The invention relates to field of biotechnology particularly to random aptamers conjugated to a silver nanoparticles.

BACKGROUND OF THE INVENTION

Metal salts and metal nanoparticles are effective antimicrobial agent that can inhibit the growth of many bacteria and other microorganism. Among a conjugation of metal salt and nanoparticle Silver and Ag NPs is well-documented and widely used nanoparticle for their antimicrobial properties. In particular silver salts the antimicrobial activity of the silver cations have been attributed to pore formation and puncturing of the bacterial cell wall by reacting with the peptidoglycan component present in both gram positive and gram negative bacteria. Silver ions also enters into the bacterial cell and are known to inhibit cellular respiration and disruption of the metabolic pathways in the bacterial cell. Silver cations also disrupt DNA and its replication cycle. The nanoparticle are less than 1000nm in size enabling it to enter the cells. The silver nanoparticles owing to their size and readily diffuse across the cell wall/ cell membrane and rupture the cell wall. AgNPs produce reactive oxygen species and free radicals which cause apoptosis leading to cell death preventing their replication. A number of factors are involved in the antimicrobial property of AgNPs such as size, concentration, pH of the medium and exposure time to pathogens.

Aptamers can be broadly defined as synthetic oligonucleotides or short sequence single nucleotide sequences that can specifically and non-covalently bind to the target sequence. These short sequence single nucleotide sequences can fold into three dimensional shapes complex shapes presents scaffolds for molecular interactions and support complex formation with protein and small-molecule target. Aptamer can bind a wide range target sequence such as surface protein of microorganism, whole micro-organisms. metal ions, chemical compounds, proteins, and cells. Both Deoxyribose nucleotide acid (DNA) or Ribose nucleotide acid (RNA) can be used as aptamers, however the single stranded (ss)DNA due its ability to form stable intricate tertiary structure are preferred as aptamers for use in various industries. The reasons for preferential use of DNA as aptamers are because DNA are more chemically and biologically stable molecule, DNA-based aptamer is more cost and time-effective, as it does not involve an extra reverse transcription step which is required for RNA-based aptamers; and also DNA is easier to synthesize and have a longer shelf life when as compared to the RNA-based aptamers. The aptamers are generated using an in vitro process known as the Systematic Evolution of Ligands by EXponential enrichment, (SELEX) first described by Tuerk and Gold (1990). SELEX is carried out in three main steps: selection, partitioning, and amplification. In SELEX an oligonucleotide library consisting of ~1016 different sequences is synthesized typically consisting of oligonucleotide of about 20-100 sequences in length. Each nucleotide is flanked by constant sequences. These constant sequences are fixed primer-binding sequences on both ends to enable amplification of the enriched sequences via PCR. The library is then incubated with the target. Out of the diverse oligonucleotide pool, some sequences will bind to the target and others will not. Sequences that have bound to the target are then separated and are copied and amplified using polymerase chain reaction. The selected sequences are then reintroduced into the process several times before being sequenced via either high throughput or low throughput sequencing. Despite of the many advantages of the technique such as chemical production by readily scalable process, non-immunogenicity, smaller size enables efficient entry into biological compartments, target specific and can be synthesizedagainst cell-surface target, reversible denaturation, conjugation chemistriesfor the attachment of dyes or functional groups can be readily introduced during synthesis, there are still very few aptamer based-drugs are in the pipeline and the only FDA approved, drug is Macugen (Pegaptanib) by Bausch and Lomb.

One of the reason for this is the labour intensive, tedious and time-consuming process of SELEX. In the ideal scenario the aptamer sequences binds to their targets through tertiary structures which is by the nucleotides from the random region of the aptamer and should be independent of the flanking primer sequences present at both ends of the aptamer. However, during the SELEX procedure, the interaction between the constant primer binding sites and sequences participate in the functional secondary structures of developed aptamers. Taking into account the significant number of primer binding sites in a SELEX library, these interactions critically influence which aptamers are selected from the random pool.

One of the alternatives to this would be delete the primer-binding site or to use a minimal-primer libraries, but in considering this the costs and time by introducing restriction and ligation enzymes has to be considered, the ligation step can also result in a significant loss of potential binder and would involve an additional steps associated with purification and separation; thus the strategy involves additional challenges before it can be implemented.

However certain challenges in the field are focus on the known aptamers rather than expending the e?orts and energies in isolating new aptamers for novel design strategies, the labour intensive and repetitious the process of isolating new aptamers, the high costs associated with SELEX, the limited abilities of the polymerases to accommodate for modi?ed and unnatural nucleic acids and short half-life in vivo due to nuclease degradation and renal clearance are faced by the researchers in the field. In addition, the selection method of aptamer is labour intensive and time consuming. There is still a need to develop selection methods that are more high-throughput and to ensure that selected aptamers have high affinities and should be, resistant to degradation and clearance.

Random aptamers are pool of short oligonucleotides with random shapes which is equivalent to the random library that’s been used for selection of aptamers against a given target in the regular SELEX process. The random aptamers possess binding ability against a wide range of targets due to the variety of shapes.

A silver nanoparticle tethered to a random aptamer can therefore be used for enhancing the antimicrobial property of the silver nanoparticle and the advantages of aptamers such as low immunogenicity, rapid tissue penetration, low toxicity and high affinity to bind with the target can be combined together to achieve an effective antimicrobial particle that can have wide implications. A number of studies have already been published in the prior art where the aptamers have been conjugated to a silver nanoparticle for applications in therapeutics. In general the conjugate has more applications which is just not limited to therapeutics and diagnostics but other biomedical applications such as devices and biosensing etc.

Some of the relevant prior arts are presented and discussed herein

CN201610506537A published on 7th December, 2016 disclose an electrochemical aptamer sensor for rapid detection of chloramphenicol. The electrochemical aptamer sensor is assembled by fixing an aptamer and silver nanoparticles on a nano-composite onto a glassy carbon electrode through a silver-sulfur bond. Chloramphenicol in a sample is quantitatively captured onto the surface of the sensor, and under the catalytic action of the nano-composite, an electrical signal is generated. In particular the invention discloses a chloramphenicol aptamer characterized in that the aptamer comprises an ACT TCA GTG AGT TGT CCC ACGGTC GGC GAG TCG GTG GTA G base sequence, and the 5' end is modified with 5'-SH-(CH2) 6. The patent however does not discloses the method or source of obtaining the said aptamer.

CN202010716473A published on 16th October, 2020 discloses a preparation method of a shell isolation nano particle and aptamer modified fluorescent probe for detecting tetracycline residues as well as a prepared fluorescent probe and application thereof. The preparation method comprises the following steps: preparation of silver nano particles, preparation of silicon-coated silver nano particles, preparation of amino-functionalized silver nano particles, preparation of cadmium telluride quantum dots, and synthesis of the aptamer-modified fluorescent probe. The aptamer used in the invention (5'-NH2-(CH2)-CGTACGGAATTCGCTAGCCCCCCGGCAGGCCACGGCTTGGGTTGGTCCCACTGCGCGTGGATCCGAGCTCCACGTG-3') has been disclosed in the detailed description and was purchased from GenScript Biotechnology Co., Ltd. This obviously add cost to the device and the method described in the invention and may be an additional hindrance in commercialization of the product/ method.

US201715771251A published on 31st March, 2020 discloses an electrochemical biosensor based on an aptamer/nano silver probe and an EXO I enzyme. Nano silver nanoparticles having the functions of identifying a target object and generating electrochemical signals and modified by an aptamer are used as a biological probe for detecting target biomolecules, under the initiation of the target object and the assistance of a complementary probe and the EXO I enzyme cyclic shear amplification, and by means of the DNA complementary pairing principle, the probe can be gathered on the surface of a gold electrode. The lysozyme aptamer 5'-ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3', and the ? interferon aptamer is: 5'-GGG GTT GGT TGT GTT GGG TGT GTC CAA CCC C-3' were used in the invention and purchased from Shanghai Bioengineering Co., Ltd.

However such a conjugate still suffers from all the problem associated with the aptamer generation by the SELEX process discussed above. There is therefore a strong need for aptamers nanoparticle conjugate which are antimicrobial in nature and binds the target (such as bacteria, fungi, viruses among other microorganism) efficiently and is devoid of the complicated process producing the same.

OBJECT OF THE INVENTION

Therefore keeping in mind the above the object of the present invention is to develop aptamer-nanoparticle conjugate that is antimicrobial.

Another object of the present invention is to developaptamer-nanoparticle conjugate that is easy to manufacture, easily scalable for industrial production, is cost-effective.
A still further object of the present invention is to develop application of the antimicrobial aptamer-silver nanoparticle conjugate.

SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts in a simplified format that is further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.

Accordingly the present invention discloses a silver-nanoparticle conjugated to an aptamer wherein the aptamer is an random oligonucleotide (AgNPs-R-Apt). The silver nanoparticle of the present invention is conjugated to a random oligonucleotide that have not been optimized to identify the target sequences of the microorganism, instead a random pool of the aptamers are used.

The present invention also discloses a method of preparation silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) comprising;obtaining the silver-nanoparticle (AgNPs) by mixing silver nitrate in a suitable reducing buffer;preparing a solution of random aptamers (R-Apt) in phosphate buffer saline;adding random aptamers (R-Apt) to silver-nanoparticle (AgNPs) to obtain silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt).

BRIEF DESCRIPTION OF FIGURES:
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:

Figure 1 depicts the method of obtaining silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) of the present invention in one embodiment;

Figure 2 depicts the Kirby Bauer method to assess the antimicrobial activity of silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) of the present invention against Acenatobacter bominii;

Figure 3 depicts the Kirby Bauer method to assess the antimicrobial activity of silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) of the present invention against Klebsiella pneumoniae; and

Figure4depicts the Kirby Bauer method to assess the antimicrobial activity of silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) of the present invention against Pseudomonas aeruginosa.

Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
DETAILED DESCRIPTION:

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method.

Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

Random aptamer- single-stranded oligonucleotides of 20-100 nucleotides in that fold into defined architectures/shapes and are not targeted against specific target.

Generation of random aptamer – aptamer generated by synthetic oligonucleotide without use of specific primers.

The present invention discloses a silver-nanoparticle conjugated to an aptamer wherein the aptamer is an random oligonucleotide(AgNPs-R-Apt). Aptamers are usually selected against a pathogen using purified protein which is the target protein. Random pool of oligomers by following the process of SELEX (Systematic evolution of ligands through hexponential enrichment) are selected against a particular antigen. In the present invention no selection steps are being followed instead random pool of oligomers may be used. The random aptamers forms a myriad of three dimensional scaffolds structures with very high probability of binding the target protein. These target proteins are usually the surface protein of the microorganism. The random aptamers also can target a large number of thus bringing the broad spectrum nature. Binding with random aptamers with the given target present such as a surface protein of a bacteria or any pathogen results due to induced fit or structure compatibility or electrostatic interactions orhydrogen bridges between the silver nanoparticle aptamer and the target molecule.

In another embodiment the present invention discloses a silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt)antimicrobial in nature. In still another embodiment the said particles may be directed against receptors of microbes including viruses, bacteria, fungi, parasites and cells. In yet another embodiment the silver-nanoparticle random aptamer conjugate binds to the target surface protein by induced fit, structure compatibility, electrostatic interactions and/ or hydrogen bridges.

In an embodiment the present application discloses a method of preparation silver nanoparticle random aptamer conjugate (AgNPs-R-Apt) comprising;obtaining the silver-nanoparticle (AgNPs) by mixing silver nitrate in a suitable reducing buffer;preparing a solution of random aptamers (R-Apt) in phosphate buffer saline;adding random aptamers (R-Apt) to silver-nanoparticle (AgNPs) to obtain silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt). In yet another embodiment the reducing buffer may besodium borohydride at pH of 2-9 and the AgNPs-R-Apt particles may be stabilized by addition of reducing agents such as TCEP (tris(2-carboxyethyl)phosphine),dithiothreitol and ß-mercaptoethanol)preferably TCEP (tris(2-carboxyethyl)phosphine).

In a one embodiment the concentration of the silver-nanoparticle (AgNPs) may in the range of 10µg/ml to 100 µg/ml and concentration of the random aptamers (R-Apt) is in the range of 50µMto 200 µM. The ratio of the silver-nanoparticle (AgNPs) to random aptamers (R-Apt) may be in the range of 10000: 10 – 500:1 preferably 5000:1. In another embodiment silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) may have a particle size of 10nm to 500nm.

In another embodiment the present application discloses an air-filter assembly comprising ofa removable filter which can fit into an air purifier device wherein the silver-nanoparticler andom aptamer conjugate (AgNPs-R-Apt) are sprayed on to the surface of the removable filter to obtain an antimicrobial filter. In yet another embodiment removable filter may be selected from a group of UV light aur filters, carbon air filters, HEPA filters preferably HEPA Filters.

In one embodiment of the present invention the air-filter assembly may reduce up to 90% of microorganism in the air after as measured by the MERV scale.

The random oligomers as reducing and stabilizing agents in the biogenic synthesis of silver nanoparticles is attractive model because of a number of advantages such as easy scaling up in an industrial set up to produce of large quantities of such aptamers, circumventing the step of selection of the specific aptamers which are time-consuming, labour-intensive and requires huge investment. The process results in high yields, easy handling and results in residues that are low in toxicity. Furthermore the synthesis process coats the nanoparticles with diverse structures derived from the random oligomers which can improve stability and confers antibacterial activity.

These nanoparticles adhere to the cell walls and membranes of the microorganism and may reach the interiors of the cell. The nanoparticles once inside damage the cellular structure and induce the production of the reactive oxygen species and alter the mechanism of signal transduction. The process leads to killing of the microorganism and reduces the load of micro-organism in an environment. This technique can therefore be effectively applied to a number of industry. Air-filters sprayed with such formulation can efficiently reduce the load of microorganisms in the environment such as offices, and industry having high microbial load such as hospitals, food industry etc.

EXAMPLE
The following examples are for illustration purposes and are not to be construed as limiting the invention disclosed in this document to only the embodiments disclosed in these examples.

Example 1
Method of Obtaining the Silver-nanoparticle- Aptamer
Silver nanoparticles was obtained by standard method. Silver Nitrate was added drop wise to an aqueous solution of chilled sodium borohydride in presence of citrate for stabilization. The mixture was stirred on a magnetic stirrer continuously. The solution turned light yellow following addition of silver nitrate. The colloid thus obtained was continuously stirred and allowed to warm to room temperature. The solution was stabilized by addition of polyvinylpyrrolidone (PVP).

The random oligonucleotide was chemically synthesized and the generated random nucleotide was conjugated to the silver nanoparticles. Briefly, Random aptamers were suspended in 60 mM phosphate buffer (pH 8.5) and treated with the reducing agent 20mM of tris (2-carboxyethyl) phosphine (TCEP) at room temperature. The pre-treatment of TCEP allowed the enables interaction of aptamers to the AgNPs by the reducing disulfide bonds of AgNPs.

The pre-treated solution of random aptamers were purified using a desalting size-exclusion column chromatography. The obtained random aptamers were resuspended in 1 mL of PBS (0.1 M with 10 mM MgCl2, pH 7.4) and submerged in a water bath in the presence of 10 mM MgCl2 for5 minutes. This step enables aptamers to fold into their appropriate tertiary structures.

The random aptamers were added to 1 mL of silver colloid at a molar ratio of 5000:1 and vigorously agitated. Citrate HCl buffer (pH 2.9, 250 mM) was added to the random aptamer-conjugated AgNP solution and mixed well for 30 mins. The silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) were washed three times to remove any unconjugated aptamer and centrifuged using PBS supplemented with MgCl2. The silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) was stored in dark until further use.

Example 2
Antimicrobial Activity of the silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt)

Bacterial culture of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acenatobacter bominii were grown overnight (16-18 hours) or to stationary phase (generally ~6 hours of growth) in 5 ml broth medium at 35-37°C. A stock solution of silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) was made in phosphate buffer saline (pH 7.4) and PBS was used as control. Small discs of What man filter paper were sterilized. On to the sterilized filter paper disc 20 µl of solution AgNPs-R-Ap in phosphate buffer saline was loaded on to each disk (5µg/20 µl). The control was also loaded on to the filter paper discs. The petri dish was prepared by adding suitable growth medium. 150 µl of the culture was added on to the petri dish and spread evenly throughout the plate. The AgNPs-R-Ap loaded disks and the control disks once fully dried, was gently placed the on top of the agar and lightly press it down with the flame-sterilize a pair of tweezers. The plates were incubated at suitable temperature in inverted position overnight or until cells have grown out completely. The next day zone of inhibition of the bacterial growth was measured with the help of a ruler. Figure 2-4 presents the zone of inhibition obtained against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acenatobacter bominii . As is evident a clear zone of inhibition visible against all three bacteria, demonstrating that the AgNPs-R-Ap of the present invention has the antimicrobial activity.

While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein. The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.

We claim:

1. A silver-nanoparticle conjugated to an aptamer wherein the aptamer is an random oligonucleotide(AgNPs-R-Apt).

2. The silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) as claimed in claim 1,wherein the silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) is antimicrobial in nature.

3. The silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) as claimed in claim 1,wherein the said particles is directed against receptors of microbes (viruses, bacteria, fungi, parasites and cells)

4. The silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) as claimed in claim1, wherein the silver-nanoparticle random aptamer conjugate binds to the target surface protein by induced fit, structure compatibility, electrostatic interactions and/ or hydrogen bridges.

5. A method of preparation silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) comprising;
a. obtaining the silver-nanoparticle (AgNPs) by mixing silver nitrate in a suitable reducing buffer;
b. preparing a solution of random aptamers (R-Apt) in phosphate buffer saline;
c. adding random aptamers (R-Apt) to silver-nanoparticle (AgNPs) to obtain silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt).

6. The method as claimed in claim 4, step(a) wherein the reducing buffer is sodium borohydride at pH of 2-6 and is stabilized by addition of polyvinylpyrrolidone (PVP).

7. The method as claimed in claim 4, wherein the concentration of the silver-nanoparticle (AgNPs) is in the range of 10 µg/ml to 100 µg/ml and concentration of the random aptamers (R-Apt) is in the range of 50µM to 200µM

8. The method as claimed in claim 4, wherein the ratio of the silver-nanoparticle (AgNPs) to random aptamers (R-Apt) is in the range of 10000: 10 – 500:1 preferably 5000:1.

9. The method as claimed in claim 4, wherein the silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) has a diameter of 50nm to 500nm.

10. An air-filter assembly comprising of a removable filter which can fit into an air purifier device wherein the silver-nanoparticle random aptamer conjugate (AgNPs-R-Apt) are sprayed on to the surface of the removable filter to obtain an antimicrobial filter.

11. The air-filter assembly as claimed in claim 8, wherein the removable filter is selected from a group of UV light aur filters, carbon air filters, HEPA filters preferably HEPA filters.

12. The air filter ad claimed 8, wherein the air filter reduces up to 90% of microorganism in the air after as measured by the MERV scale.