RESTRICTASE CASCADE EXPONENTIAL AMPLIFICATION (RCEA) DNA DETECTION - PowerPoint PPT Presentation

1 Cascades Ventures . Any bids or questions about this offering should be directed to: Mark Magas mmagas@cascadesventures.com (847) 656-3560 RESTRICTASE CASCADE EXPONENTIAL AMPLIFICATION (RCEA) DNA DETECTION A PORTFOLIO OF PATENTS EXCLUSIVELY OFFERED FOR SALE OR LICENSE BY CASCADES VENTURES . Non-confidential Marketing Package Not for public distribution without explicit permission from Cascades Ventures

2 Important Disclaimer and Notice This marketing package constitutes an offer for sale of patents on behalf of the owner by Cascades Ventures, Inc. (Cascades), an IP licensing and brokerage firm. The information is provided solely for the purpose of assisting the independent evaluation of the portfolio by prospective buyers. The information provided herein or exchanged pursuant to the sales process is not intended to be notice or accusation of infringement of any of the patents or portfolios offered for sale. Nothing in this document shall constitute or be interpreted as legal analysis regarding the scope of the patents or other intellectual property rights. Any discussion of the use or potential use of the patent portfolio is for illustrative purposes only. In making a decision regarding this sales opportunity, potential purchasers must rely on their own examination and evaluation of the patents and portfolios including the merits and risks involved. No representation or warranties regarding the patents or portfolios are provided or implied. This marketing package and any other documents or information provided by Cascades related to the patents or portfolios are intended for use by the receiving party solely for its use in engaging in the sales process and in determining whether to purchase the patents or portfolios. Any distribution of these materials or information outside of the receiving party's organization without Cascades permission is strictly prohibited. Cascades reserves the right to modify or discontinue the sales process at any time including accepting offers prior to the completion of the due diligence period.

3 Executive Summary This Intellectual Property (IP) portfolio discloses “Restrictase Cascade Exponential Amplification” (RCEA)  DNA detection technology. This disruptive technology will revolutionize the existing, laboratory-based polymerase chain reaction (PCR) market,  allowing for in-field testing and even greater reliability. This portfolio has international coverage and includes 50 total assets across five patent families:  Fourteen issued U.S. patents and five pending U.S. applications;  Twenty-five issued and six pending foreign counterparts;  All patents and applications are assigned to Cascade Biosystems.  The portfolio is technically and financially beneficial for offensive and defensive strategies for any  company in the DNA testing market.

4 DNA Testing Global Market DNA Testing Market Forecast 12 Polymerase chain reaction (PCR) 10 ➢ $10B technology dominates the current 8 DNA testing market. PCR itself is a patented technology ➢ 6 that was successfully licensed, earning billions in royalties. 4 $6.2B The DNA testing market is expected to ➢ 2 achieve significant growth , expanding to over $10 Billion by 2022. 0 2014 2022 Source: Grand View Research 2016

5 DNA Testing Collected DNA test samples often ➢ contain only trace amounts of a target DNA segment . In order for the DNA segment to be ➢ detectable, the segment generally must be amplified . PCR is currently the dominant DNA ➢ amplification technology. After amplification, detection can be ➢ performed using known techniques such as electrophoresis, hybridization, or sequencing.

6 DNA REPLICATION DURING A PCR THERMAL CYCLE Existing Step 1: Heating separates the Technology: PCR target DNA strands. Step 2: Cooling Polymerase chain reaction (PCR) is a ➢ allows primers to anneal to the laboratory-based technique that generates separated millions of copies of a target DNA sequence . strands. Primers are used in combination with DNA ➢ polymerase to exponentially amplify (copy) the target DNA segment. Step 3: DNA polymerase Primers: Short DNA fragments containing elongates the ➢ new DNA chains complementary sequences to the target DNA after the primer. segment. DNA polymerase: enzymes which “read” the ➢ existing DNA segment to produce duplicate segments. PCR generally requires thermal cycling — ➢ Then REPEAT until approximately 20-30 repeated cycles of a sufficient heating and cooling — which allows the DNA number of copies segment replication process to occur. are generated.

7 Disadvantages of PCR ➢ Must be performed in a controlled laboratory environment. ➢ Expensive due to high costs of expensive equipment and highly trained personnel. ➢ Subject to amplification bias due to the exponential nature of the reaction and altered priming events. ➢ Generally, can only be used with low-throughput assays.

8 Other PCR Alternatives Many other DNA amplification techniques have been attempted, such as: ➢ ➢ Transcription mediated amplification; ➢ Strand displacement amplification; ➢ Rolling circle amplification; ➢ Loop-mediated isothermal amplification; ➢ Florescent in situ hybridization; ➢ Etc. However, all of the alternative techniques fall short . ➢ ➢ The main problem with these techniques is a possible sequence-dependent bias , when certain sequences have much higher amplification efficiency than the other. ➢ These techniques also complicate the design and validation of the multiple primers needed.

9 How RCEA DNA Detection is Watch the following video for a detailed explanation of how RCEA DNA detection works: Different Rather than amplifying the target DNA ➢ segment like PCR, the “Restrictase Cascade Exponential Amplification” (RCEA) technology uses a series of enzymatic reactions to detect the presence of the initial non-amplified target DNA. If the target DNA is present, it will trigger a ➢ cascading reaction releasing different enzymes which can then be detected. https://youtu.be/aCSx_7YjAEU Thus, the enzymes are amplified rather ➢ than the target DNA segment ; Clicking the above image or the link will open the video in your web browser. This avoids many of the problems of PCR. ➢ A text explanation can also be found in the Appendix section.

10 Benefits of RCEA DNA Testing Technology Attribute PCR RCEA Assay cost Highly expensive Nearly 1/3 less Assay sensitivity Very high Very high Assay specificity Moderate Extremely high Use with high throughput assay Yes, but complex and expensive Yes Use with portable equipment No Yes Low sample volume requirement Poor Excellent Used for SNP assay Limited by primers Limited by endonucleases Used for cancer methylation No Yes

11 Potential Applications  Bacterial infection or pathogen detection (i.e., MRSA);  Cancer detection;  Agricultural or veterinary diagnostics;  Food safety testing;  Environmental testing (air and water);  Genetic or epigenetic testing;  Single-nucleotide polymorphism (SNP) analysis;  Forensics.

12 Research Milestones Assay development:  Completed enzyme activity assays in various formats.  Optimized protocols for testing of horseradish peroxidase (HRP) and  restrictase activity. Proof-of-concept experiments:  Developed two protocols: direct RC assay with no amplification, and RCEA  with exponential amplification. Preliminary experiments demonstrated the exponential scheme resulted in  an assay sensitivity with a 10-17 attomolar detection level, already equivalent to PCR levels. Optimization would lead to even better sensitivity. Ghindilis AL, Smith MW, Simon HM, et al., Restriction Cascade Exponential Amplification (RCEA) assay with Confirmation of signal an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR, S CI . R EP . 2015;5:7737. amplification principle doi:10.1038/srep07737 (Jan. 13, 2015). (View Publication)

13 Research Milestones Application:  Preliminary development of a Methicillin-resistant  Staphylococcus aureus (MRSA) assay. The testing successfully detected MRSA within limits similar  to PCR. The proposed assay had high specificity, was insensitive to  non-specific binding, and detected target sequences in the presence of foreign DNA. Smith MW, Ghindilis AL, Seoudi IA, Smith K, Billharz R, et al., A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus, PL O S ONE 9(5): e97826. doi:10.1371/journal.pone.0097826 (May 15, 2014). (View Publication) Typical calibration curve of the restriction enzyme assay

14 Portfolio Details This portfolio includes five patent families with 56 total assets:  Primary patent family: Detecting Nucleic Acid (22 assets);  Key patents: U.S. 8,278,048; U.S. 8,632,974; U.S. 8,865,407; and U.S. 9,677,120.  Four other patent families were created adapting the base technology to specific practice  areas: Methods and Materials for Detecting Contaminated Food Products (8 assets);  Methods and Materials for Detecting Viral or Microbial Infections (8 assets);  Methods and Materials for Detecting Genetic or Epigenetic Elements (8 assets);  Methods and Materials for Assessing RNA Expressions (4 assets).  Earliest priority date of August 5, 2008.  Patent term lasts until 2031.  A complete list of patents in this portfolio can be found in the Appendix section.