Not listed
ENRICHING AND BASE-RESOLUTION PROFILING OF 5-METHYLCYTOSINE IN DEGRADED CLINICAL SAMPLES - PROJECT SUMMARY THE EPIGENOME, CENTERED AROUND 5-METHYLCYTOSINE (5MC), CARRIES AMPLE MOLECULAR SIGNATURES FOR UNDERSTANDING, DETECTING, AND TRACKING TUMORIGENESIS. HOWEVER, 5MC PROFILING IN CLINICAL SAMPLES, SUCH AS FORMALIN-FIXED, PARAFFIN-EMBEDDED (FFPE) TISSUES AND CELL-FREE DNA (CFDNA), IS HINDERED BY THEIR LIMITED QUANTITY AND DEGRADED DNA. TO ADDRESS THESE CHALLENGES, THIS PROPOSAL DESCRIBES DEVELOPMENT AND APPLICATION OF THE FIRST GLYCOSYLASE-BASED EPIGENOME-SEQUENCING PLATFORM. WITH SUPERIOR SENSITIVITY AND FIDELITY, GLYCOSYLASE- IMPLEMENTED EPIGENOME SEQUENCING (GLIMP-SEQ) IS POISED TO UNCOVER THE MOLECULAR UNDERPINNINGS OF TUMORIGENESIS IN LOW-VOLUME DEGRADED SAMPLES. GLIMP-SEQ HARNESSES CASCADE ENZYMATIC REACTIONS INVOLVED IN MAMMALIAN ACTIVE DEMETHYLATION-5MC OXIDIZATION BY TEN-ELEVEN TRANSLOCATION DIOXYGENASE (TET) AND 5-CARBOXYLCYTOSINE (5CAC) EXCISION BY THYMINE DNA GLYCOSYLASE (TDG)-TO CONVERT 5MC INTO ABASIC SITES. THESE ABASIC SITES ARE SUBSEQUENTLY FUNCTIONALIZED BY A THYMINE MIMIC TO FACILITATE 5MC-SPECIFIC ENRICHMENT AND DIRECT MUTATION DETECTION (5MC-TO-T). GLIMP-SEQ REPRESENTS A MAJOR ADVANCEMENT OVER CURRENT EPIGENOME PROFILING METHODS, INCLUDING BISULFITE SEQUENCING (BS- SEQ), ENZYMATIC METHYL SEQUENCING (EM-SEQ), AND TET-ASSISTED PYRIDINE BORANE SEQUENCING (TAPS), BY EMPLOYING GENTLE, YET EFFICIENT, ENZYMATIC CONVERSIONS THAT PRESERVE DNA INTEGRITY. AS THE SPECIFICITY OF THE CASCADE TET/TDG REACTIONS HAS BEEN EXTENSIVELY OPTIMIZED DURING NATURAL EVOLUTION, GLIMP-SEQ ACHIEVES UNPARALLELED SPECIFICITY FOR 5MC, REDUCING THE RISK OF FALSE POSITIVE DETECTION. UNLIKE EXISTING METHODS, GLIMP-SEQ UNIQUELY SUPPORTS SEQUENCE-INDEPENDENT 5MC-SPECIFIC ENRICHMENT, THEREBY INCREASING DETECTION SENSITIVITY. THE RESULTING SUPERIOR SENSITIVITY AND FIDELITY MAKE GLIMP-SEQ PARTICULARLY SUITED FOR 5MC PROFILING IN DEGRADED CLINICAL SAMPLES. IN AIM 1, WE WILL DEVELOP AND RIGOROUSLY BENCHMARK GLIMP-SEQ, USING BOTH SYNTHETIC AND GENOMIC DNA SAMPLES. WE WILL COMPARE GLIMP-SEQ WITH BS-SEQ, EM-SEQ, AND TAPS, AND DEMONSTRATE ITS SUPERIORITY USING QUANTITATIVE METRICS. IN AIM 2, WE WILL APPLY GLIMP-SEQ TO TWO SETS OF CLINICAL SAMPLES: ~140 FFPE CANCER TISSUES FROM PATIENTS OF PRENATAL EXPOSURE TO DIETHYLSTILBESTROL (KNOWN AS DES DAUGHTERS), WHICH PROVIDES A UNIQUE OPPORTUNITY TO INVESTIGATE SYNTHETIC ESTROGEN-INDUCED EPIGENETIC INHERITANCE; AND CFDNA FROM 50 COLORECTAL CANCER PATIENTS, TO SHOWCASE NON-INVASIVE DETECTION OF CANCER-ASSOCIATED 5MC SIGNATURES. SUCCESSFUL IMPLEMENTATION OF GLIMP-SEQ WILL TRANSFORM THE CLINICAL PRACTICE OF EPIGENOME PROFILING BY FACILITATING MECHANISTIC STUDIES USING FFPE SAMPLES AND BY EXPEDITING EARLY CANCER DIAGNOSIS THROUGH CFDNA ANALYSIS. BY FREELY DISTRIBUTING GLIMP-SEQ REAGENTS AND PROTOCOLS, WE AIM TO SUPPORT CANCER RESEARCHERS BOTH LOCALLY AND GLOBALLY, FOSTERING CONTINUOUS ADVANCEMENTS IN UNDERSTANDING, DETECTING, AND TRACKING CANCER.
$0 2/12/25 Not listed
ENRICHING AND BASE-RESOLUTION PROFILING OF 5-METHYLCYTOSINE IN DEGRADED CLINICAL SAMPLES - PROJECT SUMMARY THE EPIGENOME, CENTERED AROUND 5-METHYLCYTOSINE (5MC), CARRIES AMPLE MOLECULAR SIGNATURES FOR UNDERSTANDING, DETECTING, AND TRACKING TUMORIGENESIS. HOWEVER, 5MC PROFILING IN CLINICAL SAMPLES, SUCH AS FORMALIN-FIXED, PARAFFIN-EMBEDDED (FFPE) TISSUES AND CELL-FREE DNA (CFDNA), IS HINDERED BY THEIR LIMITED QUANTITY AND DEGRADED DNA. TO ADDRESS THESE CHALLENGES, THIS PROPOSAL DESCRIBES DEVELOPMENT AND APPLICATION OF THE FIRST GLYCOSYLASE-BASED EPIGENOME-SEQUENCING PLATFORM. WITH SUPERIOR SENSITIVITY AND FIDELITY, GLYCOSYLASE- IMPLEMENTED EPIGENOME SEQUENCING (GLIMP-SEQ) IS POISED TO UNCOVER THE MOLECULAR UNDERPINNINGS OF TUMORIGENESIS IN LOW-VOLUME DEGRADED SAMPLES. GLIMP-SEQ HARNESSES CASCADE ENZYMATIC REACTIONS INVOLVED IN MAMMALIAN ACTIVE DEMETHYLATION-5MC OXIDIZATION BY TEN-ELEVEN TRANSLOCATION DIOXYGENASE (TET) AND 5-CARBOXYLCYTOSINE (5CAC) EXCISION BY THYMINE DNA GLYCOSYLASE (TDG)-TO CONVERT 5MC INTO ABASIC SITES. THESE ABASIC SITES ARE SUBSEQUENTLY FUNCTIONALIZED BY A THYMINE MIMIC TO FACILITATE 5MC-SPECIFIC ENRICHMENT AND DIRECT MUTATION DETECTION (5MC-TO-T). GLIMP-SEQ REPRESENTS A MAJOR ADVANCEMENT OVER CURRENT EPIGENOME PROFILING METHODS, INCLUDING BISULFITE SEQUENCING (BS- SEQ), ENZYMATIC METHYL SEQUENCING (EM-SEQ), AND TET-ASSISTED PYRIDINE BORANE SEQUENCING (TAPS), BY EMPLOYING GENTLE, YET EFFICIENT, ENZYMATIC CONVERSIONS THAT PRESERVE DNA INTEGRITY. AS THE SPECIFICITY OF THE CASCADE TET/TDG REACTIONS HAS BEEN EXTENSIVELY OPTIMIZED DURING NATURAL EVOLUTION, GLIMP-SEQ ACHIEVES UNPARALLELED SPECIFICITY FOR 5MC, REDUCING THE RISK OF FALSE POSITIVE DETECTION. UNLIKE EXISTING METHODS, GLIMP-SEQ UNIQUELY SUPPORTS SEQUENCE-INDEPENDENT 5MC-SPECIFIC ENRICHMENT, THEREBY INCREASING DETECTION SENSITIVITY. THE RESULTING SUPERIOR SENSITIVITY AND FIDELITY MAKE GLIMP-SEQ PARTICULARLY SUITED FOR 5MC PROFILING IN DEGRADED CLINICAL SAMPLES. IN AIM 1, WE WILL DEVELOP AND RIGOROUSLY BENCHMARK GLIMP-SEQ, USING BOTH SYNTHETIC AND GENOMIC DNA SAMPLES. WE WILL COMPARE GLIMP-SEQ WITH BS-SEQ, EM-SEQ, AND TAPS, AND DEMONSTRATE ITS SUPERIORITY USING QUANTITATIVE METRICS. IN AIM 2, WE WILL APPLY GLIMP-SEQ TO TWO SETS OF CLINICAL SAMPLES: ~140 FFPE CANCER TISSUES FROM PATIENTS OF PRENATAL EXPOSURE TO DIETHYLSTILBESTROL (KNOWN AS DES DAUGHTERS), WHICH PROVIDES A UNIQUE OPPORTUNITY TO INVESTIGATE SYNTHETIC ESTROGEN-INDUCED EPIGENETIC INHERITANCE; AND CFDNA FROM 50 COLORECTAL CANCER PATIENTS, TO SHOWCASE NON-INVASIVE DETECTION OF CANCER-ASSOCIATED 5MC SIGNATURES. SUCCESSFUL IMPLEMENTATION OF GLIMP-SEQ WILL TRANSFORM THE CLINICAL PRACTICE OF EPIGENOME PROFILING BY FACILITATING MECHANISTIC STUDIES USING FFPE SAMPLES AND BY EXPEDITING EARLY CANCER DIAGNOSIS THROUGH CFDNA ANALYSIS. BY FREELY DISTRIBUTING GLIMP-SEQ REAGENTS AND PROTOCOLS, WE AIM TO SUPPORT CANCER RESEARCHERS BOTH LOCALLY AND GLOBALLY, FOSTERING CONTINUOUS ADVANCEMENTS IN UNDERSTANDING, DETECTING, AND TRACKING CANCER.
$230.0k 1/28/25