SANTA CRUZ, CA, United States
SANTA CRUZ, CA, United States

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Disclosed herein are compositions and methods for the processing, amplification and detection of polynucleotides using target-specific oligonucleotides (TSOs). Hybridization of TSOs to target polynucleotides guides target processing into and purification of small target fragments that then can be amplified and detected with high sensitivity and reproducibility. The method is specifically beneficial for highly degraded polynucleotides found in biological samples.


Patent
Somagenics, Inc. | Date: 2016-10-06

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2OH and 2-OMe at their 3 ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2OH and 2-OMe at their 3 ends while allowing us to distinguish these two forms from each other.


Patent
Somagenics, Inc. | Date: 2015-08-25

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2OH and 2-OMe at their 3 ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2OH and 2-OMe at their 3 ends while allowing us to distinguish these two forms from each other.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 389.58K | Year: 2014

DESCRIPTION (provided by applicant): Hepatitis D virus (HDV) infection is the most severe type of viral hepatitis, often causing accelerated liver damage that leads to end-stage liver disease. About 10-15 million individuals are infected by HDV worldwide.The absence of an effective treatment for acute forms of the disease and the limited efficacy of current treatments for the chronic infection justify novel strategies towards the development of anti-HDV therapeutics. The lack of HDV-encoded druggable targets that are suitable for conventional therapeutic modalities such as small molecules and antibodies makes RNA interference an attractive alternative approach to target this virus. In this Phase I SBIR proposal, we propose to develop anti-HDV inhibitors using a platform developed by SomaGenics for its hepatitis C program in which chemically modified, synthetic small shRNAs (sshRNAs), formulated with lipid nanoparticles, produced effective viral knockdown in preclinical animal models. To effectively in


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2014

Our goal in this proposal is to develop a novel method for the direct detection of miRNAs in serum and plasma without requiring isolation of total RNA. Distinct expression profiles of microRNAs (miRNAs) have recently been associated with cancer and other diseases, implying that miRNAs could serve as diagnostic biomarkers for these diseases. Effective biomarkers could facilitate early detection of cancer, leading to improved therapeutic outcomes, and aid in monitoring progression and response to therapy. TheRT-qPCR assays currently employed to quantify circulating miRNAs are hampered by problems associated with their requirement for isolating total RNA from plasma or serum prior to miRNA quantification. RNA is typically isolated by spin-column purification or phenol extraction and ethanol precipitation. Both methods suffer from inconsistent miRNA recovery and the lack of an internal reference miRNA for data normalization. Moreover, current RNA isolation procedures do not completely remove inhibitors of enzy


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 269.38K | Year: 2014

DESCRIPTION (provided by applicant): This proposal addresses the problem of bias in the expression profiling of microRNAs (miRNAs) and other small RNAs by next-generation sequencing (NGS). Because dysregulation of miRNA expression has been implicated in cancer and certain other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for digital gene expression profiling, and is used for the discovery of novel miRNA sequences, identification of sequence variants, and the quantification of known miRNAs. Unlike other expression profiling platforms such as microarrays or RT-qPCR, NGS combines unlimited multiplexing capability, single-molecule sensitivity, a superior dynamic range, and true sequence specificity without requiring prior knowledge of miRNA sequences. However, NGS expression profiling data underestimate the amount of many miRNAs in a s


Patent
Somagenics, Inc. | Date: 2015-01-20

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2OH and 2-OMe at their 3 ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2OH and 2-OMe at their 3 ends while allowing us to distinguish these two forms from each other.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 700.00K | Year: 2015

DESCRIPTION provided by applicant Recent discoveries highlight the importance of extracellular miRNAs ex miRNAs as promising noninvasive biomarkers for many diseases It has also been reported that several ex miRNAs species might be important for cell cell signaling While there is still controversy about their biological importance it is clear that ex miRNAs hold promise as biomarkers for different pathologies It has been reported that distinct expression patterns of miRNAs are associated with cancer and other diseases and ex miRNAs may reflect these signatures Biofluids such as plasma can be accessed with minimal invasiveness unlike tissue biopsies This together with the high stability of ex miRNAs in biofluids makes them attractive for use in molecular diagnostics compared to other more labile biomolecules Despite these advantages current techniques are inadequate for specific and reliable quantification of miRNAs in biofluids Microarrays and RT qPCR are currently the preferred tools for expression profiling of ex miRNAs although each has major drawbacks Microarrays suffer from low sensitivity low dynamic range and the inability to distinguish closel related miRNA sequences while RT qPCR has limited multiplexing capability and amplification biases While next generation sequencing NGS is superior in many of these respects its reliability for ex miRNA profiling in biofluids is limited due to bias underdetection towards particular miRNAs overwhelming amounts of unrelated sequencing data the need for gel purification of amplicons and its high cost Here we propose a new approach for constructing ex miRNA sequencing libraries that addresses these problems Called miR SEQ it incorporates new enzymatic steps to simplify and increase the sensitivity of ligation reactions and it involves a targeted sequencing approach that allows for the quantification of rare ex miRNAs that would otherwise be represented by low numbers of reads making their quantification problematic and or expensive Although our method is applicable to any miRNA to establish proof of concept this Phase I proposal focuses on the ex miRNAs reported to be most abundant in plasma Once we achieve reliable quantification for known plasma miRNAs we expect to use the method to discover and validate ex miRNAs biomarkers in human plasma for Parkinsonandapos s Disease and develop miR SEQ diagnostic assays using those validated biomarkers PUBLIC HEALTH RELEVANCE The goal of this grant application is to solve a detection problem in profiling microRNAs miRNAs from biofluids using next generation sequencing NGS methods Current methods lead to serious undercounting of many miRNAs due to bias in the enzymatic reactions used to prepare the samples for NGS and because of their intrinsically low concentrations in biofluids Eliminating this bias will help to fully realize the potential of miRNAs as biomarkers of cancer and other diseases In addition this method may help reduce the cost of sequencing clinical samples making this powerful diagnostic tool more accessible to patients


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 990.25K | Year: 2016

ABSTRACT This proposal addresses the problem of sequence bias in next generation sequencing NGS of small RNAs such as microRNAs miRNAs as well as fragments of larger RNAs Because dysregulation of miRNA expression has been implicated in cancer and other diseases accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets NGS is currently the most comprehensive approach for discovery and expression profiling of small RNA sequences However NGS expression profiling data underestimate the abundance of most miRNAs in a sample some by as much as fold Knowledge of the true abundances in samples and not just the relative changes between samples is important for reliable identification of miRNAs as biomarkers or drug target candidates Other advantages of unbiased detection include the ability to discover novel RNAs and detect low abundance RNAs that cannot be detected by current NGS methods especially in samples with a low concentration of RNA The source of bias in currently available methods of preparation RNA sequencing libraries for NGS is inefficient and sequence dependent ligation of the two sequencing adapters to the sample RNAs The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and adapters which affect the ability of the ligase to access and ligate the miRNA ends Thus there is a need for new more accurate methods and most previous small RNA profiling experiments should be re evaluated To address these problems we are developing a new approach miR ACS miRNA Adapter Circularization and Sequencing for preparing unbiased sequencing libraries that is applicable to miRNAs and other small RNAs as well as small fragments of large RNAs used in general RNA Seq Key features of miR ACS include i ligation of miRNAs with only a single combo adapter CAD that combines sequences of the standard andapos and andapos adapters used for Illumina sequencing producing miRNA CAD ligation products ii circularization of the miRNA CAD products iii blocking of free CAD species that are not ligated to miRNAs and iv RT PCR amplification of the circular miRNA CAD products to produce standard sequencing amplicons containing a single RNA specific sequence insert flanked by the andapos and andapos adapter sequences In Phase I we have demonstrated the feasibility of the miR ACS approach proof of concept by greatly reducing the miRNA sequencing bias in comparison to the best current library prep methods In Phase II we will thoroughly optimize miR ACS to maximize bias reduction and to allow sequencing of a larger variety of RNAs up to nt in size with very low RNA inputs In addition we will streamline the protocol to facilitate its adoption by users and for commercial viability HEALTH RELATEDNESS NARRATIVE MicroRNAs miRNAs are promising candidates as biomarkers of cancer and other diseases that could allow early diagnosis sub typing choice of targeted treatments and monitoring of the progress of therapy Although next generation sequencing NGS is the method of choice for discovering and expression profiling of miRNAs it underestimates the abundance of most miRNAs some by as much as fold because of sequence bias The goal of this grant application is to eliminate this bias to allow discovery of new RNA species and determination of the true abundance of RNAs in biological samples


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 287.79K | Year: 2016

DESCRIPTION provided by applicant Quantification of gene expression in formalin fixed paraffin embedded FFPE tissue samples is important for the discovery and validation of cancer biomarkers for tumor classification and to assess progress during cancer treatment Because RT qPCR assays are very sensitive and sequence specific they are currently preferred for mRNA expression profiling in FFPE tissues as well as for validation of data obtained by other expression profiling methods such as microarrays and sequencing RNA seq However use of these methods to analyze FFPE samples is constrained by the RNA fragmentation that occurs in these samples and limits the sensitivity and reproducibility of these assays To overcome this problem we proposed a novel method for assaying mRNA fragments in FFPE samples called mR FQ mRNA Fragment Quantification In Preliminary Studies we developed a mR FQ prototype which can work with very short mRNA fragments of nt and demonstrated its superior sensitivity over the TaqMan RT qPCR method in quantifying two model mRNAs from FFPE samples We analyzed HER a breast cancer biomarker and GAPDH internal reference mRNAs in total RNA isolated from breast cancer and prostate cancer FFPE samples In Phase I we plan to i validate the mR FQ method using a larger number of target mRNAs and more FFPE samples ii further optimize mR FQ iii demonstrate that mR FQ reliably quantifies mRNAs in FFPE samples containing highly fragmented mRNAs that are not detectable by currently leading RT qPCR methods run in parallel iv determine the maximum mRNA fragmentation level detectable by mR FQ In Phase II we will move towards commercialization by designing mR FQ assays for BC biomarker candidates and validating them on FFPE samples having a wide range of RNA fragmentation levels with focus on currently unusable samples those containing highly fragmented RNA that cannot be assayed by standard RT qPCR methods mR FQ and RNA seq analyses will be compared and we expect that mR FQ will be able to validate RNA seq results since both methods can work with highly fragmented RNA This will allow researchers to include a wider range of FFPE samples into retrospective studies for the development and validation of improved breast as well as other types of cancer diagnostics and treatment prognostics PUBLIC HEALTH RELEVANCE This project develops new technology for studying gene expression archived tissue specimens from cancer patients We plan to use it to develop better biomarkers for breast cancer the most common cancer among women About women were diagnosed and over died from the disease last year in the US alone The goal of this grant application is to develop a method for quantitation of fragmented mRNAs from standard histology specimens FFPE blocks that will provide superior sensitivity and accuracy at a reasonable cost to facilitate research in and diagnosis of breast cancer as well as treatment prognosis

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