Nano Probes for mRNA Detection than using in-situ Hybridization and hence the use of Nano Particles in Cancer Diagnosis and Therapy

 

Mohammed Rizwan B.1, Sucharitha P.1, Jaibiba P.2

1Final Year Department of Biotechnology, Sri Venkateswara College of Engineering, Sriperumbudur

2Assistant Professor, Department of Biotechnology, Sri Venkateswara College of Engineering, Sriperumbudur

*Corresponding Author E-mail:

 

 

ABSTRACT:

In situ hybridization (ISH) is an efficient method for detecting the localization of mRNA. It has a lot of disadvantages that it doesn’t provide information about the time dependent analysis also the information of post translational modification. They also suffer from serious disadvantage that they does not allow mRNA detection in living cells. Care should be taken in the extraction of the biomolecules from living cells that severe damage to the cells should be avoided.This review reveals the development of nano-structured oligonucleotide probes that has a predominant role in the detection of mRNA in the living cells. In particular, we describe methods that emphasize on nanotechnology in diagnosis and treatment for cancer.

 

KEYWORDS:

 

 

INTRODUCTION:

Cancer is a class of diseases characterized by uncontrolled proliferation of cells. There are various types of cancer, classified based on the cells which they affect. Cancer leads to the formation of lumps of cells which are a result of unchecked growth of the damaged cells forming tumors [1-8]. Tumors can grow and interfere with the any organ of the body and alter body function. Benign tumors are those which remain in its place origin. More dangerous, or malignant, tumors form when two things occur:

1.       Invasion: A cancerous cell manages to move throughout the body using the blood or lymph systems and destroys the healthy tissue.

2.       Angiogenesis: The process of formation of new blood vessels to vascularize and nurture the newly formed tumors.

The spread of a tumor to one part of the body to others through the vascular or lymphatic system is called Metastasis. A metastasized tumor is very difficult to treat. [1-8]

The standard characteristics of cancer cells are:

 

Ψ  Loss of regulation of mitotic rate

Ψ  Loss of specialization and differentiation of the cell

Ψ  Ability to move from the original site and establish new malignant growth at other tissue sites (metastasis)

Ψ  Capacity to invade and destroy normal tissue

        Abnormalities found in cancer cells include: 

Ψ  Translocation. Part of one chromosome has broken off and relocated itself onto another chromosome.

Ψ  Inversion. Part of a chromosome is in reverse order although it is still attached to the correct chromosome.

Ψ  Deletion. Part of a chromosome is missing.

Ψ  Duplication:  Part of a chromosome has been copied and the cell contains too many copies.[1-8]

 

IMPORTANCE OF mRNA IN CANCER DETECTION :

mRNAs are molecules that carries codes from the nucleus to places to protein synthesis in thecytoplasm. In cancer, mRNA serves the purpose of a bio-marker which plays an important role in cancer detection.  RNA in the blood serves as the blood-based markers for cancers[1-8].

 

IN-SITU HYBRIDISATION IN CANCER DIAGNOSIS

Mutations can occur in the genomes of all dividing cells as a result of disincorporation during DNA replication or through exposure to exogenous mutagens such as ionizing radiation or endogenous mutagens. Cancers result from clonal proliferations that arise from an accumulation of mutations and other heritable changes that confer selective growth advantages in susceptible cells. A central aim of cancer research has been to identify the mutated genes that are causally implicated in oncogenesis[8-9]. So far, abnormalities in about 350 genes (more than 1% of our genome) have been implicated in human cancers, but the true number is unknown. This illustrates striking features in the types of sequence alteration and protein domains that are encoded in the cancer classes in which oncogenic mutations have been identified.

 

IN SITU HYBRIDIZATION

In situ hybridization of mRNA in tissues or cell preparations is a powerful technique for studying gene expression. In situ hybridization (ISH) is a technique for localization and detection of specific nucleic acid sequences within tissues and cells. DNA and RNA sequences are visualized by hybridization with labeled probes that are complementary to the sequence of interest. Hybridization histochemistry is a related term that refers specifically to RNA ISH. When carrying out this technique, cells and tissue sections are typically fixed in 4% paraformaldehyde to preserve morphology for ISH. In some cases, tissues are permeabilized with proteinase K prior to hybridization to improve tissue penetration. Probes are relatively short, labelled nucleotide sequences that are complementary to the sequence of interest. They are prepared by various enzymatic procedures with a reaction mixture that includes labelled nucleotide analogs or radioactive nucleotides, or by direct synthesis as an oligonucleotide. Probes may carry radioactive or fluorescent labels for direct detection or hapten labels for detection by various indirect methods. Once the sample has been prepared, it is incubated with the probe at elevated temperature to allow the probe to hybridize to the sequence of interest. Unhybridized probe is washed away and the remaining labelled probe is detected[8-9].

 

 

Figure 1: In situ Hybridisation- The hynridisation with labeled probe complementary o the sequence of interests

 

DISADVANTAGES OF IN-SITU HYBRIDISATION:

Ψ  It does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection.

Ψ  Chemical fixation agents that are used for permeabilization, have effects on integrity of organelles such as mitochondria.

Ψ  The fixation of cells, by either cross-linking or denaturing agents, combined with the use of proteases in ISH may not provide an accurate description of intracellular mRNA localization[8-9].

Ψ  It is also difficult to obtain a dynamic picture of gene expression in cells using ISH methods.

Ψ  It does not provide information about post translational modification.

 

 

 

ESSENTIAL CHARACTERISTICS OF NANO PROBES

The detection of specific RNAs in living cells necessitates the probes to having a number of characteristics like increased specificity and sensitivity and a high signal-to-background ratio, especially for samples with a low concentration of genes and clinical samples where only a few diseased cells are present. Additionally, for detecting any genetic alterations, the probe should be capable of recognizing single nucleotide polymorphisms (SNPs).Cellular delivery of probes with high efficiency and low degradation probe is also necessary factor. The delivery of the probe into the system and its interaction with the target sequence must be easy to comprehend[11-15].

 

REQUIREMENTS OF A PROBE: TO REFLECT THE mRNA EXPRESSION

Tagging and tracking of mRNA can be done using fluorescently labeled oligonucleotide probes. For these probes to represent the mRNA expression, they should be capable of differentiating between true and false signals, they should  identify the signal from background and must be able to carry out the conversion of its recognition into a signal that can be measured. These probes should also express fast kinetics to track all the real time gene alterations.

 

MOLECULAR BEACONS

Molecular beacons are single-stranded, stem and loop structured oligonucleotide hybridization probes[11-15].The loop contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence. One end of the arm is covalently linked to a fluorophore and the other end to a quencher. Molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce brightly.

 

 

Figure 2 Molecular Beacon action mechanism: the effect of molecular beacons over DNA or RNA target that forms a hybrid

 

In the absence of targets, the probe is dark, because the stem places the fluorophore so close to the non-fluorescent quencher that they transiently share electrons, eliminating the ability of the fluorophore to fluoresce. When the probe encounters a target molecule, it forms a probe-target hybrid that is longer and more stable than the stem hybrid. The rigidity and length of the probe-target hybrid precludes the simultaneous existence of the stem hybrid. Consequently, the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem hybrid to dissociate and the fluorophore and the quencher to move away from each other, restoring fluorescence.

 

Molecular beacons are very highly specific. They are used for the ease of discrimination of target sequences which are different from one another by a single nucleotide substitution. The hybrid formed by these structures with the target is much stronger than the stem hybrid, hence the binding of a molecular beacon to a target sequence remains intact. In the case of a mutation or any discrepancy in the target sequence, the stem hybrid is much stronger than the probe and the target sequence hybrid, thus not exhibiting any signal that demonstrates the result.

 

Molecular beacons have been considered highly sensitive and apt for detection of genes n living cells. Conventionally molecular beacons are structured with a quencher and a fluorophore pair, a recent development though explains models of these nanostructured probes with shifting molecular beacons which can fluoresce in different colors. These wavelength shifting probes much brighter than conventional probes.

 

Though the high specificity can be regarded as a merit, it suffers a major challenge that is molecular beacons can be easily broken down by the cytoplasmic nucleases or the probe might  interact non-specifically with proteins and give rise to a number of false positive signals. To overcome this drawback, probes with a donor and receptor probes are used (Dual FRET molecular beacons).

FRET is extremely sensitive to the distance between the donor and the recipient, hence probes must be bound to the same RNA molecule for a plausible positive signal of flurorescene.

 

DESIGN:

The major design parameters to be considered are sequence of the probe, the hairpin structure and the selection of quencher- fluorophore pair[21-29]. The sequence of the probe is selected to be highly specific and having a favorable melting temperature and the flurophore- quencher pair plays the important role of providing a high signal to background ratio. The stem, loop and the hairpin length are critical parameters as they control the fraction of the target bound to the structure at any given temperature. Thermodynamic and kinetic studies performed, an increased stem length, decreases the hybridization rate as it proves difficult for the hairpin loop to open. Consequently, a longer probe length will lower dissociation and specificity. Accessibility of the target is significant due to formation of ribonulceotide proteins and also RNA binding proteins[18-25].

 

Molecular beacons labeled with two fluorophores (donor and acceptor) are used for dual FRET.  The energy transfer occurs due to long range dipole- dipole interactions between donor and acceptor molecules. The acceptor absorbs at a larger wavelength than the donor. The energy transfer depends on the overlap of the emission and absorption spectra of the donor and acceptor respectively and the orientation of the donor and acceptor dipoles[18-25]. To have an increased signal to background ratio, care should be taken to avoid the direct excitation of the acceptor probe at donor excitation wavelength and to avoid donor emission at acceptor emission wavelength. Examples of FRET dye pairs include Cy3 (donor) and Cy5 (acceptor), TMR(donor) and Texas Red (acceptor), and fluorescein (FAM)(donor) and Cy3 (acceptor). Some of the quencher molecules used Organic quencher molecules such as dabcyl, BHQ2 (blackhole quencher II) (Biosearch Tech), BHQ3 (Biosearch Tech) , and Iowa Black (IDT).

 

Cellular Delivery

The ability to deliver the probes into cells for measuring the intracellular levels of RNA is an aspect to be considered. Plasma membrane proves to be barrier for transport due to its lipophilic nature which does not allow charged particles to be transported inside the cell. Hence, the hairpin oligonucleotide probe which is anpolyanionic molecule, cannot be easily traversed across this barrier. After internalization of the probes, the efficiency of this method should not be based on how many probes that internalize but on the number of probes that remain functional inside the cell. The present delivery techniques include two classifications: endocyticand non- endocytic methods. Endocytic methods include the use of liposomes and dendridimers[25-29]. The process of probe delivery takes about 2-4 hours. It has been found that mostly all the probes internalized through this method get encapsulated in endosomes, commonly is lysosomes and only 0.01 to 10% of the probes remain active after getting released from them. Non-endocytic method includes microinjection of the probe which increases accumulation in the nucleus preventing any unnecessary binding to the mRNA in the cytoplasm. However microinjection is inefficient in transferring the probe to a large cell population. Another non-endocytic delivery method is cell membrane permeabilization using toxins. For example, streptolysin, bacterial toxin that forms pores was used to introduce probes into eukaryotic cells. Its mechanism of action includes binding to cholesterol and oligomerizeinto structures that form pores. The protocol for this procedure varies for different cell types, hence needs to be optimized by varying the temperature. An advantage of this technique is that, it is reversible.  Another method of introducing biomolecules into the cells is by using cell penetrating peptides. This method proves fairly successful with very less occurance of endocytosis.

 

RNA Detection:

A carefully engineered design, access of the probe to the target and the interaction of the probe to the target sequence are the important factors for successful RNA detection in living cells. To emphasize the sensitivity of molecular beacons in mRNA design a dual FRET experiment was designed to detect K-ras and survuvinm RNAsin HDF and MIAPaCa-2 cells[21-29]. Each of the FRET pair consisted of a donor fluorophore and an acceptor fluorophore. These labeled probes were made to hybridize closer to an mRNA target, keeping the fluorophores closer (~6nm) when the actual hybridization of the probe and the target takes place. The emission of the donor at a wavelength that is a characteristic of the acceptor shows a positive result. A negative control beacon was also designed which is nothing but a random beacon whose target sequence was selected using a random walk. After delivering the FRET probes into the cells by various permeabilization techniques and incubating the cells for an hour, the resulting flurescence signal can be imaged using FRET optics[21-29].

 

ISSUES AND CHALLENGES:

Nanostructured probes such as the above mentioned find a significant place in research involving very specific and sensitive detection. Molecular beacons are increasingly used for single nucleotide detection in invitro studies, micro arrays, detection of proteins, detection of double stranded DNA and the most significant one of all is the living cell gene detection with FRET which is a powerful laboratory tool the provides a base to study gene expression in vivo.  Though quite beneficial detection RNA using molecular beacons poses a lot of challenges in designing the probe, its accessibility to target, problem of self-quenching, intracellular environmental factors, the effect of probes on normal probe function . [25-29].This being a very challenging field opens up a wide array of fields to work on and develop methods to identify the gene expression in vivo which could possibly help in diagnosis of various diseases including cancer.

 

CANCER AND NANOTECHNOLOGY:

Cancer nanotechnology is emerging as a new field of interdisciplinary research, cutting across the disciplines of biology, chemistry, engineering, and medicine providing major advances in diagnosis, treatment and therapy. Nanotechnology has recently evolved from various fields to find its stable and solid place in the field of medicine[21-29]. There are a lot of avenues like drug delivery, which includes the use of carbon nanotubes, gold Nano-rods which are used for the delivery of the chemotherapy drugs to the site of tumor, nanoparticles are also developed as antivirals, another new development is the Nano-sponges that are coated with erythrocytes these when injected in the blood stream attract toxins in the blood. Coming back to cancer, bismuth Nano-particles are used to concentrate radiation in radiation therapy for tumor therapy. Recently developed magnetic Nano-particles are in the prototype stages for development of a full-fledged therapy of cancer using Nano-particles.

 

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Received on 20.08.2013          Accepted on 15.09.2013        

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Asian J. Pharm. Tech.  2013; Vol. 3: Issue 4, Pg   213-217