VERIFICATION OF 50-MER TO 100-MER DNA AND RNA SEQUENCES WITH HIGH-RESOLUTION MASS-SPECTROMETRY

Electrospray ionization with Fourier-transform mass spectrometry achieves accurate (<50-ppm) determination of molecular weights of nucleotides, verifying structures of biological RNA and synthetic single-stranded DNA. High (10(5)) resolving power makes possible detection. of subpicomole impurities and adducts that confuse lower-resolution measurements. Molecular ions in a spectrum of 76-mer tRNA(Phe) had 34-55 Na adducts; when desalted, these show a molecular mass of 24,950.5 Da (expected, 24,950.3 Da) and minor variants at approximately -15 and +15 Da. A 5-mer DNA is characterized with <10-ppm mass error, with detection of both N + 1 and N - 1 failure sequences. Special electrospray ionization conditions are necessary for a 72-mer to minimize fragmentation in the ion source. Despite the chemical noise from this, as well as failed sequences from automated synthesis, the spectrum of a 100-mer single-standed DNA yielded a molecular mass of 30,702.4 +/- 1 Da, in good agreement with the expected value, 30,702.1 Da.