DNA templates modified with C29-methoxyls at the last two nucleotides of the 59 termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single-and double-stranded DNA templates. This strategy was used to generate several different transcripts. Two of the transcripts were demonstrated by nuclear magnetic resonance spectroscopy to be unaffected in their sequence. Transcripts produced from the modified templates can be purified with greater ease and should be useful in a number of applications.

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Methylated SEPT9 showed relatively low sensitivity in detecting early stage colorectal cancer (CRC) and advanced adenomas (AA) in plasma. Combination of multiple biomarkers was an effective strategy to improve sensitivity in early stage cancer diagnosis and screening. A new qPCR‐based assay combining the detection of methylated SEPT9 and SDC2 (ColoDefense test) was used. Methylation statuses of SEPT9 and SDC2 were examined in 40 sets of cancer tissues and paired adjacent tissues, 10 adenomatous polyps and 3 hyperplastic polyps (HP). Then evaluated with 384 plasma samples, including 117 CRC patients, 23 AA patients, 78 small polyps patients, and 166 normal individuals. The limit of detection of ColoDefense was about 25 pg per reaction. Both SEPT9 and SDC2 were shown by ColoDefense to be heavily methylated in CRC tissues when compared to paired paracancerous tissues and HP (P < .01). The sensitivities for detecting AA and stage I CRC by plasma SEPT9 methylation alone were 12.1% and 65.0%, and those by plasma SDC2 methylation alone were 43.5% and 55.0%. In comparison, the sensitivities to detect AA and stage I CRC by ColoDefense improved to 47.8% and 80.0%. The overall sensitivity of ColoDefense in detecting CRC was 88.9% (95% CI: 81.4%‐93.7%) with a specificity of 92.8% (95% CI: 87.4%‐96.0%). Detection of the combinatorial biomarker of methylated SEPT9 and/or SDC2 is a powerful, convenient and highly effective strategy for early CRC screening with high sensitivity and specificity.

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The secondary structure of a truncated P5abc subdomain (tP5abc, a 56-nucleotide RNA) of the Tetrahymena thermophila group I intron ribozyme changes when its tertiary structure forms. We have now used heteronuclear NMR spectroscopy to determine its conformation in solution. The tP5abc RNA that contains only secondary structure is extended compared with the tertiary folded form; both forms coexist in slow chemical exchange (the interconversion rate constant is slower than 1 s ؊1 ) in the presence of magnesium. Kinetic experiments have shown that tertiary folding of the P5abc subdomain is one of the earliest folding transitions in the group I intron ribozyme, and that it leads to a metastable misfolded intermediate. Previous mutagenesis studies suggest that formation of the extended P5abc structure described here destabilize a misfolded intermediate. This study shows that the P5abc RNA subdomain containing a GNRA tetraloop in P5c (in contrast to the five-nucleotide loop P5c in the tertiary folded ribozyme) can disrupt the base-paired interdomain (P14) interaction between P5c and P2.

Genetic mutation studies have revealed that abnormal expansion or contraction of certain triplet repeat (TR) sequences, such as CCG/CGG and CTG/CAG, is implicated in more than 10 neurodegenerative diseases.* 1 TR sequences are often found within or near relevant gene-coding regions16 and thus have promise as diagnostic probes and perhaps as targets for new genetic therapies for related hereditary diseases. To understand the possible mechanisms of dynamic mutations of TR sequences, we have initiated an investigation into the solution conformation of the six CXG and GXC triplets (X = A, C, G, T) using NMR methods. The presence of a stable structure associated with these TR sequences might play a role in disrupting the normal replication or transcription process.2Here we report a novel duplex motif formed by DNA CCG TR sequences in a neutral to basic pH range. The structure is characterized by NMR spectroscopy, electrophoresis, and NMRbased calculations. A series of CCG TR sequences (all sequences refer to DNA, unless otherwise noted), which include the homologous sequences CGCCG and (CCG)" (n = 2, 3, 4, and 5), the permutation isomers (CGC)2 and (GCC)2, and the linker sequence (CCG)2-xx-(CCG)2 (x = triethylene glycol linker), have been examined to supplement the results pertaining to the (CCG)2 sequence.NMR spectra of (CCG)2 in aqueous solutions containing 0.1 -0.2 M NaCl at pH 6.3-8.5 provide strong evidence for a distinct, stable Watson-Crick base paired structure. The complete !H and 31P assignments and a summary of the major spectral features of (CCG)2 are given in Tables SI and S2, respectively, in the supporting information. (CCGh forms a symmetrical duplex, which displays 'H and 31P resonances corresponding to a single strand of the hexamer (Figure SI, Table SI, supporting information).3 Twenty percent polyacrylamide nondenaturing gel electrophoresis shows that (CCG)2 and (CCG)3 duplexes migrate faster than the corresponding complementary hexamer and nanomer duplexes at 4 °C,4 excluding the possible presence of higher order structures. In the spectra recorded in 90% H2O (Figure S1A) G3 and G106 HN (imino) resonances at 13.04 and 13.48 ppm show an HN-HN NOE and HN-H2N (amino) NOEs (Figure SI A). These observations, especially the presence of the HN-HN NOE between G3 and G106 residues, are unequivocal evidence for the staggered alignment of (CCG)2 (Scheme 1).The 31P ID spectrum of (CCG)2 (Figure SIB) exhibits four well-resolved signals in a chemical shift range of 2.1 ppm [3'-P (ppm) of C2 -2.84; C5 -3.45; Cl -4.04; C4 -4.09; G3 -4.97], The 31P resonances of canonical DNA duplexes are usually detected in a ~0.6 ppm region.5 The large dispersion of31P resonances of (CCG)2 is diagnostic of an untwisted and extended duplex conformation as observed in several antibiotic-DNA complexes.6 Further evidence for a distorted (CCG)2 * To whom correspondence should be addressed.

Colorectal cancer (CRC) has become the second leading cause of new cancer cases and the fifth of cancer deaths in China, and early detection is the most effective way to reduce the incidence and mortality of CRC. A number of methylated DNA biomarkers have been found to associate with CRC and precancerous lesions in stool samples, indicating stool methylated DNA biomarkers are potential tools for CRC early detection. In this study, approximately 5 g of stool specimen was collected from 230 subjects (124 in the training set and 106 in the validation set). Stool DNA was extracted and bisulfiteconverted, followed by ColoDefense test, a multiplex qPCR assay, that simultaneously detects methylated SEPT9 (mSEPT9) and methylated SDC2 (mSDC2). Youden index was employed to determine the cut-off value of ColoDefense test for stool specimens. In the training set, the optimized cut-off value of stool ColoDefense test was: mSEPT9 analyzed with 3/3 algorithm and mean mSEPT9 Ct values of <38, or mSDC2 with 2/3 algorithm. Stool ColoDefense test achieved Youden indexes of 79.9 and 57.4% in detecting CRC and advanced adenomas (AA), respectively. Its sensitivities in the training set for AA and CRC were 66.7% (95% CI: 24.1-94.0%) and 89.1% (95% CI: 77.1-95.5%) with a 90.8% (95% CI: 80.3-96.2%) specificity, and AUC was 0.956 (95% CI: 0.924-0.988). In the validation set, its sensitivities for AA and CRC were 66.7% (95% CI: 24.1-94.0%) and 92.3% (95% CI: 78.0-98.0%) with a 93.2% (95% CI: 82.7-97.8%) specificity, and AUC was 0.977 (95% CI: 0.952-1.000). Positive detection rate of stool ColoDefense test has been found to be independent of age, gender, tumor location, and tumor size. In conclusion, stool ColoDefense test demonstrated high sensitivities and specificity for the detection of AA and CRC. Therefore, it has the potential to become a low-cost, convenient, and highly effective tool for CRC early detection.