Deoxyribozymes that recode sequence information

Abstract

Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and detected. We have engineered binary deoxyribozyme ligases whose two components are brought together by bridging oligonucleotide effectors. The engineered ligases can 'read' one sequence and then 'write' (by ligation) a separate, distinct sequence, which can in turn be uniquely amplified. The binary deoxyribozymes show great specificity, can discriminate against a small number of mutations in the effector, and can read and recode DNA information with high fidelity even in the presence of excess obscuring genomic DNA. In addition, the binary deoxyribozymes can read non-natural nucleotides and write natural sequence information. The binary deoxyribozyme ligases could potentially be used in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of short nucleic acids such as microRNAs.

Figure 1

Design of the bidirectional ligase maxizyme. (a) The DNA ligase was designed to be a binary (two black strands) enzyme with two catalytic domains fused by a common stem structure. When the binary strands associate to form the correct structure, the catalytic domains are formed and are capable of ligating two DNA substrates (green and purple). (b) An effector DNA (red) can specifically base pair with the binary enzyme, stimulating the correct folded structure and catalyzing the ligation of two substrates on the opposite end. In this way the effector oligonucleotide is recoded into a new oligonucleotide ligation product. For convention, we refer to ligation of substrates on the ‘bottom’ and on the ‘top’ of the enzyme.

Figure 2

Secondary structures of deoxyribozymes dR8, dR3 and dR3.1.5. The two oligonucleotides composing the enzymes (black) fold into the active structure (shown) in the presence of the effector (18N, red) in order to bind two substrates (green and purple, as in Figure 1). This orientation is designated as the bottom orientation, and is the standardized presentation of the ligase maxizyme.

Figure 3

Expanded base pairing in an allosteric deoxyribozyme. Construct dR8 was re-engineered to carry a non-natural nucleotide, 5-methyl-isocytidine (iso^MeC), at two positions (blue), generating dR8b. The effector was then synthesized with compensatory isoguanosine (isoG) substitutions, generating 18Nb. The adjacent gel shows the ligation activity of dR8b with different substrates. The lower band on the gel is a radiolabeled, unligated substrate, while the higher band is the ligated product. The lane labeled ‘t0’ is time 0 of the reaction and all subsequent lanes are 3.5 h reactions with effector variants engineered to make the indicated base-pairings. The extent of ligation is normalized to the most active pairing, iG–iC.

Figure 4

Temperature optimization of base pair discrimination. Temperature was increased to aid discrimination by dR8b for the correct effector, 18Nb versus the most active competitor, 18N. Fold discrimination (bars) indicates the ratio of ligated product in the presence of 18Nb to 18N. Circles indicate percent ligation of substrates in the presence of 18Nb. Ligation reactions were allowed to proceed for ∼16 h.

Figure 5

Real-time PCR detection of the ligation product formed by dR8b. (a) Each of the substrates for dR8b was extended to carry one primer-binding site. When ligated, the product becomes a template for PCR. The complementary DNA strand (cDNA, gray) formed by the first primer extension bears a site complementary to a Taqman probe. When the cDNA strand is replicated the Taqman probe is digested, liberating the fluorescent molecule from the quencher. The fluorescent readout is quantified by real-time PCR. (b) Quantification of the ligation product of dR8b by real-time PCR. dR8b was allowed to react with the modified substrates for 5 min and the product was detected as in (A). Minus and plus indicate the absence and presence of the effector molecule, 18Nb in the reaction. C_T shift indicates the decrease in number of cycles required to reach exponential amplification relative to the effector-independent background reaction plus 1000× excess DNA (left-most bar), which had an absolute C_T value of 37 ± 0.15. Error bars represent 2 SDs.