2.0 the aptamer. The designing process starts with

2.0 Introduction

Traditionally,
antibodies are used in rapid detection method. This method, however, is time
consuming as the process of synthesising monoclonal antibodies requires six
months to one year (Toh et al. 2015). Aptamer is the new approach in detection
of molecules. Nucleic acid aptamers are 20 to 80 mer long, single stranded DNA
or RNA sequences that bind to target molecules with high affinity and
specificity through their 3-dimensional structures. These aptamer are designed to
bind to specific targets from small molecules and proteins to cells, tissues
and organisms. Due to their in vitro
selection, high affinity and specificity, aptamers are valuable biotechnological
and therapeutic applications as they provide the same detection ability that
could rivals antibodies.

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Aptamers
are usually identified through systematic evolution of ligands by exponential
enrichment (SELEX) which is a process that involves multiple rounds of
selection and amplification. SELEX method starts by choosing random sequence
from large nucleic acid pool then generally involves many repetitive selection
steps, followed by an analysis of enriched nucleic acids. The process is time
consuming, costly, tedious and often fails to enrich high affinity aptamers (Ahirwar
et al.2016). In this study, in silico method is an approach of using
software and website in designing the aptamer. The designing process starts
with obtaining random tRNA sequence from nonunirandgen database. These sequence
are then altered to desired length then submitted to MFold to generate the
secondary structure and to measure the folding stability. After selecting the
suitable sequence, it will then be used to generate 3D structure of the RNA
sequence using 3d RNA. The 3D structure of RNA aptamer will then be converted
into 3D structure of DNA aptamer using VMD. Lastly, the aptamer will be
virtually binds (docking) with Streptococcus
agalactiae. This method of designing aptamer is much faster, cheaper and is
the new approach in designing aptamer which uses bioinformatics approach.

Streptococcus
agalactiae is a pathogenic bacteria that is
problematic in a lot of area including in humans and aquaculture industry. S. agalactiae infection are thought to
occur in individuals with chronic underlying disease such as cancer, diabetes
mellitus and HIV infection (Fujita et al. 2015). It is also a significant fish
pathogen causing disease and mortality in many cultured fish species in both
tropical and subtropical environments. There is a large body of literature on
the pathogenesis of this organism in a variety of fish species. In this study, S. agalactiae is being used as model for
the development of the aptamer. Antibodies that is used to detect S. agalactiae have already been
developed. The usage of antibodies for detection probe can be seen by the
process of ELISA. This research is an exploratory research to investigate the
possibility of developing chemical based detection probe against S. agalactiae using in silico approach.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.0       Objectives

The
objectives of this study are:-

1.         To design DNA aptamer using in silico process, which uses computer software and websites

2.         To measure binding affinity of DNA aptamer
using bioinformatics approach

3.         To
determine the sensitivity and specificity of designed aptamer using
electrochemical biosensor and enzyme link immunoabsorbent assay (ELISA)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4.0  
      Literature review

4.1  
Aptamer

Aptamers
are a special class of nucleic acid molecules that are beginning to be
investigated for clinical use. These small RNA or DNA molecules can form
secondary and tertiary structures capable of specifically binding proteins or
other cellular targets; they are essentially a chemical equivalent of
antibodies. Aptamers have the advantage of being highly specific, relatively
small in size, and non-immunogenic (Ni et al. 2011).

As
biorecognition elements, aptamers have some advantages over antibodies in fabrication
of the biosensors. Firstly, aptamers are obtained by in vitro screening. Preparation and synthesis process of aptamer
takes at most two weeks to be done while preparation of monoclonal antibody is
at least six months to one year. Secondly, aptamers are chemically synthesized,
thus it can be modified flexibly with a variety of groups. Besides that, antibodies
are proteins that are easy to degenerate. The instability of antibodies limit
its application scope, but the aptamers have chemical stability in the pH range
of two to 12, and have a certain thermal refolding. The composition of aptamers
is simple, generally has a few dozen nucleotides and thus the design of the
aptamers is relatively simple (Wang et al. 2012). Due to the advantages of
aptamers, the use of aptamers to design biodetection for identification and
detection of pathogenic microorganisms is of extremely importance in the field
of analysis.

 

 

 

 

 

 

 

 

 

 

4.2  
DNA
Aptamers

DNA
and RNA aptamers are functionally similar but have some differences in their structure,
stability and accessibility. Compared with DNA aptamers, RNA aptamers are
chemically unstable because of the presence of a reactive hydroxyl group (–OH)
at the 21 position of the ribose sugar in RNA nucleotides (Zhu et al. 2015).
This shows that RNA aptamer have different structure compared to DNA aptamer.
Due to these attributes, DNA aptamers can be applied in a wide range of
technologies, such as target validation, drug discovery, affinity chromatography, in vivo imaging and therapy (Ogasawara
et al. 2007). Moreover, the cost of producing DNA aptamers is lower than that
for RNA aptamers. Based on the advantages of DNA aptamer compared to RNA
aptamer mentioned previously, it has been chosen as the probe for the
biodetection in this study.

 

4.3  
Aptamers
Structure and Functionality

The
ability of aptamers to bind to non-nucleic acid targets such as metal ions,
small molecules and large macromolecules such as proteins and viruses is due to
the secondary structures that oligonucleotides can adopt. These structures are
the hairpins, loops, quadruples structure (fig.1). Aptamers are able bind a
wide range of biological targets. Although aptamers can be isolated from pools
of random sequence oligonucleotides using af?nity based selection, aptamers
with high af?nities are not always obtained. Therefore, further re?nement of
aptamers is required to achieve desired binding af?nities (Hasegawa et al.
2016). Aptamer starts with a single strand of RNA or DNA. These strand will
then be fold creating the secondary structure.

The
functionality of an aptamer is based on their stable 3D structure which depends
on the primary sequence, the length of nucleic acid molecule and the
environmental condition (Tiwari and Tiwari. 2013). The 3D structure can be
categorized into three; i) hairpin loop, ii) quadruplexes and iii) kissing
complexes. Hairpin loops occur when two regions of the same strand are
complementary to one another and can form Watson-Crick base pairs. Quadruplexes
occur in guanine rich sequences, when four guanine bases can associate through hydrogen
bonding. While kissing complexes is formed when the unpaired nucleotides in one
hairpin loop base pair with the unpaired nucleotides in another hairpin loop.
This structure usually occurs in RNA.

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