AMR cycling can be predicted - new treatment regimens with existing antibiotics
b. Methods to analyse transfer of AMR
c. Characterization of initiation of transfer process - analysis of regulatory regions

Process scheme of Horizontal gene transfer of mobile elements

Initiation Transfer Integration

Background: Transfer regions in mobile elements

Functional DNA transfer region

Pilus Groups protein binding sites

Bacterial
membrane Conservation
of protein
(gene)
defines
functional
groups

Protein complex with

DNA transfer region
Aims: Development of structure-based methods
1. Currently - sequence based
dsDNA Gene1 Gene2 Gene3
methods Regulatory
region
a. Molecular evolutionary principles
b. Inference of similar function based on
nucleotide sequence homology Original sequence: AGT CGA GGT ...
Substitution: AGT AGA GGT ...
2. However - structure based methods Insertion: ATG TCG AGG ...
a. Coevolution of enzymes and DNA Deletion: ATC GAG GTA ...

substrates ~ DNA structures

DNA sequence alignment
Aims: Development of structure-based methods
Dataset of regulatory regions

Functional Groups Protein binding sites DNA sequence

AGAGATTACGTCT...
GGTTGCAAGAGAT

CATAACAGGGGAA...
ATTGATTGAAAATA...
Functional AATATATCGCCAGC
groups
(color AGCACATGAACAAG...
TTTCGGAATGTGAT
coded)
CAATTTAAAAATTT...
ATTGACTTAGGCG...
GGCAGATACTTTAA...
Results: Analysis of DNA structural properties
1. Parametric and ML based models including
a. Thermodinamically induced duplex destabilization
b. Properties related to DNA-protein interaction
c. 64 such models

Zrimec & Lapanje 2015: Fast

prediction of melting bubbles using
DNA thermodynamic stability

dsDNA
Results: Analysis of DNA structural properties
Permutations of 5 Analysis of
nucleotides: Prediction of 64 principal
AAAAA
AAAAC
structural properties components
AAAAG
...
and clustering

Properties of one
DNA DNA clusters
permutation
sequence structure

2 bits (4 clusters)
3 .
4 (16 clusters) PC3
2 bits
information 5 .
6 .
7 .
8 (256 clusters) PC2
PC1
Results: Analysis of DNA structural properties
Dataset of regulatory regions

Functional Groups Protein binding sites DNA sequence Structural

representation
AGAGATTACGTCT...
GGTTGCAAGAGAT

CATAACAGGGGAA...
ATTGATTGAAAATA...
Functional AATATATCGCCAGC Vectors of
groups values of
(color AGCACATGAACAAG... structural
coded) TTTCGGAATGTGAT variables

CAATTTAAAAATTT...
ATTGACTTAGGCG...
GGCAGATACTTTAA...
Results: Statistical analysis of DNA representations
1. Variance ~ distance between elements
a. structures Euclidean distance
b. sequences p-distance = 1 - sum(identity)

nbins = 100

Frequency

FBootstra
p
Anderson 2001
Results: Statistical analysis of DNA representations

Funaligned = 0.525, p = 0.475

Faligned = 0.728, p = 0.029

Fstructure = 1.000, p < 10E-03

Results: Statistical analysis of DNA representations

Structure

Sequence

Conservation
of data in
groups (F)

Amount Size of
of bits Size of region
region
Results: Prediction of transfer properties
- Models built using Neural Networks and
- 64 experimental regions, tested with 140 new regions:
accuracy = 0.975 0.004
- 10-fold cross validation (90% training and 10% testing examples)
with all 204 regions: accuracy = 0.945 0.008

- Prediction of potential host repertoires of mobile elements

- Based on published data in Barcia et al. 2011
- Implemented in a webtool at www.dnatools.eu
Results: Prediction of location of transfer regions
query ... known regions structural representation (64)
target mobile elements structural representation (~ 600)

Frequency
Pseudocode: nbins = 100
for i = 1 : size(query)
{ for j = 1 : length(target)

Distance d
{ d(i,j) = dist(query,target)
} % dist is Euclidean distance
}
find min(d(:,:) < treshold)
% treshold is set by statistical significance
dBoostrap

Position in target

Using this method we find new regulatory transfer regions with p < 10-6 in over 60% of targets.
Conclusions and further work
- New structural representation (code) for DNA
- Significant improvement with functional regulatory regions

- Algorithm for structural alignment of DNA

- finds new functional sites and regions
- up till now possible only experimentally

- Enables development and adaptation of algorithms

- Resolving other regulatory regions - e.g. promoters
- Synthetic biology - design of DNA with similar functionality

- Webtools at www.dnatools.eu
- prediction of transfer properties and hosts
Thank you for listening

Acknowledgements
Dr. Ale Lapanje
Dr. Toma Rijavec
Dr. Tatjana Zrimec
Dr. Ale Beli
and others