PROJECT

OUR AIM

Novel molecules are constantly being discovered and developed to find better means of managing the fatal diseases, like cancer in multiple forms. Recent studies have shown the potential DNAzymes to serve as drugs both in cell-based assays and preclinical models of cancer. Some of the properties like serum stability, inexpensive, relatively easy synthesis, self-sufficient catalysis do not require the recruitment of catalytic cellular machinery (such as Dicer/ Risc) to degrade target substrate and higher sequence specificity amuse our attentiveness over the development of DNAzymes that can prey the anti-apoptotic protein mRNAs like BCL-2 family proteins in the cancer cell by an unique In vitro evolution of Bcl-2 mRNA cleaving DNAzymes by an unique in vitro evolution strategy.

OUR STRATEGY

The general failure of DNAzymes evolved through In Vitro selection when tested in cellular environments and animal models lead us to envisage a strategy where target mRNA was used in in vitro selection for first time to provide a proof of concept. our strategy 5’ end of the Bcl-2 mRNA of length 50 ntds was tagged with the Biotin and DNA library molecules of length 70 ntds with randomized 40 ntds and 3’ and 5’ Primer binding sites of length 15 bases each were ordered commercially. The 5’ Primer binding site of the library molecules were designed in such a way that they are complimentary with the 10 bases of the Bcl-2 mRNA molecule at the 3’ end. This allowed binding of DNA library molecules with the Bcl-2 mRNA by Watson and crick base pairing and facilitated the DNA library molecules to fold around the mRNA and perform the mRNA cleaving chemical reaction under different metal ions at different concentration and temperature conditions. The library molecules which cleaved mRNA were collected from the flow through and were enriched by SELEX approach whereas the inactive molecules remained in the column. The selected active molecules were transfected in the cell lines and we have evaluated the Bcl-2 mRNA expression studies, apoptosis regulation and cell death to understand the efficacy of the evolved DNAzyme in In Vitro conditions.

Results

In vitro evolution of Bcl-2 mRNA cleaving DNAzymes through Biotin-Streptavidin selection approach

In this strategy 5! Of the Bcl-2 mRNA of length 50 bp was tagged with the Biotin and DNA library molecules of length 70 bp with randomized 40 bp and 3! And 5! Primer binding sites of length 15 bp each were ordered commercially. The 5! Primer binding site of the library molecules were designed in such a way that they are complimentary with the 3! 10 bp of the BCL- 2 mRNA molecule. This will allow binding of DNA library molecules with the BCL2-mRNAby Watson and crick base pairing and will facilitate the DNA library molecules to coil around the mRNA and perform the mRNA cleaving chemical reaction under different metal ions at different concentration and temperature conditions. The active molecules which will cleave mRNA will be washed out though flow throw will be enriched by selex approach whereas the inactive molecules will be remained in the column. The selected active molecules will be sequenced by NGS platform and will be studied further both Invitro as well as in vivo -vitro and in vivo studies.

After the standardisation of the amount of mRNA bind to the Avidin beads we have concluded that 1 ul of Bcl-2 mRNA from 100um concentration was sufficient to saturate the Avidin binding sites in the 50 ul of Avidin magna beads , 1 ul of 100ul concentration of mRNA was incubated with the 50ul of Avidin magna beads for a period of 15 minutes at 370C temperature, washed the beads with wash buffer contains 0.1m NaOH and 0.5m NaCl for 3 times to remove the un bound mRNA molecules from the beads .Added the 1 ul of 10 um concentration of library molecules after heating at the 950C for 5 minutes to the mRNA binded Avidin beads. Incubated mRNA binded Avidin beads and library molecules for a period of 15 minutes at 370C temperature to felicitate the binding of 5! End library molecules to the 3! Of the mRNA molecules by Watson and Crick base pairing. After the incubation we have performed 10 number of washes, each wash elute was performed pcr with the respective library primers to standardise the no. of washes, from the above experiment unbound library molecules were washed out in the first and second washes, where as no library molecules were amplified in the wash 3 and the other respective washes, from this experiment we have concluded that 1 to 4 washes are sufficient to remove the unbound library molecules.

For the conformation of binding of DNA library molecules to the 3! End of the Bcl-2 mRNA after 10 washes heated the beads at 950c for 15 minutes in the dry bath. At 950c temperature DNA library molecules if binds to the mRNA will be denatured and wash out in the elute, in the above figure lane 2 And 3 were the eluted pcr products of library molecules .This experiment reveals that DNA library molecules are binding to the 3! End of the Bcl-2 mRNA molecules.

Evolution of BCL-2 mRNA Dnazymes with metal ions concentrations 150mmkcl+2mm MgCl2 After the conformation of binding of DNA library molecules at 3! Bindingsite of Bcl-2 mRNA. We have started the evolution process of DNAzymes to the Bcl-2 mRNA under the metal ions KCl and MgCl2 at the concentrations 150mm and 2mm respectively. We have incubated the mRNA attached library complex in 100ul of above mentioned concentrations meata ions for a period of 6 hours at 370C at 150 rpm. After 6 hours took out the elute and did the pcr with the respective library primers , lane 2 shows the first round single stranded cutters of DNA Library molecules , where as in the lane 2 control reaction without the metal ions. For the above experiment we have confirmed that, the salts of KCl and MgCl2 at the concentrations of 150mm and 2mm may have ability to act as co-factors for the some of the DNA library molecules to induce Bcl-2mRNA cleaving ability.

EVOLUTION OF DNAZYMES BY SELEX

Bcl-2 mRNA cleaving DNAzymes have been evolved by using the selex method , the cutter molecules for the every round was enriched by using pcr with spacer 18 reverse primer and forward primer molecules , at the end of the pcr, the amplified molecules were denatured into single strand by running in the denatured urea page gels. Two strands get separated due to different in the molecular size upper band as antisense strand and the lower band sense strand. Sense strand molecules in each round was extracted from the gel by diffusion method and concentrated by alcohol precipitation method. This was performed for 10 round, the selected cutters in the 10 round were clones by using TA cloning method.

Cloning:

10 th round selected DNAzymes are cloned into TA cloning vector

Insert preparation:

PCR with Forward and reverse primers of long final extension 15 min with TAQ pol it will adds 3’Adenylation of respected insert.

PCR cycle:

94°C	94°C	55°C	72°C	72°C	4°C
5min	30sec	30sec	30sec	15min	∞

Ligation reaction with TA cloning vector: Ligation is done by commercially provided PURE gene TA cloning kit

Reaction

TA cloning vector - 2ul

T4 DNA ligase – 1ul

T4 DNA ligase buffer A-1ul

T4 DNA ligase buffer B-1ul

DNAzyme insert – 3:1 ratio

NF water upto – 10ul

Mix all and incubate at 40C for overnight, then transform whole reaction into Ecoli competent cells and plated into LB agar ampicillin and IPTG, X-GAL plates.

Clones were confirmed by blue white screening:

In this TA cloning vector having the β-gal reporter gene in positive clones insert will disrupt β-gal gene so no blue colour colonies appear in the presence of IPTG and X-GAL.

Figure: shows both blue and white colonies in this white colonies are positive clones which is having insert.

Picked the positive white colonies and grow into LB broth and isolate the recombinant plasmid by using thermo scientific commercial plasmid isolation kit.

Agarose gel confirmation of plasmids

Confirmation of insert from the plasmid

Validation of evolved Deoxyribozymes and their modifications for the RNA cleaving property in Human solid and non-solid Cancer cell lines