thiouracil and 2-6-diaminopurine

Serinol nucleic acid (SNA) is a promising candidate for nucleic acid-based molecular probes and drugs due to its high affinity for RNA. Our previous work revealed that incorporation of 2,6-diaminpurine (D), which can form three hydrogen bonds with uracil, into SNA increases the melting temperature of SNA-RNA duplexes. However, D incorporation into short self-complementary regions of SNA promoted self-dimerization and hindered hybridization with RNA. Here we synthesized a SNA monomer of 2-thiouracil (sU), which was expected to inhibit base pairing with D by steric hindrance between sulfur and the amino group. To prepare the SNA containing D and sU in high yield, we customized the protecting groups on D and sU monomers that can be readily deprotected under acidic conditions. Incorporation of D and sU into SNA facilitated stable duplex formation with target RNA by suppressing the self-hybridization of SNA and increasing the stability of the heteroduplex of SNA and its complementary RNA. Our results have important implications for the development of SNA-based probes and nucleic acid drugs.

Topics: 2-Aminopurine; Base Pairing; Hydrogen Bonding; Nucleic Acid Hybridization; Oligonucleotides; Phase Transition; Propanolamines; Propylene Glycols; RNA; Thiouracil; Transition Temperature

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this 'double-duplex invasion', a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the alpha-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G-C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.

Topics: 2-Aminopurine; AT Rich Sequence; Base Pair Mismatch; Base Sequence; DNA; GC Rich Sequence; Peptide Nucleic Acids; Static Electricity; Stereoisomerism; Thiouracil

Pseudo-complementary peptide nucleic acid (pcPNA) is a DNA analog in which modified DNA bases 2,6-diaminopurine (D) and 2-thiouracil (U(s)) 'decorate' a poly[N-(2-aminoethyl)glycine] backbone, together with guanine (G) and cytosine (C). One of the most significant characteristics of pcPNA is its ability to effect double-duplex invasion of predetermined DNA sites inducing various changes in the biological and the physicochemical properties of the DNA. This protocol describes solid-phase synthesis of pcPNA. The monomers for G and C are commercially available, but the monomers for D and U(s) need to be synthesized (or can be ordered to custom synthesis companies). Otherwise, the procedure is the same as that employed for Boc-strategy synthesis of conventional PNA. This protocol, if the synthesis of D and U(s) monomers is not factored in, takes approximately 7 d to complete.

Topics: 2-Aminopurine; Molecular Structure; Peptide Nucleic Acids; Thiouracil

Chiral PNA monomers (PNA = peptide nucleic acid), in which nucleobases are attached to N-(aminoethyl)-D-lysine, were introduced to PNAs bearing pseudo-complementary nucleobases (2,6-diaminopurine and 2-thiouracil). When these highly cationic PNAs targeted double-stranded DNA, they invaded there much more efficiently than conventional pseudo-complementary PNAs composed of achiral PNA monomers. Although introduction of N-(aminoethyl)-D-lysine backbone was effective for promotion of strand invasion, L-isomer never promote it. Simple incorporation of lysine groups to the termini of PNA was also ineffective, indicating that introduction of positive charges into PNA backbone is important. Even highly G-C rich sequence, which conventional pseudo-complementary PNAs never invade, was successfully targeted based on this strategy.

Topics: 2-Aminopurine; DNA; Electrophoretic Mobility Shift Assay; GC Rich Sequence; Isomerism; Lysine; Peptide Nucleic Acids; Static Electricity; Thiouracil

The propensity of RNA to fold into higher-order structures poses a major barrier to the use of short probes (<15 nucleotides) by preventing their accessibility. Introduction of the pseudo-complementary bases 2-aminoadenine (nA) and 2-thiouracil (sU) and the destabilizing base 7-deazaguanine (cG) into RNA provides a partial solution to this problem. While complementary in hydrogen bonding groups, nA and sU cannot form a stable base pair due to steric hindrance, and are thus pseudo-complementary. Each, however, recognizes the regular T/U and A complements, allowing pairing with oligonucleotides. Short pseudo-complementary RNAs can be prepared by in vitro transcription. Relative to standard transcripts, the modified transcripts possess reduced secondary structure and increased accessibility to short (8-mer) probes in the locked nucleic acid (LNA) configuration. They also hybridize to complementary probes with increased specificity and thermostability. Practical application of this strategy to oligonucleotide-based hybridization assays will require engineering of RNA polymerase for more efficient utilization of pseudo-complementary nucleoside triphosphates.

Topics: 2-Aminopurine; Base Pairing; Guanine; Hypoxanthine; Nucleic Acid Conformation; Nucleic Acid Hybridization; Oligonucleotides; RNA; RNA Probes; Temperature; Thiouracil

Secondary structure in RNA targets is a significant barrier to short DNA probes. However, when such targets are the end product of an in vitro amplification scheme, it is possible to carry out transcription in the presence of nucleoside triphosphate analogues that reduce secondary structure of the RNA without impairing subsequent hybridization. Here we show that nucleoside triphosphates of 2-aminoadenine (nA) and 2-thiouracil (sU) are taken up by T7 RNA polymerase and that the resulting RNA possesses reduced secondary structure and improved accessibility to DNA probes. The hybridization properties of short RNA transcripts were studied using a new gel mobility shift assay from which melting temperatures were determined. RNA hairpins that contained nA and sU were able to hybridize to DNA probes under conditions where the unmodified hairpins did not. DNA-RNA hybrids that contained nA and sU in the RNA strand exhibited enhanced specificity, increased stability, and greater equality of base pairing strength than the same hybrids without modifications. Substitution of guanine (G) with inosine (I) further reduced secondary structure, but RNA with this base hybridized nonselectively. The high stability of nA-T and A-sU base pairs in DNA-RNA hybrids, combined with the destabilizing effect of the nA-sU couple in RNA targets, accounts for the improved hybridization properties. These results suggest that incorporation of nA and sU during in vitro transcription is a promising strategy for enhancing the performance of oligomeric DNA probes with an RNA target.

Topics: 2-Aminopurine; Bacteriophage T7; Base Composition; Base Pair Mismatch; Base Pairing; DNA Probes; DNA-Directed RNA Polymerases; Electrophoretic Mobility Shift Assay; Enzyme Stability; Hot Temperature; Inosine; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes; Nucleic Acid Hybridization; RNA, Viral; Thiouracil; Viral Proteins