DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003

COMPLETE SPECIFICATION
(See Section 10 and Rule 13)

Title of invention:
METHOD FOR CIRCULAR PERMUTATION OF SINGLE STRANDED POLYNUCLEOTIDES

Applicant:
Tata Consultancy Services Limited
A company Incorporated in India under the Companies Act, 1956
Having address:
Nirmal Building, 9th Floor,
Nariman Point, Mumbai 400021,
Maharashtra, India

The following specification particularly describes the invention and the manner in which it is to be performed.
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY

[001] The present application claims priority from Indian provisional patent application no. 202021002718, filed on January 21, 2020. The entire contents of the aforementioned application are incorporated herein by reference.

TECHNICAL FIELD
[002] The disclosure herein generally relates to the field of molecular biology, and, more particular, to a method for circular permutation of single stranded polynucleotides.

BACKGROUND
[003] Circular permutation represents a form of macromolecular isomerization when normal termini are covalently linked and new termini are introduced by breaking the backbone at a different location. Generally circular permutations are used for studying structure and dynamics of polynucleotides like DNA (Deoxyribose Nucleic Acid) and RNA (Ribose Nucleic Acid).
[004] Conventional methods mainly focus on circular permutation of double stranded polynucleotides and less focus is given on circular permutation of single stranded polynucleotides due to instability of the polynucleotides and lack of viable methods. Due to the instability of single stranded polynucleotides, generating all possible permutations of n number of polynucleic acid fragment using circular permutation is a challenging task. Further, conventional methods fail to generate all possible permutations of the single stranded polynucleotides when the number of fragments increase exponentially. Hence synthesizing all possible permutations of single stranded polynucleotides is a challenge both experimentally and economically.

SUMMARY

[005] Embodiments of the present disclosure present technological improvements as solutions to one or more of the above-mentioned technical problems recognized by the inventors in conventional systems. For example, in one embodiment, a method for circular permutation of single stranded polynucleotides is provided. The method includes generating a circularized single stranded polynucleotide template by circularizing a linear single stranded polynucleotide based on a thermostable ligase, wherein the circularized single stranded polynucleotide template includes a plurality of polynucleotide components stitched together with a plurality of common polynucleotide sequences in between each of the plurality of polynucleotide components, wherein the plurality of common polynucleotide sequences are uniformly distributed. The method further includes amplifying the circularized single stranded polynucleotide template to obtain a linear single stranded polynucleotide fragment using an amplification technique, wherein a plurality of primers are designed to anchor to a starting point of the plurality of common polynucleotide sequences during amplification. Finally, the method includes obtaining a plurality of permutated fragments of interest by gel purifying the linear single stranded polynucleotide fragment.
[006] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[007] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate exemplary embodiments and, together with the description, serve to explain the disclosed principles:
[008] FIG. 1 illustrates a flow diagram of a method for circular permutation of single stranded polynucleotides, according to some embodiments of the present disclosure.
[009] FIG. 2 is an example diagram illustrating the method for circular permutation of single stranded polynucleotides, in accordance with some embodiments of the present disclosure.

DETAILED DESCRIPTION OF EMBODIMENTS

[010] Exemplary embodiments are described with reference to the accompanying drawings. In the figures, the left-most digit(s) of a reference number identifies the figure in which the reference number first appears. Wherever convenient, the same reference numbers are used throughout the drawings to refer to the same or like parts. While examples and features of disclosed principles are described herein, modifications, adaptations, and other implementations are possible without departing from the scope of the disclosed embodiments. It is intended that the following detailed description be considered as exemplary only, with the true scope being indicated by the following embodiments described herein.
[011] Embodiments herein provide a method for circular permutation of single stranded polynucleotides, for example, Deoxyribose Nucleic Acid (DNA) and Ribose Nucleic Acid (RNA). Initially, a circularized single stranded polynucleotides template is generated by circularizing a linear single stranded polynucleotide based on a thermostable ligase. Further, the circularized single stranded polynucleotide are amplified using Rolling Circle Amplification (RCA) to obtain a linear single stranded polynucleotide fragment. Here a plurality of primers are designed to anchor to a starting point of the plurality of a plurality of common polynucleotide sequences associated with the circular template during RCA. Further, a plurality of permuted fragments of interest are obtained by gel purification and the plurality of gel purified permuted components are deciphered using polynucleotide sequencing.
[012] The plurality of permuted fragments of polynucleotides can be used as single stranded probe to detect and bind complementarily to the single stranded polynucleotides of a virus/bacteria and help in identification of strain in a given sample.
[013] In an embodiment, single stranded polynucleotides are considered as elemental material with an intriguing application potential in the field of DNA/RNA nanotechnology. For example, single stranded polynucleotides provided a feasible option for creating the desired sequence design and for using in experiment validation The method for circular permutation of single stranded polynucleotides aims in designing of the RCA template (linear single stranded polynucleotide fragment) and utilize the multiple priming sites of the RCA template for generating all possible permutations. Priming site is a region of a nucleotide sequence wherein a single stranded RNA or DNA primer binds to start replication. A primer is a short nucleic acid sequence that provides a starting point for DNA or RNA synthesis. A primer suitable for the amplification of all possible permutation of sequences is designed and synthesized before performing the RCA.
[014] The method for circular permutation of single stranded DNA and RNA is described further in detail with reference to FIGS. 1 and 2. FIG. 1 illustrates a flow diagram for the method for circular permutation of single stranded DNA and RNA, in accordance with some embodiments of the present disclosure. Now referring to FIG. 1 the method includes a circular template designing procedure 102, a Rolling Circle Amplification (RCA) 104, a purification procedure 106 and a sequencing procedure 108.
[015] In an embodiment, the template designing procedure 102 of FIG. 1 generates a circularized single stranded polynucleotide template by circularizing a linear single stranded polynucleotide based on a thermostable ligase. The circularized single stranded polynucleotide template includes a plurality of polynucleotide components stitched together with a plurality of common polynucleotide sequences in between each of the plurality of polynucleotide components. The plurality of common polynucleotide sequences are uniformly distributed.
[016] Initially, a linear ssDNA template (ssDNA sequence with 5' Phosphate and 3' hydroxyl (OH) groups) is circularized using the thermostable ligase that catalyzes an intramolecular ligation like circularization. For example, the thermostable ligase utilized in circularization is CircLigase™. The circularization is followed by an enzyme inactivation. Further, a plurality of un-circularized templates are removed from circularized mixture by incubating the circularized mixture with exonuclease I and exonuclease III.
[017] In an embodiment, the circularization is con?rmed by Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis is used in biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. The ssDNA/ssRNA template once circularized can generate all possible permutations of fragments of interest.
[018] In an embodiment, the RCA procedure 104 of the FIG. 1, amplifies the circularized single stranded polynucleotide template to obtain a linear single stranded polynucleotide fragment (RCA template) using an amplification technique. The plurality of primers are designed to anchor to a starting point of the plurality of common polynucleotide sequences during amplification. For example, the plurality of primers designed to anchor to the start of the designed common sequences can be 12-18 nucleotides long DNA/RNA. The plurality of primers amplify the whole circular template starting and ending from the anchoring site.
[019] In an embodiment, the RCA amplification technique is used for amplification. RCA is a process of unidirectional nucleic acid replication that can rapidly synthesize multiple copies of circular molecules of polynucleotides. Further, RCA is an isothermal enzymatic process where a short DNA or RNA primer amplifies a template of interest to form a long single stranded DNA or RNA.
[020] In an embodiment, the purification procedure 106 of the FIG. 1, obtains a plurality of permutated fragments of interest by gel purifying the linear single stranded polynucleotide fragment obtained from RCA. Here, the linear ssDNA/ssRNA fragments are gel purified in order to screen for the ideal permutations. Here, all the ssDNA/ssRNA fragments are allowed to pass through gel electrophoresis in order to isolate fragments of interest.
[021] In an embodiment, a Polymerase Chain Reaction (PCR) amplification using a single specific primer pair (Reverse primer of common primer that is used for RCA. The RCA provides double stranded polynucleotides.
Table I
Component Volume per 50 µl RXN Final Concentration
NEBNext High Fidelity 2x PCR Master Mix 25 µl 1x
10 µM Forward Primer 2.5 µl 0.5 µM
10 µM Reverse Primer 2.5 µl 0.5 µM
Template DNA* Variable Variable
Nuclease-free water To 50 µl

Table II
Step Temp Time Cycles
Initial Denaturation 98 degree Celsius 30 seconds 1
Denaturation 98 degree Celsius 5-10 seconds 25-35
Annealing 50-72 degree Celsius 10-30 seconds
Extension 72 degree Celsius 20-30 seconds/kb
Final Extension 72 degree Celsius 2 minutes 1
Hold 4-10 degree Celsius Infinity 1

[022] The PCR is a method widely used to rapidly make millions to billions of copies of a specific DNA sample. This is followed by enrichment of dsDNA using specific primer pairs. After this size selection is performed using gel purification to isolate the fragments of interest. PCR is carried using master mix kits like NEBNext® High-Fidelity 2X PCR Master Mix. Here, the reaction mixture is gently mixed and the liquid at the bottom of a tube is obtained by a quick spin if necessary. Further, the mixture is transferred to PCR tubes of a PCR machine for thermocycling. PCR products are purified using QIAquick® Gel Extraction Kit. Table I illustrates reagents used foo PCR and Table II illustrates the thermocycling conditions during amplification.
[023] The sequencing procedure 108 of the FIG. 1 deciphers the purified fragments. Here, the purified/isolated fragments from gel electrophoresis are checked for the nucleic acid sequence using DNA sequencing. In an embodiment, the sequencing procedure is explained below: The sequencing involves two steps
Step 1: Fragment Analysis using analyzers for validating size (bp): This step is performed using Fragment Analyzers like Agilent High Sensitivity DNA ScreenTape Analysis using Agilent 4200 TapeStation instrument.
Step 2: Sanger/Next Generation Sequencing (NGS) Sequencing to validate the DNA sequence: Samples are enriched using specific target primers designed against specific DNA fragments. For Sanger sequencing reactions for ABI capillary-based methods are used. For NGS method, libraries are prepared using Illumina amplicon chemistry. The quality and quantity of libraries are checked using Agilent TapeStation (see protocol above). Selected libraries are sequenced using MiSeq sequencer using 2 x 75 bp and 2 x 100 bp sequencing mode.
[024] The present disclosure is explained with the help of the following example. Initially, a circular template is created by stitching together 4 components, A,B,C,D, by keeping a common sequence in between consecutive components. Now to generate the permutations involving the 4 components (A,B,C,D), RCA is set targeting the primers to the common sequence regions. Multiple priming sites are available for the primer as the common sequence present between any two consecutive components. This enables multiple amplified linear products (CS-A-CS-B-CS-C-CS-D; CS-B-CS-C-CS-D-CS-A, CS-A-CS-B etc.). Further, the multiple amplified linear products are separated by gel electrophoresis and the correct fragments based on the right size i.e. involving 4 components separated by the common sequence (CS-A-CS-B-CS-C-CS-D) are isolated and purified. The purified fragments are then sent for sequencing to ensure that the fragment chosen by the right size involves all the 4 unique components (CS-A-CS-B-CS-C-CS-D).
[025] FIG. 2 is an example diagram illustrating the method for circular permutation of single stranded DNA and RNA, in accordance with some embodiments of the present disclosure. Now referring to FIG. 3, the entire process is explained using an example with 4 input sequences A,B,C,D. As shown in the diagram these 4 sequences are separated by a stretch of common sequence (i.e. 202 between A and B, 204 between B and C, 206 between C and D, 208 between D and A. The primers for generating the permutations 210, 212, 214, 216, bind to the common sequences (between A and B, B and C, C and D, D and A) and enable amplification that will result in all possible permutations for example : ABCD, BCDA, CDAB, DABC.
[026] In an embodiment, the experimentation of the circularization procedure of present disclosure is performed as explained below: Initially, the linear ssDNA template (0.5 M ?nal concentration) is circularized using CircLigase™ (ssDNA ligase) at 60 degree Celsius for 3 hours in a reaction buffer containing 10× CircLigase™ ssDNA ligase buffer, 2.5 miiliMolarity Manganese Chloride (mM MnCl2), 0.05 mM Adenosine Tri-Phosphate (ATP), 5 Units of ligase and double distilled water (ddH2O). This is followed by enzyme inactivation at 80 degree Celsius for 10 min. The un-circularized templates were removed by incubating with 2 units of exonuclease I and 20 units of exonuclease III at 37 degree Celsius for 45 minutes, followed by enzyme inactivation at 60 degree Celsius for 15 minutes and 80 degree Celsius for 10 minutes. The circularization was con?rmed by polyacrylamide gel electrophoresis.
[027] In an embodiment, the experimentation of the RCA procedure of present disclosure is performed as explained below: A 10 µL (microLitre) or 2 µL aliquot of 1 µM circular DNA template prepared by CircLigase™ was combined with an equivalent molar amount of primer in a total of 50 µL containing 40 mM Trisamaenomethane-Hydrochloric acid (Tris-HCl) with pH 7.5 at a temperature of 23°C, 50 mM Potassium Chloride (KCl), 10 mM Magnesium Chloride (MgCl2), 5 mM Ammonium Sulphate ((NH4)2SO4), and 4 mM Dithiothreitol (DTT). To ensure binding of the primer to the template, an annealing procedure was performed by stepwise 2-min incubations at 80°C, 60°C, 45°C, and 23°C. After annealing, 1 µL of 10 mM deoxy Nucleoside Tri-Phosphates (dNTPs), 1 µL of 10 mg/mL Bovine Serum Albumin (BSA), and 2 µL of 10 unit/µL Phi 29 DNA polymerase were added to initiate DNA synthesis. The reaction was incubated at 30°C for 4 hours and stopped by inactivating the enzyme at 65°C for 10 minutes.
[028] The written description describes the subject matter herein to enable any person skilled in the art to make and use the embodiments. The scope of the subject matter embodiments is defined herein and may include other modifications that occur to those skilled in the art. Such other modifications are intended to be within the scope of the present disclosure if they have similar elements that do not differ from the literal language of the present disclosure or if they include equivalent elements with insubstantial differences from the literal language of the embodiments described herein.
[029] The embodiments of present disclosure herein addresses unresolved problem of circular permutation of single stranded DNA and RNA. The method generates a RCA template which can be further utilized as probes in various molecular biology techniques. It can vary from detection of mutated bacteria, virus to identification of specific antibodies present in a sample. It can also be exploited to generate RNAi (RNA interference) screening library for closely associated genes.
[030] The illustrated steps are set out to explain the exemplary embodiments shown, and it should be anticipated that ongoing technological development will change the manner in which particular functions are performed. These examples are presented herein for purposes of illustration, and not limitation. Further, the boundaries of the functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternative boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed. Alternatives (including equivalents, extensions, variations, deviations, etc., of those described herein) will be apparent to persons skilled in the relevant art(s) based on the teachings contained herein. Such alternatives fall within the scope of the disclosed embodiments. Also, the words “comprising,” “having,” “containing,” and “including,” and other similar forms are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. It must also be noted that as used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
[031] It is intended that the disclosure and examples be considered as exemplary only, with a true scope of disclosed embodiments being indicated by the following claims.
,CLAIMS:
1. A method comprising:
generating a circularized single stranded polynucleotide template by circularizing a linear single stranded polynucleotide based on a thermostable ligase, wherein the circularized single stranded polynucleotide template comprises a plurality of polynucleotide components stitched together with a plurality of common polynucleotide sequences in between each of the plurality of polynucleotide components, wherein the plurality of common polynucleotide sequences are uniformly distributed;
amplifying the circularized single stranded polynucleotide template to obtain a linear single stranded polynucleotide fragment using an amplification technique, wherein a plurality of primers are designed to anchor to a starting point of the plurality of common polynucleotide sequences during amplification; and
obtaining a plurality of permutated fragments of interest by gel purifying the linear single stranded polynucleotide fragment.

2. The method as claimed in claim 1, further comprising deciphering the plurality of circularly permutated fragments of interest by using polynucleotide sequencing.

3. The method as claimed in claim 1, wherein a plurality of un-circularized single stranded polynucleotide templates are removed by incubating with exonuclease.

4. The method as claimed in claim 1, wherein the circularization is con?rmed by Polyacrylamide gel electrophoresis.